(Methods in Enzymology 13) John M. Lowenstein (Eds.) - Citric Acid Cycle-Academic Press (1969)

Contributors to Volume X I I I
Article numbers are shown in parentheses followlng the names of contributors.

Affiliations listed are current.
S. J. AJL (55), Department o] Bio- vania School o] Medicine, Philadelphia,

chemistry, Albert Einstein Medical Pennsylvania
Center, Philadelphia, Pennsylvania Boa B. BUCHANAN (30), Department o]
NANCY W. ALOOCK (61), Sloan Kettering Cell Physiology, University o] Cali-
Institute ]or Cancer Research, New ]ornia, Berkeley, Cali]ornia
York, New York J. L. ChNovAs (45), Instituto de BioIogia
S. H. G. ALLEN (33, 41), Department o] Cellular, C ~.I.C., Madrid, Spain
Biochemistry, Albany Medical College, Louis N. CECi (70, 78), The Murray
Albany, New York Strassman Memorial Laboratory, De-
DANmL I. ARNON (30), Department o] partment o] Microbiology, Albert
Cell Physiology, University o] Cali- Einstein Medical Center, Philadelphia,
]ornia, Berkeley, Cali]ornia Pennsylvania
R. E. BAaVEN (7), Department o] Bio- SUNOMAN CHA (13), Department o] Bio-
chemistry, University o] Wisconsin, logical and Medical Sciences, Brown
Madison, Wisconsin University, Providence, Rhode Island
H. A. BARKER (50, 51, 52), Department H. C. CHANa (42), Biochemistry De-
o] Biochemistry, University o] CaIi- partment, Central Research, Shulton,
]ornia, Berkeley, Cali]ornia Inc., Cli]ton, New Jersey
MOSHE BENZXMAN (22), Laboratory o] J. F. A. CHASE (60), Department o]
Organic and Biological Chemistry, The Biochemistry, University o] Cam-
Hebrew University o] Jerusalem, Jeru- bridge, Cambridge, England
salem, Israel W. W. CLELANI) (7), Department o] Bio-
JNANENDRA K. BHATTACHARJEE (78), The
chemistry, University o] Wisconsin,
Madison, Wisconsin
Murray Slrassman Memorial Labora-
R. A. Cook (9), Department o] Bio-
tory, Department o] Microbiology,
chemistry, University o] Cali]ornia,
Albert Einstein Medical Center, Phila-
delphia, Pennsylvania Berkeley, California
R. A. COOPER (48), Department o] Bio-
EITAN BOGIN (4), Department o] Plant
chemistry, University o] Leicester,
Biochemistry, University o] Cali]ornla, Leicester, England
Los Angeles, Cali]ornia
BARBARAE. CORKEr (65), Graduate De-
P. D. BORER (14), Molecular Biology partment o] Biochemistry, Brandei.~
Institute, University o] Cali]ornia, Los University, Waltham, Massachusetts
Angeles, Cali]ornia G. F. Cox (10), School o] Biological
RALPH A. BRaDSHAW (17), Department Sciences, University o] East Anglia,
o] Biochemistry, University o] Wash- Norwich, England
ington School o] Medicine, Seattle, S. DAOLEY (28, 67), Department o] Bio-
Washington chemistry, College o] Biological Sci-
W. A. BRIDGER (14), Department o] Bio- ences, University o/ Minnesota, St.
chemistry, University o] Alberta, Ed- Paul, Minnesota
monton, Alberta, Canada DAVID D. DAVIES (26), School o] Biologi-
HAROLD J. BRIaHT (53), Department o] cal Sciences, University o] East Anglia,
Biochemistry, University o] Pennsyl- Norwich, England
vi CONTRIBUTORS TO VOLUME XIII
JUDXTH J. DAvls (47), Department o] chemistry, Dental School, Osaka Uni-

Psychiatry, Stan]ord Medical Center, versity, Osaka, Japan
Pals Alto, Cali]ornia BmGIT JACORSON (36), Department o]
D. V. DERVARTANIAN (16), Institute ]or Biochemistry, Case Western Reserve
Enzyme Research, University o] Wis- University School o] Medicine, Clevc-
consin, Madison, Wiscor~sin land, Ohio
ROBERT J. DUMMEL (79), Department o] WM. P. JENCKS (15), Graduate Depart-
Pharmacology, University o] CaliIornia ment o] Biochemistry, Brandeis Uni-
School o] Medicine, San Francisco, versity, Waltham, Massachusetts
Cali]ornia HAKUJI KATSURA (76), Faculty o] Sci-
DANIEL I)UPOURQUE (20), Deparlment o] ence, Osaka University, Osaka, Japan
Pharmacology, University o] Cali]ornia, YOSHITO KAzmo (31), Department o]
San Francisco Medical Center, School Chemistry, Institute o] Medical Sci-
o] Medicine, San Francisco, Cali]ornia ence, University o] Tokyo, Minatoku,
R. L. E^STEm)AY (43), Pharmacia Fine Tokyo, Japan
Chemicals, Inc., Piscataway, New R. W. KELLERMEYER (33, 35), Depart-
Jersey ment o] Medicine, Case Western Re-
SASHA ENOLARD(18, 21, 74), Biochemistry serve University School o] Medicine,
Department, Albert Einstein College Cleveland, Ohio
o] Medicine, Bronx, New York LEO KESNER (62), Department o] Bio-
B. FANSLEa (6), The Institute ]or Cancer chemistry, College o] Medicine, Down-
Research, Philadelphia, Pennsylvania state Medical Center, State University
P. B. GA~HD (2, 72), Department o] o] New York, Brooklyn, New York
Biochemistry, The Medical School, G. BARRIE grrTO (19, 25), Department o]
Bristol, England Chemistry, Clayton Foundation Bio-
BRENDA I. G~RWIH (36), Department o] chemical Institute, University o] Texas,
Biochemistry, Case Western Reserve Austin, Texas
University School o] Medicine, Cleve- HELMUT R. KLOTZSCH (59), Boehringer
land, Ohio Mannheim Corp., New York, New
KENNETH R. HANSON (74), The Con- York
necticut Agricultural Experiment Sta- H. L. KORNDERO (45, 48), Department o]
tion, New Haven, Connecticut Biochemistry, University o] Leicester,
Lovm B. HERSH (15), Laboratory o] Bio- Leicester, England
chemistry, National Heart Institute, ERNEST KUN (20, 79), Department o!
National Institutes o] Health, Be- Pharmacology, University o] California,
thesda, Maryland San Francisco Medical Center, School
ROBERT L. HILL (17), Department o] Bio- o] Medicine, San Francisco, Cali]ornia
chemistry, Duke University, Durham, M. DANIEL LANE (42, 43), Department
North Carolina o] Biochemistry, New York University
MvawLv. W. HSnNO (53), Department o] School o] Medicine, New York, New
Biophysics, University o] Chicago, York
Chicago, Illinois I-IENRY A. LARDY (37, 49), Institute ]or
R. Y. Hsv (37), Department o] Bio- Enzyme Research, The University o]
chemistry and Microbiology, College Wisconsin, Madison, Wisconsin
o] Agriculture and Environmental Sci- DONALD J. LARTIGUE(54), Department o]
ence, Rutgers, The State University, Biochemistry, Louisiana State Uni-
New Brunswick, New Jersey versity, Baton Rouge, Loui~.iana
KuN-YEN I-IuANO (64), Department o] Y. S. LEwis (77), Central Food Techno-
Microbiology, Naval Medical Research logical Research Institute, Mysore,
Institute, Bethesda, Maryland India
HIDEO INOUE (27), Department o] BIO- W. D. LooM1S (73), Department o] Bio-
CONTRIBUTORS TO VOLUME X I I l vii
chemistry and Biophysics, Oregon State versity School o] Medicine, Cleveland,

University, Corvallis, Oregon Ohio
JOH~ M. LOWENSTFaN (6, 66), Graduate R. PARVIN (3), Biochemistry Division,
Department oS Biochemistry, Brandeis Department o] Biochemistry, Univer-
University, Waltham, Massachusetts sity o] Cali]ornia, Los Angeles, CaIi-
F. A. McELRoY (71), Department o] Bio- Jornia
chemistry, University o] Toronto, P. J. R. PHIZACKERLEY (23), Nuffeld De-
Toronto, Canada partment o] Clinical Biochemistry,
BRUCE A. McFADDEN (29), Department University o] Ox]ord, Ox]ord, England
o] Chemistry, Washington State Uni- G. W. E. PLAUT (8), Department o] Bio-
versity, Pullman, Washington chemistry, R~tgers Medical School,
P. MAERA (44), Laboratory o] Molecu- New Brunswick, New Jersey
lar Biology, University o] Wisconsin, J. R. QUAYLE (46, 57), Department o]
Madison, Wisconsin Microbiology, The University o] She]-
H. MARUYAMA (43), Research Institute, field, Sheffield, England
St. Joseph Hospital, Lancaster, Penn- R. RABIN (55), Department o] Bio-
sylvania chemistry, Albert Einstein Medical
RAJARSHI MAZUMDER (32, 34), Depart- Center, Philadelphia, Pennsylvania
ment o] Biochemistry, All-India Insti- R. F. RAMALEY (14), Department o]
tute o] Medical Sciences, New Delhi, Microbiology, Indiana University,
India Bloomington, Indiana
ROBERT S. MILLER (42), Department o] LESTER J. REED (12), Department o]
Biochemistry, New York University Chemistry, Clayton Foundation Bio-
School o] Medicine, New York, New chemical Institute, University o] Texas,
York Austin, Texas
BARID B. MUKHERJEE (12), Corporate I~. C. REEVES (55), Department o] Bio-
Research, Tenneco Chemicals, Inc., chemistry, Albert Einstein Medical
Piscataway, New Jersey Center, Philadelphia, Pennsylvania
EDWARD MUNTWYLER (62), Department D. R. SANAUI (11), Department o] BIO-
o/ Biochemistry, College o] Medicine, energetic Research, Retina Foundation,
Downstate Medical Center State Uni- Boston, Massachusetts
versity o] New York, Brooklyn, New B. D. SANWAL (9, 44), Department o]
York Microbiology, University o] Manitoba,
WILLIAM H. MURPHEY (25), Department Winnipeg, Canada
o~ Pediatrics, State University o] New M. C. SCRU~ro~ (38), Department o]
York at Buf]alo Medical School, Bu]- Biochemistry, Rutgers University
/alo, New York School o] Medicine, New Brunswick,
WILLIAM F. MYERS (64), Department o] New Jersey
Microbiology, University o] Maryland W. SEUBERT (40), Institut ]iir Vegetative
Medical School, Baltimore, Maryland Physiologie der Universit~it, Chemisch-
DEXTER B. NORTHROP (36), Department Physiologisches Institut der Stadt
o/ Biochemistry, Case Western Reserve Frank]urt, Frank]~lrt/Main, Germany
University School o] Medicine, Cleve- D. SHEPHERD (2), Shell Centre, London,
land, Ohio England
SEVERO OCHOA (34), Department o] Bio- LEWIS SIEGEL (18), Biochemistry Depart-
chemistry, New York University, ment, Albert Einstein College o] Medi-
School o] Medicine, New York, New cine, Bronx, New York
York PAUL A. SREaE (1), Veterans Administra-
M. R. OLMSTED (38), Department o] Bio- tion Hospital. D~dlas, Texas
chemistry, Case Western Reserve Uni- MURRAY STRASSMAN" (70), $ Department
* Deceased.
o , °
Vlll CONTRIBUTORS TO VOLUME XIII
o] Microbiology, Albert Einstein Medi- serve University School o/ Medicine,

cal Center, Philadelphia, Pennsylvania Cleveland, Ohio
FuJIo SuzuxI (27), Department o] Bio- W. S. WEOENER (55), Department o/
chemistry, Osaka University Dental Biochemistry, Albert Einstein Medical
School, Osaka, Japan Center, Philadelphia, Pennsylvania
YOSHIRO TAKEDA (27), Department of H. WEICKER (40), Institut fiir Vegetative
Biochemistry, Osaka University Dental Physiologie der Universittit, Chemisch-
School, Osaka, Japan Physiologisches In~titut der Stadt
V. W. THOMPSON (7), Department o] Frank]urt, Frank]urt/Main, Germany
Biochemistry, University o] Wisconsin, P. D. J. WEIVZMAN (5, 56), Department
Madison, Wisconsin o] Biochemistry, University o] Leices-
BERNADINE TOLBERT (39), Department o] ter, Leicester, England
Biochemistry, Case Western Reserve JAMES M. WILLAaD (47), Department o]
University School o] Medicine, Cleve- Biochemistry, Case Western Reserve
land, Ohio University School o] Medicine, Cleve-
P. K. TUBBS (72), Department o] Bio- land, Ohio
chemistry, University o] Cambridge, G. R. WILL,MS (71), Department o]
Cambridge, England Biochemistry, University o] Toronto.
ANTHONY F. TvccI (78), The Murray Toronto, Canada
Strassman Memorial Laboratory, De-
VIaCINXA R. WILLIAMS (54), Department
partment o] Microbiology, Albert Ein-
of Biochemistry, Louisiana State Uni-
stein Medical Center, Philadelphia,
versity, Baton Rouge, Louisiana
Pennsylvania
M. F. UTTER (3~, 39), Department of JOHN R. WILLIAMSON(65), Johnson Re-
Biochemistry, Case Western Reserve search Foundation, Department o] Bio-
University School o] Medicine, Cleve- physics and Physical Biochemistry,
land, Ohio University o] Pennsylvania Medical
C. V~-EGER (16, 69), Department o] Bio-
School, Philadelphia, Pennsylvania
chemistry, Agricultural University, HAE[aNI) G. WOOD (33, 35, 36, 47), De-
Wageningen, The Netherlands partment o] Biochemistry, Case West-
H. B. VXCKZRY (75), The Connecticut ern Reserve University School o]
Agricultural Experiment Station, New Medicine, Cleveland, Ohio
Haven, Connecticut AKmA YOSHIDA (24), Medical Genetics,
R. W. VoN KoR~ (63, 68), Friends o] Department o] Medicine, University
Psychiatric Research, Incorporated, o] Washington School o] Medicine,
Baltimore, Maryland Seattle, Washington
AaTHUE W^LL^CZ (4), Department o] M. R. YOUNG (39), Department o] Bio-
Plant Biochemistry, University o] Cali- chemistry, Case Western Reserve Uni-
]ornia, Los Angeles, California versity School o] Medicine, Cleveland,
C. C. W^N(~ (51), Department o] Bio- Ohio
chemistry, University o] California, W. P. ZEYLEMAKEE (16, 69), Laboratory
Berkeley, Cali]ornia of Biochemistry, B. C. P. Jansen Insti-
LESLIE W. WEBSTER,JR. (58), Department tute, University o] Amsterdam, Am-
o] Pharmacology, Case Western Re- sterdam, The Netherlands
Preface
The citric acid cycle represents the terminal stage for the oxidation of
the major foodstuffs and energy stores in many organisms. In addition,
the cycle plays an important part in the synthesis of many cell constit-
uents from simple precursors. This volume of "Methods in Enzymology"
deals w.ith the reactions of the citric acid cycle and with a number of
reactions leading to and from the cycle. In deciding what to include in
the present volume, boundaries had to be drawn between various areas
of metabolism and the citric acid cycle. These boundaries are of necessity
somewhat arbitrary. The aspartase reactiofi is included. On the other
hand, enzymes which might have been included here, such as certain
transaminases and glutamate dehydrogenase, will be covered in a future
volume devoted to amino acid metabolism. A few of the methods which
are included should logically have appeared in earlier volumes. These are
warranted by the importance of the topic and the time it would take for
them to appear in future revisions of recent volumes.
The help and cooperation of the contributors is greatfully acknowl-
edged. I am indebted to Drs. H. L. Kornberg, H. A. Lardy, and H. Vickery
for useful suggestions, to Mrs. Janice Bright, Miss Geraldine Conner, and
Miss Kathryn Rader for their skilled and patient secretarial help, and to
the Staff of Academic Press for their cooperation. Much of the work
involved in organizing this volume was performed while I was at the
Johnson Research Foundation of the University of Pennsylvania. My
thanks and appreciation go to Dr. Britton Chance for his hospitality.
March, 1969
JOHN M. LOWENSTEIN
METHODS IN ENZYMOLOGY
EDITED BY
Sidney P. Colowick and Nathan O. Kaplan
VANDERBILT UNIVERSITY DEPARTMENT OF CHEMISTRY
SCHOOL OF MEDICINE UNIVERStITY OF CALIFORNIA
NASHVILLE~ T~NNESSEE AT SAN DIEGO
LA JOLLA, CALIFORNIA
I. Preparation and Assay of Enzymes

II. Preparation and Assay of Enzymes
III. Preparation and Assay of Substrates
IV. Special Techniques for the Enzymologist
V. Preparation and Assay of Enzymes
VI. Preparation and Assay of Enzymes (Continued)
Preparation and Assay of Substrates
Special Techniques
VII. Cumulative Subject Index
METHODS IN ENZYMOLOGY
EDITORS-IN-CHIEF
Sidney P. Colowick Nathan O. Kaplan
VOLUME VIII. Complex Carbohydrates
Edited by ELIZABETH F. NEUFELD AND VICTOR GINSBURG
VOLUME IX. Carbohydrate Metabolism

Edited by WILLIS A. WOOD
VOLUME X. Oxidation and Phosphorylation

Edited by RONALDW. ESTABROOKAND MAYNARDE. PULLMAN
VOLUME XI. Enzyme Structure

Edited by C. H. W. Hms
VOLUME XII. Nucleic Acids (Parts A and B)

Edited by LAWaENCEGROSSMAN AND KIVIE MOLDAVE
VOLUMEXIII. Citric Acid Cycle

Edited by J. M. LOWENSTEIN
VOLUME XIV. Lipids

Edited by J. M. LOWENSTEIN
VOLUME XV. Steroids and Terpenoids

Edited by RAYMONDB. CLAYTON
VOLUMEXVI. Fast Reactions

Edited by KENNETH KUSTIN
VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B)

Edited by HERBERTTABORANDCELIAWHITE TABOR
VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C)

Edited by DONALDB. MCCORMICKAND LEMUELD. WRIGHT
VOLUMEXIX. Proteolytic Enzymes

Edited by GERTRUDEE. PERLMANNAND LASZLOLORAND
xviii
METHODS IN ENZYMOLOGY xix
VOLUME XX. Nucleic Acids and Protein Synthesis (Part C)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN
VOLUME XXI. Nucleic Acids (Part D)

Edited by LAWRENCE GROSSMANAND KIVIE MOLDAVE
VOLUME XXlI. Enzyme Purification and Related Techniques

Edited by WILLIAM B. JAKOBY
VOLUME XXlII. Photosynthesis (Part A)

Edited by ANTHONY SAN PIETRO
VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B)

Edited by ANTHONY SAN PIETRO
VOLUME XXV. Enzyme Structure (Part B)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME XXVI. Enzyme Structure (Part C)

VOLUME XXVII. Enzyme Structure (Part D)

VOLUME XXVIII. Complex Carbohydrates (Part B)

Edited by VICTOR GINSBURG
VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE
VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F)

VOLUME XXXI. Biomembranes (Part A)

Edited by SIDNEY FLEISCHER AND LESTER PACKER
VOLUME XXXII. Biomembranes (Part B)

VOLUME XXXIII. Cumulative Subject Index Volumes I-XXX

Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS
XX METHODS IN ENZYMOLOGY
VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B)

Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK
VOLUME XXXV. Lipids (Part B)

Edited by JOHN M. LOWENSTEIN
VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN
VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones)

VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides)

Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY
VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and

Organ Systems)
Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY
VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function)

VOLUME XLI. Carbohydrate Metabolism (Part B)

Edited by W. A. WOOD
VOLUME XLII. Carbohydrate Metabolism (Part C)

Edited by W. A. WOOD
VOLUME XLIII. Antibiotics

Edited by JOHN H. HASH
VOLUME XLIV. Immobilized Enzymes

Edited by KLAUS MOSBACH
VOLUME XLV. Proteolytic Enzymes (Part B)

Edited by LASZLO LORAND
VOLUME XLVI. Affinity Labeling

Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK
VOLUME XLVII. Enzyme Structure (Part E)

METHODS IN ENZYMOLOGY xxi
VOLUME XLVIII. Enzyme Structure (Part F)

VOLUME XLIX. Enzyme Structure (Part G)

VOLUME L. Complex Carbohydrates (Part C)

Edited by VICTOR GINSBURG
VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism

Edited by PATRICIA A. HOFFEE AND MARY ELLEN JONES
VOLUME LII. Biomembranes (Part C: Biological Oxidations)

VOLUME LIII. Biomembranes (Part D: Biological Oxidations)

VOLUME LIV. Biomembranes (Part E: Biological Oxidations)

VOLUME LV. Biomembranes (Part F: Bioenergetics) (in preparation)

Edited by SIDNEY FLEISCI-IERAND LESTER PACKER
VOLUME LVI. Biomembranes (Part G: Bioenergetics) (in preparation)

Edited by S I D N E Y FLEISCHER AND LESTER PACKER
VOLUME LVII. Bioluminescence and Chemiluminescence

Edited by MARLENE A. DELUCA
VOLUME LVIII. Cell Culture

Edited by WILLIAM B. JAKOBY AND IRA H. PASTAN
VOLUME LIX. Nucleic Acids and Protein Synthesis (Part G)

Edited by KIVlE MOLDAVE AND LAWRENCE GROSSMAN
VOLUME LX. Nucleic Acids and Protein Synthesis (Part H) (in prepa-
ration)
xxii M E T H O D S IN E N Z Y M O L O G Y
VOLUME 61. Enzyme Structure (Part H) (in preparation)

VOLUME 62. Vitamins and Coenzymes (Part D) (in preparation)

Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT
VOLUME 63. Enzyme Kinetics and Mechanism (Part A) (in preparation)

Edited by DANIEL L. PURICH
PREVIOUSLY PUBLISHED ARTICLES FROM METHODS IN EXZY~~OLOGY
RELATED TO SECTIOX I
Vol. I [199]. Deacylases (Thiol E&erase). John Gergely.

Vol. I [114]. Crystalline Condensing Enzyme from Pig Heart. Sever0 Ochoa.
Vol. I [115]. Aconitase from Pig Heart Muscle. Christian B. Anfinsen.
Vol. I [116]. Isocitric Dehydrogenase Syste.m (TPN) from Pig Heart. Sever0 Ochoa.
Vol. I [117]. Isocitric Dehydrogenase from Yeast (TPN). Arthur Kornberg.
Vol. I [118]. Isocitric Dehydrogenase from Yeast (DPN). Arthur Kornberg.
Vol. I [119]. Diphosphopyridine Nucleotide Isocitric Dehydrogenase from Animal
Tissues. G. W. E. Plaut and S.-C. Sung.
Vol. I [lw)]. a-Ketoglutaric Dehydrogenase System and Phosphorylating Enzyme
from Heart Muscle. Seymour Kaufman.
Vol. I [121]. Succinic Dehydrogenase. Walter D. Bonner.
Vol. I [122]. Fumarase. Vincent Massey.
Vol. I 11231. Malic Dehydrogenase from Pig Heart. Sever0 Ochoa.
Vol. V [82]. Succinic Dehydrogenase. Paul Bernath and Thomas P. Singer.
Vol. V [83]. Aconitic Hydrase from Aspergillus niger. Nora E. Nielson.
Vol. V [89]. Isocitric Dehydrogenase (TPN-Linked) fro.m Pig Heart (Revised Pro-
cedure). G. W. E. Plaut.
Vol. V [go]. Formation and Breakdown of Acyl Lipoic Acids. Ernest Knight, Jr. and
I. C. Gunsalus.
Vol. V [116a]. N-Succinyl-L-diaminopimelic Deacylase. S. H. Kindler.
Vol. VI [123]. Histochemical Methods for Dehydrogenases. Arnold M. Seligman.
Vol. VI [127]. The Use of Starch Electrophoresis in Dehydrogenase Studies. I. H.
Fine and L. A. Costello.
Vol. IX [59]. Purification and Resolution of the Pyruvate Dehydrogenase Complex
(Escherichia coli). Lester J. Reed and Charles R. Willms.
Vol. IX [52]. Lipoyl Dehydrogenase from Pig Heart. Vincent Massey.
Vol. IX [59]. o-a-Hydroxy Acid Dehydrogenase. Terenzio Cremona and Thomas P.
Singer.
Vol. X [l]. Enzyme Profiles in Mitochondria. Martin Klingenberg.
Vol. X [2]. Criteria of Homogeneity and Purity of Mitochondria. Christian de Duve.
Vol. X [39]. Preparation and Properties of Succinic-Cytochrome c Reductase (Com-
plex II-III). Howard D. Tisdale.
Vol. X [40]. Preparations of Succinate-Cytochrome c Reductase and the Cytochrome
b-cl Particle, and Reconstitution of Succinate-Cytochrome c Reductase. Tsoo E.
King.
Vol. X [42]. Preparation and Properties of Succinate Dehydrogenase-Coenzyme Q
Reductase (Complex II). D. Ziegler and J. S. Rieske.
Vol. X [58]. Preparation of Succinate Dehydrogenase and Reconstitution of Succinate
Oxidase. Tsoo E. King.
Vol. X [72a]. Isolation of a Mitochondrial Membrane Fraction Containing the Citric
Acid Cycle and Ancillary Enzymes. David W. Allmann and Elisabeth Bachmann.
Vol. X [112]. Energy-Linked Reduction of NAD’ by Succinate. Lam Ernster and
Chuan-pu Lee.
[1] CITRATE SYNTHASE 3
[ 1] C i t r a t e S y n t h a s e 1, 2
[EC 4.1.3.7 Citrate oxaloacetate-lyase (CoA-acetylating)]
B y P. A. SRERE
Acetyl-CoA -t- oxaloacetate 2- -t- H20 ~ citrate ~- -t- CoASH ~- H + (1)
Assay Methods
Utilization o] Acetyl Phosphate

The assay of citrate synthase can be performed by coupling it to the
transacetylase reaction. 2
Acetyl phosphate -t- CoASH ~ acetyl-CoA + P~ (2)
The disappearance of acetyl phosphate can be followed by a hydrox-
amate 3 method and the formation of citrate by the pentabromoacetone 4
method.
Utilization of Acetyl-CoA
Acetyl-CoA has an adsorption band at 233 m s due to the thioester
bond. As the citrate synthase reaction proceeds and acetyl-CoA is used,
there is a decrease in absorption at this wavelength. Oehoa 5 and Srere 6
have presented the details of the method. I t is the most straightforward
of all assays in t h a t only reaction components are present. The high
absorbancy of proteins at 233 m s makes this method unsuitable for
assay of crude tissue extracts. Eggerer 7 has pointed out that the extinc-
tion coefficient for this reaction changes at higher pH's due to increase
of formation of CoAS-, which has considerable absorption at this wave-
length.
Utilization o] Oxaloacetate
The most commonly used assay is the spectrophotometric coupled
assay described by 0choa. 2 The malate dehydrogenase catalyzed reaction
L-Malate 2- -t- D P N + ~ oxaloacetate 2- -{- D P N H -t- H + (3)
1Also known as the condensing enzyme; the citrate-condensing enzyme; acetyl

CoA-oxaloacetate condensing enzyme; oxaloacetate transacetase; and citrogenase.
2S. Ochoa, Vol. I, p. 685.
F. Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945).
'S. Natelson, J. B. Pincus, and J. K. Lugovy, J. Biol. Chem. 175, 745 (1948).
5S. Oehoa, Biochem. Prep. 5, (1957).
' P. A. Srere and G. W. Kosicki, J. Biol. Chem. 236, 2557 (1961).
~W. Buckel and H. Eggerer, Biochem. Z. 343, 29 (1963).
4 REACTIONS ON THE CYCLE [1]
is used to generate the oxaloacetate for the citrate synthase reaction. The
formation of D P N H is followed at 340 m~ or with greatly enhanced
sensitivity by fluorometry.8 In this assay the steady state concentration
of oxaloacetate is low and cannot be varied, and reaction rates lower than
for other methods are obtained2 The coupled nature of this assay leads
to systematic errors when this system is used for the stoichiometric
determination of acetyl-CoAJ,"
Formation oJ Citrate
Another method 1° for assaying citrate synthase uses 14C-acetyl-CoA
and measures its incorporation into "C-citrate, which is isolated as a
silver salt. Citrate formation can be measured colorimetrically as men-
tioned above.
Formation oJ CoASH
Citrate synthase can be followed by measuring the appearance of the
free SH group of the released CoASH; three such methods have appeared
in the literature.
One method is to measure the oxidation of the CoASH by dichloro-
phenol-indophenol, which is accompanied by a decrease in absorbancy at
578 m~. ~° Still another method measures the CoASH polarographically."
The third method measures the SH by use of 5,5'-dithiobis-(2-nitro-
benzoate) (DTNB) (Ellman's reagentl~,~").
O,N~ S--S--~N02+ RSH
coo" coo-
O,N ~~_ S--S--R + -S--~NO~+ H+

COO"
' D. Shepherd and P. B. Garland, Bioehem. Biophys. Res. Commun. ~2, 89 (1966).
'D. J. Pearson, Biochem. $. 95, 23c (1965).
"O. Wieland, L. Weiss, and I. Eger-Neufeldt, Bioehem. Z. 339, 501 (1964).
"P. D. $. Weitzrnan, Bioehem. J. 99, 18p (1966).
"P. A. Srere, H. Brazil, and L. Gonen, Aeta Chem. 8cand. 17, $129 (1963).
"G. L. Ellman, Arvh. Biochem. Biophys. 82, 70 (1959).
This reaction is easily followed at 412 n ~ where the mercaptide ion

has a strong absorption (E = 13,600); none of the starting materials
absorb at this wavelength. The pH range that can be studied is limited
to 7.4-9.0.
When either acetyl-CoA or oxaloaeetate is used in limiting amounts
in this assay, it can be used as a rapid and sensitive assay for the limiting
compound.
Reagents
DTNB, 1 mM: 3.9 mg of DTNB (free acid) is dissolvcd in l0 ml
of 1 M Tris-HC1, pH 8.1
Acetyl-CoA, 10 mM: 10 mg of AcCoA dissolved in H20 or 10 mg
of CoA q- 0.9 ml H~O -[- 0.1 ml 1 M KHCO3 -[- 0.013 ml acetic
anhydride
0xaloacetate, 10 mM: 1.32 mg OAA dissolved in 1 ml of Tris-HC1
buffer 0.1 M (prepared fresh daily)
Procedure. Add the following to a cuvette: DTNB, 0.1 ml; acetyl-

CoA, 0.03 ml; enzyme solution, 0.05 ml adjusted by dilution to contain
0.1-0.4 unit of enzyme per milliliter; and H20, 0.77 ml.
The absorption at 412 m~ is followed for 3 minutes to measure
possible acetyl-CoA deacylase activity. The citrate synthase reaction is
then started by the addition of 0.05 ml of oxaloacetate. Linear rates are
obtained for at least 3 minutes.
Purification Procedure
Preparation froqn Pig Heart

Extraction. Fresh pig hearts, packed in ice, were trimmed of fat and
connective tissue and cut into l~-inch cubes. One hundred-gram portions
of the tissue were weighed into plastic bags; these were sealed and
placed in the deep freeze because more enzyme can be extracted from
frozen tissue than from fresh tissue. No loss in activity occurred upon
3-6 months storage of the frozen tissue. Unless otherwise noted, all
operations were performed at 3 ° .
One hundred grams of frozen pig heart was placed in a cold Waring
blendor containing 400 ml of 0.4 M KC1 in 20% ethanol (4 volumes of
0.5 M KCl plus one volume of absolute alcohol) at --10 ° and homog-
enized for 10 minutes. Tile temperature rises to 20 ° during homogeniza-
tion, with no loss of activity. Tile homogenate was centrifuged for 15
minutes at 23,000 g, and the supernatant fluid was dialyzed against 8
liters of cold 2 mM potassium phosphate, pH 7.4, for 2 hours. The out-
side fluid was then changed and dialysis continued for another 2 hours.
The dialyzate was centrifuged at 23,000 g for 15 minutes and the pre-
cipitate was discarded. This dialyzate is stable overnight at 3 °.
Ammonium Sulfate Fractionation. Ammonium sulfate, 31.3 g, was
added to each 100 ml of dialyzate (approximately 50% saturation), and
the mixture was stirred for 15 minutes. The precipitate was removed by
centrifugation at 23,000 g for 15 minutes; 13.5 g of ammonium sulfate
was added to each 100 ml of the supernatant fluid (approximately 70%
saturation), and this mixture was stirred for 15 minutes. The precipitate,
collected by centrifugation, was dissolved in a small amount of cold
H20 (10-20 ml). This fraction was stable overnight at 3% The salts were
removed either by dialysis against 2 mM potassium phosphate or by
use of a Sephadex (G-25) column. We routinely placed the fraction on a
column containing 100 g of Sephadex (4.5 cm X 35 cm) and collected the
H20 eluate until the resistance of the solution (measured in a conduc-
tivity cell) was equal to the resistance of a 2 mM potassium phosphate,
pH 7.4, solution. The removal of salt with Sephadex was carried out at
room temperature. The Sephadex-treated enzyme was stable overnight.
DEAE-Cellulose. The enzyme in 2 mM potassium phosphate is added
to 100 g of DEAE-cellulose which is suspended in 1 liter of 2 mM potas-
sium phosphate, pH 7.4. After filtration the cellulose was washed with
two l-liter portions of 2 mM potassium phosphate, pH 7.4, two l-liter
portions of 8 mM potassium phosphate, pH 7.4, and then with seven
l-liter portions of 18 mM potassium phosphate, pH 7.4. The cellulose
was collected by filtration on a Biichner funnel after each elution.
The fractions containing the bulk of the activity were combined, and
a 5% volume of calcium phosphate gel (17 mg/ml) was added. The
mixture was allowed to stand overnight and the gel collected either by
centrifugation or by decantation and centrifugation. The enzyme was
eluted from the gel with two washings of 50-75 ml of 50% saturated
ammonium sulfate (neutralized to pH 7.4). Occasionally we have con-
centrated the enzyme in a flash evaporator at room temperature without
loss of activity.
Ammonium Sulfate Precipitation and Crystallization. Solid ammo-
nium sulfate was added to the ammonium sulfate eluate to 70% satura-
tion (specific gravity 1.165). The precipitate was collected by centrifuga-
tion (23,000 g for 15 minutes), dissolved in potassium phosphate, 20 mM,
pH 7.4, with a protein concentration above 20 mg/ml. Crystallization was
induced by slow addition of ammonium sulfate, in either a solid or
saturated solution. Crystallization was allowed to proceed for 3 days at
3 ° . Crystals could be obtained (as judged by the "silkiness" of the
precipitate) from solutions containing as little as 2 mg of protein per
TABLE I
PURIFICATION OF PIG I-IEART CITRATE SYNTHASE
Total activity
Fraction (units) ~ Specific activityb
KC1-ETOH extract 2350 0.35

50-70% ammonium sulfate 2170 1.6
Combined DEAE eluate 1460 10 5
Calcium phosphate 1110 25 8
Second crystals
Supernatant 270 33
Precipitate 425 33
Units are expressed in terms of the coupled malate dehydrogenase assay (340 mu),
25 ° for 100 g
b Units per milligram of protein.
milliliter. W h e n the p r o t e i n c o n c e n t r a t i o n was low, p r e c i p i t a t i o n was

n e v e r complete a n d a d d i t i o n a l a m m o n i u m sulfate was added to the
s u p e r n a t a n t fluid to recover the a c t i v i t y . T h e results of a complete
p u r i f i c a t i o n are shown in T a b l e I. Several r e c r y s t a l l i z a t i o n s are necessary
to remove c o n t a m i n a t i n g m a l a t e dehydrogenase.
Preparation o] Enzyme ]rom Pigeon Breast

T h e m e t h o d used for the p u r i f i c a t i o n of this e n z y m e was i d e n t i c a l to
t h a t described for the pig h e a r t enzyme. T h e o n l y difference in b e h a v i o r
was observed d u r i n g the D E A E - c e l l u l o s e step. T h e pigeon b r e a s t e n z y m e
TABLE II
PURIFICATION OF CITRATE SYNTItASE FROM PIGEON BREAST M U S C L E a
Total activity
Fraction (units) Specific activity
KC1-ETOH extract 2900 0.08

50-70% ammonium sulfate 2600 O. 58
DEAE eluate 1540 --
Combined calcium phosphate eluate 4750 S. 9

70% ammonium sulfate precipitate 4250 16.1
First crystals 2420 30
Residue 900 10
Second crystals 1750 50
" The top half of the table represents a typical purification from 100 g of pigeon
breast muscle. The bottom half of the table represents a purification where the
eluates from three separate 100 g runs were combined and purified together. Units
are the same as in Table I.
is eluated with 8 m M potassium phosphate buffer, pH 7.4, whereas the

pig heart enzyme is not eluted until 18 m M potassium phosphate buffer,
pH 7.4. A summary of a typical purification is shown in Table II.
Preparation o] Enzyme ]rom Moth Muscle

Fifty grams of thoraces are homogenized for 2 minutes in 500 ml of
ice cold water, using a Waring blendor. The homogenate is centrifuged
for 15 minutes at 20,000 g. The supernatant fluid is poured through
cheesecloth. DEAE-cellulose, 200 g wet weight, equilibrated with 2 m M
potassium phosphate, pH 7.4, arc taken up in 500 ml of H20 and added
TABLE III
PURIFICATION OF CITRATE SYNTHASE FROM MOTH
(Samia cynthia) FLIGHTMUSCLE
Total activity
Fraction (units) = Specific activity
H20 Extract 4200. 0.83 b

DEAE eluate 3500 2.1 b
Calcium phosphate eluate 3850 8.9
80% ammonium sulfate precipitate 2580 15
Crystals 40¢
° Units as in Table I.
b These extracts have materials which absorb strongly at 260 mg so that Lowry's
method is used for protein determination at these steps.
c The crystals reported here represent the reprecipitation of a number of ammonium
sulfate precipitates. A 30-50~ yield in the crystallization step is obtained.
to the extract. The purification of citrate synthase then follows the pro-
cedure outlined for the pig heart enzyme except in the last ammonium
sulfate step. While the pig heart enzyme is recovered with 70% ammo-
nium sulfate saturation, it is necessary to have 80~5 ammonium sulfate
saturation, pH 6.5, in order to precipitate all the moth enzyme. A sum-
mary of the purification procedure is shown in Table III.
In addition to the crystalline enzymes reported here, the enzyme has
been partially purified from liver, yeast, Escherichia coli, and lemons.
Characteristics of the Pig Heart Enzyme

Substrates. In addition to acetyl-CoA and oxaloacetate both fluoro-
acetyl-CoA 14 and fluoroxaloacetate 15 have been shown to be substrates
for citrate synthase.
Citryl-CoA, a postulated intermediate, is both hydrolyzed to citrate
"R. O. Brady, J. Biol. Chem. 217, 213 (1955).
'~D. W. Fanshier, L. K. Gottwald, and E. Kun, J. Biol. Chem. 239, 3588 (1962).
and CoA ~G,'7 and clcaved to acetyl-CoA and oxaloacetate. 1~ Only one of
the citryl-CoA isomers undergoes this reaction, presumably the S isomer.
S-Malyl-CoA is hydrolyzed by the enzyme but R-malyl-CoA Is is not.
Eggerer has shown by tritium exchange experiments 1~ that, in the
presence of S-malate (but not R-malate), the enzyme will catalyze a slow
exchange between the protons of the medium and the hydrogens of the
methyl carbon of the acetyl group of acetyl-CoA. Using nuclear magnetic
resonance 2° techniques it was shown that a-ketoglutarate also can act as
an inducer for this reaction.
Compounds that are not substrates include glycolyl-CoA, proprionyl-
CoA, butyryl-CoA, glyoxalate, pyruvate, monoethyl oxaloacetate, a-keto-
glutarate, ketomalonate, and a-ketobutyrate. 21 B-Hydroxyl B-methyl
glutaryl-CoA, R-malyl-CoA, succinyl-CoA, glutaryl-CoA, and malonyl-
CoA are among the compounds not hydrolyzed by the enzyme. TM Neither
acetylpantetheine 22 nor acetylacyl carrier protein ~3 can serve as sub-
strafes for the pig heart enzyme.
Inhibitors. The enzyme is inhibited when it is acetylated by acetic
anhydride 17 or iodinated by iodine. ~4 Sulfhydryl reagents such as N-
ethylmaleimide, iodoacetate, or ferricyanide do not inhibit the enzyme
activity. The sulfhydryl groups can be titrated with t t g ++, Ag ++, and
pCMB without inhibition, but aggregation will occur after a short time,
leading to loss of activity. 24
Long-chain acyl-CoA derivatives inhibit the enzyme. ~5-~6 This can
be prevented by oxaloacetate but not reversed by this compound. ~6 On
the other hand the inhibition can be reversed by palmitoylcarnitine 2:
and albumin. The palmitoyl-CoA inhibition is seen in enzyme derived
from moth, ~6 pigeons, ~6 rat liver, TM and Escherichia coll. A T P is a com-
petitive inhibitor for acetyl-CoA 2s in the reaction, with A D P and AMP
,o H. Eggerer and U. Remberger, Biochem. Z. 337, 202 (1963).
"P. A. Srere, Biochim. Biophys. Acta 77, 693 (1963).
'sH. Eggerer, U. Remberger, and C. Grunewalder, Biochem. Z. 330, 435 (1964).
1~H. Eggerer, Biochem. Z. 343, 111 (1965).
,o p. A. Stere, Biochem. Biophys. Res. Commun. 26, 609 (1967).
.~1p. A. Srere, J. Biol. Chem. 241, 2157 (1966).
2.oj. R. Stern, in "The Enzymes," (P. D. Boyer, H. Lardy, and K. Myrbgck, eds.),
Vol. 5, p. 367. Academic Press, New York, 1961.
"A. W. Alberts, personal communications (1966).
'~P. A. Srere, Biochem. Biophys. Res. Commun. 18, 87 (1965).
2~p. K. Tubbs, Biochim. Biophys. Aeta 70, 608 (1963).
~" O. Wieland and L. Weiss, Biochem. Biophys. Res. Commun. 13, 26 (1963).
P. A. Srere, Biochim. Biophys. Acta 106, 445 (1965).
"I. Fritz, Bioehem. Biophys. Res. Commun. 22, 744 (1966).
"J. A. Hathaway and D. E. Atkinson, Biochem. Biophys. Res. Commun. 20, 661
(~5).
having only small effects. Metal ions can overcome the ATP inhibitionJ ~
The A T P inhibition has been shown to be operative in the citrate syn-
thase from liver, yeast, and lemons. The enzyme from E. coli does not
seem to be inhibited by ATP, but is inhibited by NADH 3° and is very
sensitive to palmitoyl-CoA21
Desulfo-CoA has been shown to be a competitive inhibitor for acetyl-
CoA in the enzyme from pig heart22
Treatment of the enzyme with diisopropyl fluorophosphate, potassium
cyanate, acetylimidazole, or peroxide does not result in a loss of activity.
Physical Properties. The molecular weight of the pig heart enzyme
determined by light scattering, osmotic pressure, and sedimentation
equilibrum measurements is about 87,000. The sedimentation coefficients
of the pig heart and pigeon breast enzyme is about 5.9, while that of the
moth flight muscle enzyme is about 5.0. Optical rotatory dispersion
measurement gives values of a ° = 100 and b ° = 250 indicating about
30% helix content for the pig heart enzyme.
TABLE IV
AMINO ACID COMPOSITION a OF CITRATE SYNTHASESb
Amino Amino
acid- acid
residue Pig Pigeon Moth residue Pig Pigeon Moth
Lys 5.9 6.7 8.9 G|y 8.0 8.5 8.0

His 3.3 2.6 2.6 Ala 8.4 9.9 8.1
Arg 4.3 5.0 3.7 Val 6.4 7.5 7.8
Asp 9.3 10.2 9.8 Met 3.4 3.3 2.2
TAr 5.5 4.9 4.9 Ile 4.4 6.0 4.6
Ser 6.5 5.1 5.5 Leu 11.9 9.3 10.2
Glu 10.0 10.0 10.6 Tyr 4.2 2.7 3.6
Pro 5.7 4.9 6.0 Phe 3.0 3.4 3.5
Mole percent.
b We are indebted to Dr. C. Yanofsky for these amino acid analyses.
The stability of the enzyme depends greatly on the ionic strength of
the solution. At low ionic strengths the enzyme loses activity even at 0 °
in a few hours. At high ionic strength (~ = 0.2) the enzyme is quite
stable. The stability of the enzyme can be greatly enhanced if oxalo-
acetate is added. The binary complex that is formed has a good heat
s t a b i l i t y a n d is r e s i s t a n t to u r e a d e n a t u r a t i o n .
'9 G. W. Kosicki and L. P. K. Lee, J. Biol. Chem. 241, 3571 (1966).
~op. D. J. Weitzman, Biochim. Biophys. Acta 128, 213 (1966).
sip. A. Srcre and N. Whissen, Federation Proc. 26, 559 (1967).
32j. F. A. Chase, B. Middleton, and P. K. Tubbs, Biochem. Biophys. Res. Commun.
23, 208 (1966).
[2] CITRATE SYNTHASE FROM RAT LIVER 11
Chemical Composition. Amino acid analyses of the three crystalline

synthases are shown in Table IV. End group studies have not been re-
ported, nor are there available data on possible subunit composition.
Distribution. The enzyme has been detected in all aerobic cells that
have been examined. I t has also been detected in the obligate anaerobe
Clostridium kluyveri? ~,3~ There is a striking correlation between the
respiratory capacity of a cell and its citrate synthase content.
In cells containing mitochondria the enzyme is exclusively in that
compartment. Bachmann et al. ~s report t h a t the enzyme is localized in
the outer membrane of pig heart mitochondria.
~sE. Gottschalk and H. A. Barker, Biochemistry 5, 1125 (1966).
3,j. R. Stem, C. S. Hegre, and G. Bambers, Biochemistry 5, 1119 (1966).
E. Bachmann, D. W. Allmann, and D. E. Green, Arch. Biochem. Biophys. 115, 153
(1~),
[2] Citrate Synthase from Rat Liver 1

B y D. SHEPHERD and P. B. GARLAND
Acetyl-S-CoA + oxaloacetate 2- + H20 ~ - citrate 3- + CoASH + H +
Citrate synthase has been well characterized previously from a

variety of sources, including pig heart, 2 pig liver, s pigeon breast muscle, ~
moth flight muscle, 4 yeast 5 and Escherichia coli, e and a regulatory func-
tion has been postulated 1, 5, 6 since it is effected allosterically by adenine
nucleotides 1,~ or N A D H . 6 Evidence that citrate synthase controls the
flow of acetyl-CoA into the tricarboxylic acid cycle has been presented
only in the case of rat liver, and the enzyme m a y not have a regulatory
role in those tissues where its activity is in considerable excess (20- to
40-fold) of the maximal rates of acetyl-CoA production or isocitrate
oxidation. 7
1D. Shepherd and P. B. Garland, Biochem. Biophys. Res. Commun. 22, 89 (1966).
~S. Ochoa, J. R. Stern, and M. C. Schneider, J. Biol. Chem. 193, 691 (1951); see
Vol. I [114].
O. Wieland, L. Weiss, and I. Eger-Neufeldt, Biochem. Z. 339, 501 (1964).
' P. A. Stere, H. Brazil, and L. Gonen, Acta Chem. Scan& 17, S129 (1963).
~J. A. Hathaway and D. E. Atkinson, Biochem. Biophys. Res. Commun. 20, 661
(I~).
op. D. J. Weitzman, Biochim. Biophys. Acta 128, 213 (1966).
'P. B. Garland, D. Shepherd, D. W. Yates, D. G. Nicholls, and P. Ann Light, in
"Control of the Citric Acid Cycle" (J. M. Lowenstein, ed.). Dekker, New York,
in press.
Chemical Composition. Amino acid analyses of the three crystalline

synthases are shown in Table IV. End group studies have not been re-
ported, nor are there available data on possible subunit composition.
Distribution. The enzyme has been detected in all aerobic cells that
have been examined. I t has also been detected in the obligate anaerobe
Clostridium kluyveri? ~,3~ There is a striking correlation between the
respiratory capacity of a cell and its citrate synthase content.
In cells containing mitochondria the enzyme is exclusively in that
compartment. Bachmann et al. ~s report t h a t the enzyme is localized in
the outer membrane of pig heart mitochondria.
~sE. Gottschalk and H. A. Barker, Biochemistry 5, 1125 (1966).
3,j. R. Stem, C. S. Hegre, and G. Bambers, Biochemistry 5, 1119 (1966).
E. Bachmann, D. W. Allmann, and D. E. Green, Arch. Biochem. Biophys. 115, 153
(1~),
[2] Citrate Synthase from Rat Liver 1

B y D. SHEPHERD and P. B. GARLAND
Acetyl-S-CoA + oxaloacetate 2- + H20 ~ - citrate 3- + CoASH + H +
Citrate synthase has been well characterized previously from a

variety of sources, including pig heart, 2 pig liver, s pigeon breast muscle, ~
moth flight muscle, 4 yeast 5 and Escherichia coli, e and a regulatory func-
tion has been postulated 1, 5, 6 since it is effected allosterically by adenine
nucleotides 1,~ or N A D H . 6 Evidence that citrate synthase controls the
flow of acetyl-CoA into the tricarboxylic acid cycle has been presented
only in the case of rat liver, and the enzyme m a y not have a regulatory
role in those tissues where its activity is in considerable excess (20- to
40-fold) of the maximal rates of acetyl-CoA production or isocitrate
oxidation. 7
1D. Shepherd and P. B. Garland, Biochem. Biophys. Res. Commun. 22, 89 (1966).
~S. Ochoa, J. R. Stern, and M. C. Schneider, J. Biol. Chem. 193, 691 (1951); see
Vol. I [114].
O. Wieland, L. Weiss, and I. Eger-Neufeldt, Biochem. Z. 339, 501 (1964).
' P. A. Stere, H. Brazil, and L. Gonen, Acta Chem. Scan& 17, S129 (1963).
~J. A. Hathaway and D. E. Atkinson, Biochem. Biophys. Res. Commun. 20, 661
(I~).
op. D. J. Weitzman, Biochim. Biophys. Acta 128, 213 (1966).
'P. B. Garland, D. Shepherd, D. W. Yates, D. G. Nicholls, and P. Ann Light, in
"Control of the Citric Acid Cycle" (J. M. Lowenstein, ed.). Dekker, New York,
in press.
Assay Method
Spectrophotometric DTNB Assay

Principle. The method 4 is based on the chemical coupling of CoASH,
released from acetyl-CoA during the enzymatic synthesis of citrate, to
Ellman's s reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The re-
lease of the absorbing mercaptide ion is followed at 412 m#, with 530 or
355 m~ as a reference in a double-beam instrument; the molar absorb-
ancy index is 13,600. The double-beam spectrophotometer a has high
sensitivity with a noise level of about 0.0001 optical density units and a
response time of 0.25 second.
Reagents
Tris-HCl buffer, 0.1 M pH 8.0
DTNB 10 mM (4 mg/ml) in 0.1 M Tris-HC1, pH 8.0 l°
Potassium oxaloacetate 50 mM 11
Acetyl-CoA, ca. 5 mM 12
Procedure. The reaction mixture (in 2.0 ml ceils; d---- 1.0 cm) con-
tains 2.0 ml of buffer, 10 t~l of oxaloacetate (0.5 micromole), 20 ~l of
D T N B (0.2 micromole), 20 ~l of acetyl-CoA (0.1 micromole), and about
0.03 unit of the enzyme. The reaction is carried out at 25 ° and is started
by the addition of citrate synthase. D T N B and acetyl-CoA must be
added initially, and in that order, to estimate the CoASH blank. This
assay can also be used to calibrate acetyl-CoA because 0.1 mieromole
gives an optical density change of about 0.68 under the above conditions.
The reaction is linear for about 2 minutes. This is a routine assay used
during the purification procedure and has the added advantage that if
the reaction is initiated by the addition of oxaloacetate, the rate prior
to this is a measure of the deacylase activity.
Units. One unit of enzyme is defined as that amount which, under
the conditions of the above assay, catalyzes the synthesis of 1 micromole
of citrate per minute at 25 ° . Specific activity is defined as units per
milligram protein.
FIuorimetric Assay ~
Principle. This is a more sensitive assay, applicable to kinetic studies,
based on the coupled malic dehydrogenase assay described by Ochoa?
SG. L. Ellman, Arch. Biochcm. Biophys. 82, 70 (1959).
*B. Chance, Rev. •ci. Instr. 22, 634 (1951).
1, This solution deteriorates with age and should be freshly prepared.
"A freshly prepared solution of oxaloacetie acid is neutralized before use with
potassium hydroxide, pH 7.5.
=Prepared by the method described by S. Ochoa, see Vol. I [114].
The reaction may be carried out using any temperature-stabilized fluorim-

eter which incorporates a recorder fitted with a zero suppression device,
necessary for zeroing after oxaloacetate production. The exciting wave-
length for N A D H assay is 340 m~ (or 366 m~ with a mercury vapor
lamp), and the secondary filter should be 420-3000 m~ (e.g., Wratten
2E).
Reoge~ts
Tris-HC1 buffer, 0.1 M, pH 7.4
Tris malate, 1.0 M, pH 7.4
Acetyl-CoA, ca. 5 mM
NAD+, 50 mM
NADH, 0.5 mM (standardized)
Malic dehydrogenase (specific activity 720; l0 mg/ml)
Procedure. The reaction mixture (in 2.0 ml cells) contains 2.0 ml

of buffer, 20 ~l of malate (20 micromoles), 20 ~l of NAD ÷ (1 micromole),
5/~l of malic dehydrogenase (36 units), 20 #l of acetyl-CoA (0.1 micro-
mole), citrate synthase (10 -5 to 10-3 unit), the additions being made in
this order. When the malie dehydrogenase reaction has equilibrated, the
recorder deflection giving the oxaloacetate concentration (about 20
under the conditions given), the recorder is returned to zero and acetyl-
CoA and citrate synthase are added. Under these conditions, fluorescence
increases linearly with time for 2 minutes. The fluorimeter is calibrated
using N A D H which has been standardized spectrophotometrically.
Units. These are defined as for the previous assay.
Citrate synthase in rat liver is localized intramitochondrially, and
hence the firststep in the purification of the enzyme is the preparation of
mitochondria, since this reduces the amount of starting protein by 50-
60% without any loss in activity.
Step 1. Preparation o/ Rat Liver Mitochondria. Mitochondria are
prepared from the livers of 30 male Wistar rats, 200-300 g body weight,
by a procedure similar to that of Lardy and Wellman, 13 except for the
two final washings, which are omitted.
Step ~. Sonication o] Mitochondria. The mitochondrial pellet is re-
suspended in four times its own volume of 0.1 M potassium phosphate
buffer, pH 7.4; 25 ml batches of this 20% suspension are sonicated in
stainless steel centrifuge tubes, cooled by an ice-salt mixture, for 8
minutes using an M.S.E. 60 W ultrasonicator at 20 kc/sec. The sonicate
lSH. A. Lardy and It. Wellman, J. Biol. Chem. 201, 357 (1953).
is spun at 78,000 g for 90 minutes at 4 ° in a Beckman L2 centrifuge,

using the 30 head {12 X 38 ml), and the precipitates are discarded.
Step 3. Ammonium Sul]ate Precipitation. The sonic supernatants are
pooled to give 0.8 liter of a clear red solution, which is brought to 50%
saturation with solid ammonium sulfate (31.2 g/100 ml). The salt is
added slowly over a period of an hour at 0 ° with mechanical stirring, and
the solution is allowed to stand for a further 30 minutes, before centrif-
ugation at 23,000 g for 20 minutes at 4 °. The precipitate is discarded.
The supernatant is brought to 75% saturation with solid ammonium
sulfate (17.2 g/100 ml) added slowly over a period of 30 minutes, and
centrifuged as above, after standing. The supernatant is discarded. The
precipitate is taken up in 15 ml of 0.1 M potassium phosphate buffer pH
7.4, and the solution is dialyzed against 5 liters of 2 mM potassium
phosphate buffer, pH 7.4, overnight at 4 °. The volume increases to 45 ml
during dialyses.
Step ~. Fractionation on a DEAE-Cellulose Column. The enzyme
solution is placed on a DEAE-cellulose column (4.0 X 20.0 cm) equili-
brated previously with 2 mM potassium phosphate, pH 7.4; the column
is washed with 1 liter of 2 mM potassium phosphate buffer, pH 7.4. No
citrate synthase activity is eluted during this washing, but 150 mg of
protein are removed, including the deacylase activity. The column is
next washed with 1 liter of 8 mM potassium phosphate buffer, pH 7.4.
No citrate synthase activity is eluted during this washing. The column
is washed finally with 18 mM potassium phosphate buffer, pH 7.4. Frac-
tions of 250 ml are collected, and the bulk of the citrate synthase
activity is eluted in the second and third fractions, which are pooled.
Step 5. Adsorption on Calcium Phosphate Gel. To the pooled fractions
from the column procedure is added 2 ml per 100 ml of 18 mg of
calcium phosphate gel per milliliter, prepared according to Dixon and
Webb, ~4 and the 2% suspension is stirred mechanically at 0 ° for 1 hour
before centrifugation at 2500 g for 15 minutes. The supernatant is dis-
carded. The precipitate is suspended in 10 ml of 0.1M potassium
phosphate buffer, pH 7.4, and stirred mechanically for 20 minutes before
centrifugation at 2500 g for 15 minutes. Four more elutions of the gel
are carried out in a similar manner until the bulk of the citrate synthase
activity is removed from the gel.
Step 6. Ammonium Sul]ate Concentration o] the Calcium Phosphate
Gel Eluate. The pooled eluates from step 5 (50 ml) are brought to 5 0 ~
saturation with solid ammonium sulfate in a manner similar to that
described in step 3, and the precipitate is discarded. The supernatant is
~4M. Dixon and E. C. Webb, (eds.), "Enzymes" 2nd ed., p. 42. Academic Press,
New York, 1964; see also Vol. I [11].
brought to 75% saturation, and the precipitate is centrifuged and dis-

solved in a minimal amount (ca. 2 ml) of 0.1 M potassium phosphate
buffer, pH 7.4. The enzyme solution is dialyzed overnight at 4 ° against
5 liter of 2 m M potassium phosphate buffer, pH 7.4, and the precipitate
is removed by centrifugation at 23,000 g for 15 minutes.
Notes to Purification Procedure. The specific activity of citrate
synthase prepared as described is 15, and by comparison with the
specific activity of crystalline citrate synthase from other sources it
would appear to be about 30% pure. I t is likely that with a larger amount
of starting material, crystals could be obtained. D a t a on the purification
of the enzyme are summarized in the table.
PURIFICATION PROCEDURE FOR CITRATE SYNTHASE FROM RAT LIVER
Volume
of Total Purifi-
solution activity Protein Specific cation" Yield
Step (ml) (units) (mg) activity (fold) (%)
2. Sonic supernatant 810 635 5780 0.11 1 100

3. 50-75 Am~SO4 16 143 540 0,27 2.4 22
After dialysis 45 280 540 0,5 4.5 44
4. DEAE eluate 570 151 35 4,3 39 24
5. Ca3(PO4)~gel eluate 50 97 7.5 13 120 15
6. 50-75 Am~SO4 2 53 5.5 10 90 8
After dialysis 3 40 2.7 15 135 6
a The specific activity of citrate synthase in broken mitochondria is about 0.05
unit/rag and hence the overall purification by this procedure is about 300 from
mitochondria or about 750 from whole liver.
Properties
Stability. Citrate synthase is very stable. Dilute solutions in 0.1 M
potassium phosphate buffer, p H 7.4, can be kept at --15 ° for at least 4
months without any change in activity, although repeated freezing and
thawing leads to loss of activity.
Purity. Citrate synthase prepared as described is only about 30%
pure by comparison with the specific activities of crystalline citrate
synthase from other sources. The main contaminant is malic dehydro-
genase (specific activity 33 units/mg). The enzyme has no N A D H
oxidase or acetyl-CoA deacylase activity. ATPase activity is 0.1 unit/rag,
but only in the presence of Mg *÷.
Other Properties. Citrate synthase has a high affinity for both
acetyl-CoA and oxaloacetate with K,~ values of 14 and 4 t~M, respec-
tively, although high substrate inhibition occurs for concentrations of
oxaloacetate in excess of about 10 ~M. One of the more interesting
16 REACTIONS ON THE CYCLE ~]
properties of the enzyme is the inhibition by adenine nucleotides. ~ This

inhibition is most marked for ATP, less extensive for ADP, and least
of all for AMP. The inhibition is competitive with acetyl-CoA; e.g., in
the presence of 4 mM ATP, the K,~ is raised from 14 to 160/~M, and also
with oxaloacetate although potential relief of inhibition by high oxalo-
acetate concentrations is masked by substrate inhibition. The enzyme is
inhibited by palmitoyl-CoA2 The pH optimum of the enzyme is 9.0, but
this becomes more acidic in the presence of adenine nucleotides. The
apparent equilibrium constant of the enzymatic reaction at pH 7.2 and
22 ° is 8.38 )< 103.15
,sj. R. Stern, B. Shapiro, E. R. Stadtman, and S. 0choa, J. Biol. Chem. 193, 703
(1951); J. R. Stern, S. Ochoa, and F. Lynen, ibid. 198, 313 (1952); see also Vol. I
[114].
[3] C i t r a t e S y n t h a s e f r o m Y e a s t
[EC 4.1.3.7 Citrate oxaloacetate-lyase (CoA aeetylating)]
By R. PARVIN
Acetyl-SCoA + oxaloacetate ~ citrate + C o A S H (I)
Assay Method
Principle.Several assay methods for citrate synthase are available.I-3
The method of Srere3 is used in the following purification. C o A S H
liberated in reaction (I) reacts with 5,5r-dithiobis-(2-nitrobenzoicacid)
( D T N B ) to form a mercaptide ion which absorbs light at 412 m ~ with
a molar extinction coefficientof 13,600.
Reagents
Tris[ (hydroxymethyl)aminomethane]-HCl,0.5 M, pH 8.0
DTNB, 2.5 raM, dissolved in 20 mM Tris-HCl, pH 8.0
Oxaloacetate, 2 mM, freshly prepared and neutralized
Acetyl-CoA, 1 mM
Procedure
Add to a spectrophotometer cell of 1 cm lightpath: Tris-HC1, 0.2 ml;
DTNB, 0.1 ml; oxaloacetate, 0.1 ml; acetyl-CoA, 0.1 ml; and water to 1.0
' P. A. Srere and G. W. Kosciki, J. Biol. Chem. ~36, 2557 (1961).
~P. A. Srere, H. Brazil, and L. Gonen, Acta Chem. ~cand. 17, S129 (1963).
16 REACTIONS ON THE CYCLE ~]
properties of the enzyme is the inhibition by adenine nucleotides. ~ This

inhibition is most marked for ATP, less extensive for ADP, and least
of all for AMP. The inhibition is competitive with acetyl-CoA; e.g., in
the presence of 4 mM ATP, the K,~ is raised from 14 to 160/~M, and also
with oxaloacetate although potential relief of inhibition by high oxalo-
acetate concentrations is masked by substrate inhibition. The enzyme is
inhibited by palmitoyl-CoA2 The pH optimum of the enzyme is 9.0, but
this becomes more acidic in the presence of adenine nucleotides. The
apparent equilibrium constant of the enzymatic reaction at pH 7.2 and
22 ° is 8.38 )< 103.15
,sj. R. Stern, B. Shapiro, E. R. Stadtman, and S. 0choa, J. Biol. Chem. 193, 703
(1951); J. R. Stern, S. Ochoa, and F. Lynen, ibid. 198, 313 (1952); see also Vol. I
[114].
[3] C i t r a t e S y n t h a s e f r o m Y e a s t
[EC 4.1.3.7 Citrate oxaloacetate-lyase (CoA aeetylating)]
By R. PARVIN
Acetyl-SCoA + oxaloacetate ~ citrate + C o A S H (I)
Assay Method
Principle.Several assay methods for citrate synthase are available.I-3
The method of Srere3 is used in the following purification. C o A S H
liberated in reaction (I) reacts with 5,5r-dithiobis-(2-nitrobenzoicacid)
( D T N B ) to form a mercaptide ion which absorbs light at 412 m ~ with
a molar extinction coefficientof 13,600.
Reagents
Tris[ (hydroxymethyl)aminomethane]-HCl,0.5 M, pH 8.0
DTNB, 2.5 raM, dissolved in 20 mM Tris-HCl, pH 8.0
Oxaloacetate, 2 mM, freshly prepared and neutralized
Acetyl-CoA, 1 mM
Procedure
Add to a spectrophotometer cell of 1 cm lightpath: Tris-HC1, 0.2 ml;
DTNB, 0.1 ml; oxaloacetate, 0.1 ml; acetyl-CoA, 0.1 ml; and water to 1.0
' P. A. Srere and G. W. Kosciki, J. Biol. Chem. ~36, 2557 (1961).
~P. A. Srere, H. Brazil, and L. Gonen, Acta Chem. ~cand. 17, S129 (1963).
[3] CITRATE SYNTttASE FROM YEAST 17
ml. The reaction is started at room temperature (25-28 °) by the addition

of enzyme, and increase in absorbancy is measured at intervals of 15 or
30 seconds. Under these conditions the reaction is linear for absorbancy
increases up to 0.5.
Units. A unit of enzyme is defined as that amount capable of forming
1 micromole of CoASH per minute under the described assay conditions.
Specific activity is expressed as enzyme unit(s) per milligram of protein.
Protein Determinatio~s. Protein is determined either by differential
(A) absorbancy biuret method/ or phenol method:
Starting Material. Fresh commercial bakers' yeast (Fleischmann)
in cake form is crumpled and blended with an equal volume of chilled
water in a Waring blendor at low speed for about 10 seconds. Frothing
is avoided at this and other steps. The resulting suspension is poured into
12 volumes of chilled acetone (--16 °) with constant stirring and then is
filtered immediately by suction on a Biichner funnel at room temperature.
The cake obtained is washed with 5-6 volumes of chilled acetone while
on the funnel. After being dried by suction for 5-10 minutes, the powder
is thinly spread on filter paper at l'oom temperature (2-3 hours).
Step 1. Extractions. For enzyme extraction, 200 g (obtained from 1.5
pounds of yeast) of freshly prepared acetone powder is suspended and
stirred in 10 volumes of 0.05 M Tris-HC1 (pH 8.0) for 16 hours at room
temperature (26-28°). It is then chilled and centrifuged for 20 minutes
at 13,000 g and the residue is discarded.
All subsequent steps are carried out at 0-4 ° .
Step ~. Fractional pH Precipitation. As promptness is necessary at
this step, it may be advantageous to divide the supernatant solution
from the above step into smaller lots and to treat each lot separately.
The pH of the supernatant solution is lowered to 5.6 by slow addition
of 0.5 N acetic acid with adequate stirring. Without delay the precipitate
is removed by centrifugation for 10 minutes at 13,000 g. The pH of the
supernatant liquid is then lowered to 5.0 with 0.5 N acetic acid, and the
precipitate obtained on centrifugation for 15 minutes at 13,000 g is
dissolved in 50 mM Tris-HC1 (pH 8.0) to a protein concentration of
about l0 mg/ml. Total volume is 415 ml.
Step 3. Protamine Sulfate TreatnzeT~t. To the enzyme preparation
'R. Parvin, S. V. Pande, and T. A. Venkitasubramanian, Anal. Biochem. 12, 219
(1965).
O. It. Lowry, N. J. Rosebrough, A. L. Parr, and R. J. Randall, J. Biol. Chem.
193, 265 (1951).
]8 :REACTIONS ON THE CYCLE [3]
from the above step, add with stirring 0.09 to 0.1 volume of 2% protamine
sulfate (pH 7.5). The precipitate is removed by centrifugation.
Step ~. Ammanium SulIate Fractionation. Solid ammonium sulfate
is added gradually over a period of 1 hour to the supernatant from step 3
to bring the salt concentration to 70% saturation with slow stirring.
Separated protein is removed by centrifugation at 13,000 g for 30 minutes.
More ammonium sulfate is gradually added to the supernatant solution
to raise salt concentration to 85% saturation. After 1 hour of stirring,
the separated protein is recovered by centrifugation as above and dis-
solved in a minimal volume of 50 mM Tris-HC1 (pH 8.0). Total volume
at this step is 4.0 ml.
Step 5. Fractionation on Sephadex G-200. The above enzyme prepara-
tion is applied to a 1.5 X 40 cm Sephadex G-200 column (in 50 mM
Tris-HC1, pH 8.0). The same buffer is used for elution. Flow rate is
adjusted to 5 ml per hour, and 2 ml fractions are collected. The fractions
containing enzyme at the highest specific activity (typically tubes 15
to 21) are pooled and brought to 50% saturation by adding solid am-
monium sulfate. Any precipitate appearing is removed by centrifugation,
and the ammonium sulfate concentration is raised to 85% saturation.
The precipitate appearing between 50 and 85% ammonium sulfate satura-
tion is dissoh'ed in a minimal volume (about 2 ml) of 50 mM Tris-HC1
(pH 8.0) and then again passed through the same washed Sephadex
G-200 column described above. Peak activity (and specific activity) is
found about fractions 16 and 17. The specific activity of the final prepara-
tion is 160 with overall purification of 430-fold.
The purification scheme is summarized in the table.
PURIFICATION OF CITRATE SYNTHASE FROM YEAST
Specific
Total Total activity
volume Protein activity (units/mg Yield
Step (m|) (mg/ml) (units) a protein) (%)
1. Acetone powder extract 1,750 15.3 10,030 0.375 100

2. pH 5 precipitation 415 9.5 6,460 1.64 64
3. Protamine sulfate 405 6.1 5,810 2.35 58
4. Ammonium sulfate 4.0 49.1 2,610 13.3 26
70-85% saturation
5. (i) Sephadex G-200 eluate 2.3 11.0 1,490 59.0 15
(after 50-85% ammonium
sulfate saturation)
(ii) Sephadex G-200 eluate 7.25 0.49 570 160 5.7
a Unit = amount capable of forming 1 micromole of CoASH per minute under

conditions described.
[4] CITRATE SYNTHASE FROM LEMON FRUIT 19
Properties 6, 7
Purity. The purified citrate synthase shows a single band on poly-
acrylamide disc eleetrophoresis. It is free of the following enzyme
activities: NAD- and NADP-speeific isocitrate dehydrogenases, citrate
cleavage enzyme, acetyl-CoA deacylase, and oxaloaeetate decarboxylase.
Stability. The purified enzyme preparation is fairly stable at --16 °
in 50 mM Tris-HCl (pH 8.0) when protein concentration is maintained
above 450 /~g/ml. In dilute solutions, the enzyme is extremely labile. As
the purification progresses, the enzyme preparation becomes susceptible
to inactivation on ~tialysis against water or l0 mM Tris-HC1 (pH 8.0).
Bovine serum albumin (1 mg/ml) stabilizes the dilute enzyme solution.
The enzyme is most stable at pH 8.0; it is labile above pH 8.5 and
below pH 5.0.
Kinetic Properties. The K,~ value for acetyl-CoA is 2-4/~M and that
of oxaloacetate is 1-3 ~M.
Effectors. The enzyme activity is inhibited by ATP > ADP > PP
AMP in the order given, when each compound is tested at 5 mM. Mg ÷÷
above 5 mM also inhibits enzyme activity. Inhibition by ATP and pyro-
phosphate is competitive with respect to acetyl-CoA.
pH Optimum. The enzyme shows a rather broad pH optimum from
7.0 to 8.0 with somewhat higher activity at pH 7.5.
cj. A. Hathaway and D. E. Atkinson, Biochem. Biophys. Res. Commzm. 20, 661
(1965).
7R. Parvin and D. E. Atkinson, unpublished observations.
[4] C i t r a t e S y n t h a s e f r o m L e m o n F r u i t
[EC 4.1.3.7 Citrate oxaloaeetate-lyase (CoA-acetylating)]
By EITAN BOGIN and ARTHURWALLACE
Acetyl-S-CoA ~ oxaloacetate + H~.O ~ citrate + CoASH
Assay Method 1
Principle. The condensation of oxaloacetate and acetyl-S-CoA results

in the formation of citrate and the release of CoASH. The reaction
is based on the reaction of CoASH with Ellman's reagent, 2 5,5'-dithiobis-
(2-nitrobenzoic acid) (DTN'B), forming a mercaptide which absorbs
~P. Srere, H. Brazil, and L. Gonen, Acta Chem. Scand. 178, 129 (1963).
2G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959).
Properties 6, 7
Purity. The purified citrate synthase shows a single band on poly-
acrylamide disc eleetrophoresis. It is free of the following enzyme
activities: NAD- and NADP-speeific isocitrate dehydrogenases, citrate
cleavage enzyme, acetyl-CoA deacylase, and oxaloaeetate decarboxylase.
Stability. The purified enzyme preparation is fairly stable at --16 °
in 50 mM Tris-HCl (pH 8.0) when protein concentration is maintained
above 450 /~g/ml. In dilute solutions, the enzyme is extremely labile. As
the purification progresses, the enzyme preparation becomes susceptible
to inactivation on ~tialysis against water or l0 mM Tris-HC1 (pH 8.0).
Bovine serum albumin (1 mg/ml) stabilizes the dilute enzyme solution.
The enzyme is most stable at pH 8.0; it is labile above pH 8.5 and
below pH 5.0.
Kinetic Properties. The K,~ value for acetyl-CoA is 2-4/~M and that
of oxaloacetate is 1-3 ~M.
Effectors. The enzyme activity is inhibited by ATP > ADP > PP
AMP in the order given, when each compound is tested at 5 mM. Mg ÷÷
above 5 mM also inhibits enzyme activity. Inhibition by ATP and pyro-
phosphate is competitive with respect to acetyl-CoA.
pH Optimum. The enzyme shows a rather broad pH optimum from
7.0 to 8.0 with somewhat higher activity at pH 7.5.
cj. A. Hathaway and D. E. Atkinson, Biochem. Biophys. Res. Commzm. 20, 661
(1965).
7R. Parvin and D. E. Atkinson, unpublished observations.
[4] C i t r a t e S y n t h a s e f r o m L e m o n F r u i t
[EC 4.1.3.7 Citrate oxaloaeetate-lyase (CoA-acetylating)]
By EITAN BOGIN and ARTHURWALLACE
Acetyl-S-CoA ~ oxaloacetate + H~.O ~ citrate + CoASH
Assay Method 1
Principle. The condensation of oxaloacetate and acetyl-S-CoA results

in the formation of citrate and the release of CoASH. The reaction
is based on the reaction of CoASH with Ellman's reagent, 2 5,5'-dithiobis-
(2-nitrobenzoic acid) (DTN'B), forming a mercaptide which absorbs
~P. Srere, H. Brazil, and L. Gonen, Acta Chem. Scand. 178, 129 (1963).
2G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959).
20 a E A c T I o N s oN THe. c v c L r [4]
light at 412 n~. The molar absorbancy index is 13,600. Measurement of

this enzyme can also be followed by coupling citrate synthase with
malate dehydrogenase and measuring NADH2 formation at 340 m# as
described by Ochoa2 The measurement is carried out in the Hitachi
spectrophotometer using corex or silica cells of 1.0 cm light path.
Reagents
Tris-HC1, 0.2 M, pH 8.0
Oxaloacetate, 50 mM
Acetyl-S-CoA, 50
DTNB, 0.1 mM
Procedure. The reaction mixture in corex or silica cells (d = 1.0 cm)

consists of Tris-HC1 buffer, 0.5 ml, pH 8.0; DTNB, 0.2 ml; acetyl-S-
CoA, 0.1 ml; oxaloacetate, 0.1 ml (5.0 micromoles) ; enzyme, 0.05 ml;
and water to a final volume of 3.0 ml. The assay is performed at room
temperature (23-25°). The reaction is started by the addition of either
oxaloacetate or enzyme. Readings are made against a blank containing
all components except acetyl-S-CoA and recorded with a Photovolt
linear-log recorder attached to the spectrophotometer. The increase in
optical density between 15 and 30 seconds after the start of the reaction
is used to calculate the enzyme activity. The amount of enzyme used
is adjusted so that the rate of increase of optical density for the period
between 15 and 30 seconds does not exceed 0.050.
Units. One unit of enzyme is defined as that amount of protein
required to condense 1 micromole of acetyl-S-CoA with oxaloacetate per
minute. Specific activity is expressed as units per milligram of protein.
Homogenization. 4 Young, green lemon fruits 3-5 cm in diameter are
cooled and then peeled. Six hundred grams of the peeled tissue is grated
in 1 liter of 0.5 M sucrose, 0.25 M Tris-HCl solution, pH 8.0. During the
grating of the tissue in the cold solution the pH of the homogenate should
not fall below 7.2. The pH is kept at about 7.5 by adding slowly and
periodically 1 N KOH solution while mixing the homogenate.
Precipitation o] the Mitochondria. The homogenate is passed through
four layers of cheesecloth and centrifuged at 1000 g for 15 minutes. The
supernatant is then centrifuged at 18,000 g for 20 minutes. The resulting
pellet, which contains mitochondria, is dispersed by homogenization with
'~S. Ochoa, Biochcm. Prep. 5, 19 (1957).

' E . Bogin and L. C. Erickson, Plant Physiol. 40, 566 (1963).
an Elvehjem mortar and pestle in 100 ml of 0.5M sucrose 50 raM

Tris-HC1, pH 7.5. The suspension is recentrifuged at 18,000 g for 20
minutes. The pellet is then suspended in 30 ml of 50 mM Tris-HC1,
pH 7.5. Most of the citrate synthase activity (92%) is found in the
mitochondrial fraction. Since acetyl-CoA does not easily penetrate intact
mitochondria, a meaningful assay cannot be made until they have been
broken.
Breaking o] the Mitochondria2 Mitochondria are broken with a cell
homogenizer Model MSK (Bronwill Scientific, Inc., Rochester, New
York). Glass beads, 10 ml, are mixed with 30 ml of the mitochondria
suspension and shaken for 60 seconds (15 seconds' shaking interrupted
by 30 seconds' rest). The temperature is kept at 2-4 ° by cooling the
homogenizing cell with a liquid COs cooling device. The solution is
centrifuged at 20,000 g for 10 minutes, and the precipitate is discarded.
PURIFICATION OF CITRATE SYNTHASE FROM LEMON
Specific
activity
Volume Units (umoles
of (~moles AeSCoA/
solution Protein AcSCoA mg Yield
Fraction or step (ml) (rag) utilized) protein) (%)
Homogenate 300 2870 344 0.12 100
Mitochondria 30 120 316 2.64 91.8
MSK cell homogenization 28 98 290 2.96 84.3
and centrifugation
Freezing and thawing 28 51 216 4.24 62.8
4 times
(NI~)2SO~, 40-65%, 10 19 164 8.64 47.7
after dialysis
Freezing and Thawing. The supernatant is frozen at --30 ° for 5

minutes and then thawed at room temperature. This procedure is
repeated four times and the suspension is then centrifuged at 20,000 g
for 10 minutes.
Ammonium Sulfate Fractionation. To 28 ml of the supernatant, 18 ml
of ice cold (NH,)2S04 solution, adjusted to pH 7.5 with Tris base,
saturated at 0 ° is added slowly while stirring, and the mixture is allowed
to stand for 10 minutes at a temperature of 2-4 °. The solution is then
centrifuged at 30,000 g for 20 minutes; the precipitate is discarded. To
the supernatant, 38 ml of ice cold (NH4)~SO, solution, saturated at 0 °,
is added slowly and allowed to stand for 10 minutes and recentrifuged
E. Bogin and A. Wallace, Biochim. Biophys. Acta 128, 190 (1966).
at 30,000 g for 20 minutes. The precipitate is suspended in 10 ml of 10

mM Tris-HCl buffer, pH 7.2, and dialyzed against 4 liters of 1 mM
Tris-HC1 buffer, pH 7.0, containing 1 mM cysteine for 2 hours at 2-4 °.
The dialyzing solution is changed once, after 1 hour. The suspension is
then recentrifuged at 10,000 g for 10 minutes and kept frozen at --20 °.
In this state the enzyme retains up to 70% of the original activity for one
month. A summary of the purification procedure is shown ill the table.
[ 5 ] C i t r a t e S y n t h a s e f r o m Escherichia coli
[EC 4.1.3.7 Citrate oxaloaeetate-lyase (CoA-aeetylating)]
By P. D. J. WEITZMAN
Aeetyl-S-CoA + oxaloacetatd- + H~O ~ citrate s- + CoASH + H +
Assay Method
Principle. The enzyme may be assayed conveniently by following the
formation of CoASH either by a polarographic or a spectrophotometric
method. In the former (method A) the anodic wave produced at the
dropping mercury electrode by CoASH is monitored directly with a
recording polarograph. 1 Alternatively (method B), the CoASH produced
may be allowed to react with the chromogenic reagent 5,5'-dithiobis-
(2-nitrobenzoic acid) (DTNB) : and the rate of change in extinction be
measured at 412 mt~. With this method of assay it is necessary to raise the
ionic strength of the buffer in order to avoid the inactivation of the
enzyme by DTNB which would otherwise occur.
Method A
The reagents required and full details of the procedure are described
elsewhere in this volume [56].
Method B
Reagents
Acetyl-S-CoA, 8 mM, prepared as described by Stadtman:'
Sodium oxaloacetate, l0 mM
DTNB, 10 mM
1p. D. J. Weitzman, Biochem. J. 99, 18P (1966) ; this volume [56].
" P. A. Srere, It. Brazil, and L. Gonen, Acta Chem. Scand. 17, S129 (1963),
~E. R. Stadtman, "Col. I I I [137].
at 30,000 g for 20 minutes. The precipitate is suspended in 10 ml of 10

mM Tris-HCl buffer, pH 7.2, and dialyzed against 4 liters of 1 mM
Tris-HC1 buffer, pH 7.0, containing 1 mM cysteine for 2 hours at 2-4 °.
The dialyzing solution is changed once, after 1 hour. The suspension is
then recentrifuged at 10,000 g for 10 minutes and kept frozen at --20 °.
In this state the enzyme retains up to 70% of the original activity for one
month. A summary of the purification procedure is shown ill the table.
[ 5 ] C i t r a t e S y n t h a s e f r o m Escherichia coli
[EC 4.1.3.7 Citrate oxaloaeetate-lyase (CoA-aeetylating)]
Aeetyl-S-CoA + oxaloacetatd- + H~O ~ citrate s- + CoASH + H +
Assay Method
Principle. The enzyme may be assayed conveniently by following the
formation of CoASH either by a polarographic or a spectrophotometric
method. In the former (method A) the anodic wave produced at the
dropping mercury electrode by CoASH is monitored directly with a
recording polarograph. 1 Alternatively (method B), the CoASH produced
may be allowed to react with the chromogenic reagent 5,5'-dithiobis-
(2-nitrobenzoic acid) (DTNB) : and the rate of change in extinction be
measured at 412 mt~. With this method of assay it is necessary to raise the
ionic strength of the buffer in order to avoid the inactivation of the
enzyme by DTNB which would otherwise occur.
Method A
The reagents required and full details of the procedure are described
elsewhere in this volume [56].
Method B
Reagents
Acetyl-S-CoA, 8 mM, prepared as described by Stadtman:'
Sodium oxaloacetate, l0 mM
DTNB, 10 mM
1p. D. J. Weitzman, Biochem. J. 99, 18P (1966) ; this volume [56].
" P. A. Srere, It. Brazil, and L. Gonen, Acta Chem. Scand. 17, S129 (1963),
~E. R. Stadtman, "Col. I I I [137].
[5] CITRATE SYNTHASE FROM E . coli 23
Procedure. Into a cuvette (1 cln light path, approximately 1.5 ml

volume) are pipetted 0.93 ml of Tris buffer, 0.02 ml of aeetyl-S-CoA, 0.01
ml of DTNB, and 0.02 ml of enzyme. The extinction at 412 m~ i~
recorded against a blank cuvette containing water to indicate the pres-
ence of any deacylase activity in the enzyme preparation. In the absence
of any such activity, 0.02 ml of sodium oxaloacetate is added and the
linear rate of increase in extinction at 412 mt~ is recorded. Should the
enzyme preparation contain any measurable deacylase activity, the
activity of citrate synthase may be measured by incorporating a blank
cuvette containing all the components of the assay mixture with the
exception of sodium oxaloacetate. The molar extinction coefficient of the
measured species at 412 m~ is 13,600.
Units. One unit of citrate synthase is defined as that quantity of en-
zyme which catalyzes the formation of 1 micromole of CoASH per
minute under the above assay conditions. Specific activity is expressed
as enzyme units per milligram of protein. Protein is determined by the
method of Lowry et al. 4
The following procedure has been used ~ to purify the enzyme from
E. coli, strain K12.
Growth o] Organisms. Fifteen liters of medium 6 containing 50 mM
sodium acetate as carbon source are made up in a glass carboy and
inoculated with 200 ml of an actively growing culture of bacteria which
has been grown in the same medium at 37 ° . The carboy is kept in a
30°-warm room and the contents are vigorously aerated. After 18-24
hours of growth, when the cell density is 0.7-0.9 mg/ml, dry weight, the
cells are harvested and washed with cold water.
Step 1. Preparation of Sonic Extract. The washed cells are suspended
to a density of approximately 40 mg/ml, dry weight, in the following
buffer: 20 mM Tris-HC1, 10 mM MgC12, and 1 mM EDTA, pH 8.0.
This suspension, in cooled batches of 50 ml, is treated in a 20 kc sonic
oscillator (Dawe Soniprobe) at 4 amp for 11~ minute. The sonic extracts
are combined and centrifuged for 20 minutes at 25,000 g, the precipitated
material being discarded. This step, and all subsequent steps, are carried
out at 0-4 ° .
Step 2. Treatment with Protamine Sulfate. To the supernatant solu-
tion is added 2% (w/v) protamine sulfate (1 mg for each 10 mg of
40. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193,
265 (1951).
P. D. J. Weitzman and P. Dunmore, unpublished.
~H. L. Kornberg, P. J. R. Phizackerley, and J. R. Sadler, Biochem. 3.77, 438 (1960).
24 aEACTIONS ON THE CYCLE [5]
protein), and the mixture is stirred continuously. After 30 minutes the

precipitate is removed by centrifugation for 20 minutes at 25,000 g.
Step 3. Fractionation with Ammonium Sul]ate. To each 100 ml
of the supernatant solution is added slowly 35.1 g of finely ground solid
(NH4)2SO~ (55% saturation). After the preparation has stood for 30
minutes with continuous stirring, the precipitate is removed by centrif-
ugation for 20 minutes at 25,000 g. The supernatant solution is now
brought to 70% saturation by the addition of further solid (NH,)2S04
(10.3 g/100 ml) followed by stirring for 30 minutes. The precipitate is
collected by centrifugation for 20 minutes at 25,000 g, dissolved in 10 ml
of buffer, pH 8.0, containing 20 mM Tris-HC1, 1 mM EDTA {hereafter
referred to as Tris buffer) and dialyzed overnight against a solution of
this buffer containing 0.1 M KC1. (The KCI is included here since, on
occasions, it has been found that loss of enzyme activity during this
dialysis step may be prevented by the presence of 0.1 M KCI.) Any
insoluble material precipitated during the dialysis is removed by centrif-
ugation.
Step ~. Chromatography on DEAE-Cellulose. A column of DEAE-
cellulose (1.5 >( 25 cm) is prepared in the usual way and equilibrated at
4 ° with Tris buffer. The dialyzed enzyme solution is diluted with an
equal volume of buffer (to reduce the KC1 concentration to 50 mM) and
applied to the column. When all the material has entered the column,
it is washed with Tris buffer until no more protein is eluted. The column
is then washed with Tris buffer containing 0.1 M KC1, again until no
more protein is eluted. Finally, a solution of Tris buffer containing 0.15 M
KCI is applied to the column and the emuent is collected in 5 ml frac-
tions. The citrate synthase is eluted from the column by this solution.
Those fractions containing the enzyme at a specific activity greater than
20 are combined, and the protein is precipitated with (NH~)2S04 to 80%
saturation. The precipitate is collected by centrifugation and dissolved
in 1 ml of Tris buffer.
Step 5. Gel Filtration on Sephadex G-200. A column (1.5 X 30 cm)
of Sephadex G-200 is prepared in the usual way and equilibrated at 4 °
with Tris buffer. The product from step 4 is applied to the column, al-
lowed to enter the gel bed, and washed in with 1-2 ml of buffer. The
protein is then eluted from the column at a flow rate of approximately 10
ml per hour, 1-ml fractions being collected. The citrate synthase emerges
early and is present in several fractions. Those with a specific activity
above 40 are pooled.
The degree of purity of citrate synthase prepared in this way has
been examined5 by electrophoresis on acrylamide gels. The enzyme is
located in a single band which represents 90% or more of the protein
[5] CITRATE SYNTHASE FROM E. coli 25
PURIFICATION OF CITRATE SYNTHASE FROM Escherichia coli
Specific
Volume enzyme protein (units/rag Recovery
Step (ml) (units) (mg) protein) (%)
1. Sonic extract 450 1830 2200 0.83 --

2. Supernatant from 450 1790 1885 0.95 98
protamine sulfate
3. Ammonium sulfate 10.9 970 236 4.1 53
precipitate, 55-70%
4. Pooled selected fractions 15 492 20.3 24.3 27
from DEAE-cellulose
column
5. Pooled selected fractions 3 275 6.1 45 15
from Sephadex G-200
column
present. The enzyme preparation thus appears to be of high purity. D a t a

on the purification of the enzyme are summarized in the table.
Properties
Stability and p H Optimum. The enzyme appears to be very stable.
The solution of citrate synthase obtained in step 5 of the purification
procedure m a y be kept at 2 ° for several weeks without loss of activity.
The enzyme exhibits an optimum activity at approximately p H 8.0.
Kinetic Properties. At p H 8.0, the Km for acetyl-S-CoA was measured
to be 0.5 m M ; t h a t for oxaloaeetate was 14 p.M.
Allosteric Ef]ectors. T h e citrate synthase from E. coli differs from the
enzyme isolated from yeast, 7 plants, s and m a m m a l s 9,1° in its relative
insensitivity to inhibition by A T P ; instead, the enzyme is inhibited
powerfully by N A D H ? 1,12 This inhibition is dependent on the concentra-
tion of acetyl-S-CoA and is most marked in solutions of low salt content.
Thus, in a buffer containing 20 m M Tris-HC1, 1 m M E D T A , at p H 8.0,
80-90% inhibition is produced by 0.1 m M N A D H . The inhibition by
N A D H is specific; no inhibition is produced by any of the following
'J. A. Hathaway and D. E. Atkinson, Biochem. Biophys. Res. Commun. 20, 661
(1965).
*E. Bogin and A. Wallace, Biochim. Biophys. Acta 128, 190 (1966).
' D. Shepherd and P. B. Garland, Biochem. Biophys. Res. Commun. 22, 89 (1966).
10 N . O. Jangaard, J. A. Hathaway, and D. E. Atkinson, Federation Proc. 25, 220
( 19{}6).
lip. D. J. Weitzman, Biochim. Biophys. Acta 128, 213 (1966).
P. D. J. Weitzman, Biochem. J. 101, 44c (1966).
substances at a concentration of 1 mM: NAI), NADP, NADPH, AMP,

ADP, ATP. Furthermore, the inhibition appears to be of the allosteric
type, since the enzyme may be completely desensitized to NADH either
at pH values above 8 or in the presence of 0.2 M KC1, without loss of
enzymatic activity. It is suggested that NADH exerts a fine control over
the activity of E. coli citrate synthase consistent with the energy require-
ments of the cell. It has also been reported that palmityl-S-CoA13 and
a-ketoglutarate 1. may act as additional allosteric inhibitors of the
enzyme.
,3p. A. Srere and N. Whisscn, Federation Proc. 26, 559 (1967).
'*J. A. Wright, P. Maeba, and B. D. Sanwal, Biochem. Biophys. Res. Commun. 29,
34 (1967).
[6] A c o n i t a s e f r o m P i g H e a r t 1
[EC 4.2.1.3 Citrate (isocitratc) hydro-lyasc]
By B. FANSLERand J. M. LOWENSTEIN
Citrate ~ c/s-Aconitate ~ Isocitrate
In fresh extracts of heart high activities of aconitase can usually be

demonstrated without activating the enzyme. The enzyme loses activity
in the course of purification and on storage in the purified form. In the
presence of protecting agents, such as citrate or tricarballylate, the loss
in activity is reversible. It becomes irreversible only on very prolonged
storage. In the absence of protecting agents the purified enzyme undergoes
irreversible loss of activity relatively quickly. Reversibly inactivated
enzyme can be reactivated by incubation with ferrous ions and a thiol.
The preparation to be described is a simplification of the alcohol
fractionation method of Morrison, 2 followed by column chromatography.
Assay Method
Principle. The activity of the enzyme is measured spectrophotomet-
rically by following the disappearance of cis-aconitate at 240 m~ as a
function of time2
'The new work described in this article was carried out by the authors at the
Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsyl-
vania.
' J . F. Morrison, Biochem. J. 56, 99 (1954).
E. Racker, Biochim. Biophys. Acta 4, 211 (1950).
substances at a concentration of 1 mM: NAI), NADP, NADPH, AMP,

ADP, ATP. Furthermore, the inhibition appears to be of the allosteric
type, since the enzyme may be completely desensitized to NADH either
at pH values above 8 or in the presence of 0.2 M KC1, without loss of
enzymatic activity. It is suggested that NADH exerts a fine control over
the activity of E. coli citrate synthase consistent with the energy require-
ments of the cell. It has also been reported that palmityl-S-CoA13 and
a-ketoglutarate 1. may act as additional allosteric inhibitors of the
enzyme.
,3p. A. Srere and N. Whisscn, Federation Proc. 26, 559 (1967).
'*J. A. Wright, P. Maeba, and B. D. Sanwal, Biochem. Biophys. Res. Commun. 29,
34 (1967).
[6] A c o n i t a s e f r o m P i g H e a r t 1
[EC 4.2.1.3 Citrate (isocitratc) hydro-lyasc]
By B. FANSLERand J. M. LOWENSTEIN
Citrate ~ c/s-Aconitate ~ Isocitrate
In fresh extracts of heart high activities of aconitase can usually be

demonstrated without activating the enzyme. The enzyme loses activity
in the course of purification and on storage in the purified form. In the
presence of protecting agents, such as citrate or tricarballylate, the loss
in activity is reversible. It becomes irreversible only on very prolonged
storage. In the absence of protecting agents the purified enzyme undergoes
irreversible loss of activity relatively quickly. Reversibly inactivated
enzyme can be reactivated by incubation with ferrous ions and a thiol.
The preparation to be described is a simplification of the alcohol
fractionation method of Morrison, 2 followed by column chromatography.
Assay Method
Principle. The activity of the enzyme is measured spectrophotomet-
rically by following the disappearance of cis-aconitate at 240 m~ as a
function of time2
'The new work described in this article was carried out by the authors at the
Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsyl-
vania.
' J . F. Morrison, Biochem. J. 56, 99 (1954).
[5] ACONITASE FROM PIG HEART 27
Reagents ]or Activation

Thiomalate-Tris buffer. Dissolve 30 mg thiomalic acid in about 7
ml water, add 0.4 ml 1 N NaOH, add 1.0 ml 1.0 M Tris-HCl
buffer, pH 7.8, and make to 10 ml with water. The resulting
solution is 20 mM with respect to thiomalate. Gas the solution
with nitrogen for 10 minutes and seal until used.
Ferrous ammonium sulfate, 4 mM (16 mg per 10 ml). Gas the
solution with nitrogen for 10 minutes and seal until used.
Reagents ]or Assay

cis-Aconitate, 10 mM when using a light path of 0.5 mm, or 1 mM
when using a light path of 1 cm (c/s-aconitic acid neutralized
with NaOH).
Sodium chloride, 0.5 M
Activation o] Enzyme. Immediately before starting the activation mix

equal volumes of the thiomalate-Tris buffer and ferrous ammonium
sulfate solutions. One volume of the resulting activation mixture is added
to one volume of the enzyme solution and the whole is incubated at 37 °
for 25 minutes. The solution is then kept in an ice bucket. Enzyme acti-
vated in this manner usually remains fully active for at least 6 hours.
Assay Procedure. The reaction rate is near maximum in the presence
of 2 mM c/s-aconitate. This co~centration requires a light path of 0.5 mm
at 240 m~. The reaction mixture contains 0.1 ml of 0.1 M Tris-HC1 buffer,
0.1 ml of 0.5 M NaC1, 0.1 ml of 10 mM of cis-aconitate, and water and
enzyme to give a final volume of 0.5 ml. The reaction mixture is pipetted
into a standard cuvette with a light path of 1 cm; the reaction is started
by adding the enzyme, and a quartz insert is placed quickly into the
cuvette to reduce the light path to 0.5 mm. The decrease in absorbancc
at 240 m~ is then followed for 2 to 5 minutes. The assay is run at 24 °.
If a quartz insert to reduce the light path is not available, the assay
can be performed in the presence of 0.1 mM c/s-aconitate. The reaction
rate is considerably below maximum under these conditions. The reaction
mixture contains 0.6 ml of 0.1 M Tris-HC1 buffer, 0.6 ml of 0.5 M NaC1,
0.3 ml of 1 mM cis-aconitate, and water and enzyme to give a final
volume of 3.0 ml. The reaction is started by adding the enzyme, and the
decrease in absorbance at 240 m~ is followed with time.
it is also possible to assay the enzyme in the presence of 2 mM cis-
aconitate using a light path of 1 em by increasing the wavelength of the
light.
Units. The disappearance of cis-aconitate is proportional to the time
over a wide variety of conditions. The millimolar extinction coefficient at

240 m~ is 4.88 (by definition this is for a light path of 1 cm). One unit of
aconitase is defined as the amount of enzyme which causes the disappear-
ance of 1 ~mole of c/s-aconitate at 24 ° in 1 minute under the conditions
described. Specific activity is expressed in units per milligram of protein.
Step 1. Extraction. Fresh pig hearts are obtained and placed on ice
immediately after slaughter of the animals. All operations are performed
at 0--4° unless otherwise indicated. One kg of hearts is cut into 1-inch
cubes,~ and placed in a large Waring blendor (capacity 5 liters). This is
followed by 3000 ml of 15 mM tricarballylato--Tris buffer, pH 7.8
(prepared by neutralizing a solution of tricarballylic acid with Tris base).
This solution is hereafter referred to as "buffer." Chloroform (650 ml) is
added, and the mixture is homogenized for 4 minutes at medium speed.
The mixture is then centrifuged at about 10,000 g for 15 minutes. The
resulting precipitate is discarded. The supernatant has a volume of 2660
ml.
Step ~. Ethanol Fractionation. The supernatant from step 1 is placed
in a large flask which is supplied with a stirrer. The flask is supported
in a bath containing about 40% ethanol-water (v/v). Ice cold ethanol
(721 ml) is now run into the solution of supernatant with stirring. During
the addition of ethanol the temperature is gradually lowered to --7 ° by
adding dry ice to the bath. Freezing of the contents of the flask should be
avoided. This can be guarded against by checking the temperature in the
flask and in the bath periodically. The addition of the ethanol takes 15-
20 minutes. The mixture is centrifuged at a temperature of --6 ° at about
20,000 g for 15 minutes. The precipitate is discarded. The supernatant
has a volume of 3250 ml.
The supernatant is returned to a large flask which is supplied with a
stirrer. The flask is supported in the alcohol-water bath at --7 ° . Ice cold
ethanol (975 ml) is now run into the solution with stirring, and the
temperature is gradually lowered to --12 ° by adding dry ice to the bath.
The addition of the ethanol takes 15-20 minutes. The mixture is centri-
fuged at a temperature of --12 ° at about 20,000 g for 15 minutes. The
supernatant is discarded. The precipitate is washed out of the centrifuge
bottles with small portions of buffer. The final volume of the resulting
solution is 85 ml. The solution is dialyzed against 4 liters of buffer at 0 °
for 12 hours. The resulting solution contains 43 mg protein per milliliter.
,Step 3. Gel Filtration. A Sephadex G-100 column, 3 cm in diameter
The meat may be stored at --15 ° at this stage.

[5] ACONITASE FROM PIG HEART 29
with a bed height of 36 cm, is packed and is washed overnight with buffer.
A sample from step 2 containing 80 mg of protein is applied to the bottom
of the gel by the reverse flow technique. The column is then eluted with
buffer and fractions of 7.5 ml are collected. The fractions are analyzed
for protein and for aconitase activity. The enzyme is retarded in relation
to the main protein peak. About two-thirds of the activity placed on the
column is recovered in the peak fractions. The purification in this step is
about threefold.
The procedure is summarized in the table.
PURIFICATION OF PIG HEART ACONITASE
Total
protein Total Specific Purifi- Yield
Fraction obtained after (mg) units activity cation (%)
Centrifugation of 8740 17400 2.0 1.0 100

homogenate (Step 1)
Alcohol precipitate dissolved 2040 12100 5.9 3.0 70
and dialyzed (Step 2)
Sephadex G-100 967 8700 10.4 b 5.2 48
chromatography (Step 3) ~
This step is performed on aliquots obtained from step 2. The results shown assume
that all the material from step 2 was used for step 3.
b Average of three fractions which contained 96% of total activity recovered. The
highest specific activity in one of these fractions was 14.3 (yield = 15% in terms
of material from step 1).
Properties
Stability. The dialyzed alcohol fraction from step 2 can be stored
frozen for many months without irreversible loss of activity. Since re-
peated freezing and thawing causes irreversible loss of activity it is
recommended that the enzyme is stored frozen in aliquots of convenient
size which can be thawed separately. The enzyme is best stored in the
presence of citrate. However under these conditions an equilibrium mix-
ture of citrate, c/s-aconitate, and isocitrate results which interferes with
the assay. Tricarballylate (15 mM) can be used as a protecting agent in
place of citrate. This substance is not a substrate of the enzyme. Tri-
carballylate is such a weak inhibitor of the aconitasc reaction (K~ ~ 47
mM with respect to cis-aconitate) that its presence in the assay does not
interfere under most circumstances. Storage in the presence of citrate or
tricarballylate results in a reversible loss of activity. Reactivation of the
enzyme is achieved by incubation with thiomalate and ferrous ions (see
activation of enzyme).
Other Properties. A number of different thiols serve to activate the

enzyme in the presence of ferrous ions. 5,~ The optimum conditions for the
activation depend on the thiol and on the concentration of ferrous ions.
Maximum activation can be achieved with thioglycolate, cysteine, and
thiomalate. We prefer to use thiomalate as a matter of practical choice.
The activation is strongly temperature dependent. The enzyme is acti-
vated slightly by NaC1, the optimum concentration is 25-50 mM. Higher
concentrations are inhibitory. 7
S. R. Diekman and A. A. Cloutier, Arch. Biochem. Biophys. 25, 229 (1950).

Gj. F. Morrison, Biochem. J. 58, 685 (1954).
JR. A. Peters [Biochem. J. 79, 261 (1961)] showed that NaCl and KCI inhibit the
reaction slightly.
[7] Isocitrate Dchydrogenase (TPN-Specific) from Pig Heart

[EC 1.1.1.42 threo-D~-Isocitrate:NADP oxidoreductase (decarboxylating)]
B y W. W. CLELAND,V. W. THOMPSON,and R. E. BARDEN
Mg++ or Mn++
threo-Ds-Isocitrate -b T P N + , * a-ketoglutarate
Keq -- 4.0 M, pH 6.8
-b HCO3- q- T P N H q- H+
The preparation from pig heart of TPN-specific isocitric dehy-

drogenase of very high specific activity (~12,000 units/mg) has been
described previously (Vol. V [89]). This report describes a simple pro-
cedure for isolating TPN-specific isocitric dehydrogenase which has a
specific activity of approximately 5000 units/mg.
Assay Method
Enzymatic activity is measured by recording the increase in absorb-
ance at 340 m~. The following assay mixture is employed: 1.0 ml of a
solution containing 1 m M E D T A , 0.3 m M dithiothreitol acid, and 100
m M Tris-HC1 buffer, pH 7.4; 20 m M MnSO4, 0.2 ml; 1.5 m M T P N , 0.2
ml; 80 m M threo-D~Ls-isocitrate, 0.05 ml; enzyme solution, 0.03-0.1 ml;
and water to a total volume of 3.0 ml. The reactions are carried out at
25 ° in silica cuvettes with a 1 cm light path. Under these conditions a unit
of enzyme has been defined as the amount of enzyme that will give a
change of 0.01 OD per minute (Vol. I [116]). Protein concentration is
Other Properties. A number of different thiols serve to activate the

enzyme in the presence of ferrous ions. 5,~ The optimum conditions for the
activation depend on the thiol and on the concentration of ferrous ions.
Maximum activation can be achieved with thioglycolate, cysteine, and
thiomalate. We prefer to use thiomalate as a matter of practical choice.
The activation is strongly temperature dependent. The enzyme is acti-
vated slightly by NaC1, the optimum concentration is 25-50 mM. Higher
concentrations are inhibitory. 7
S. R. Diekman and A. A. Cloutier, Arch. Biochem. Biophys. 25, 229 (1950).

Gj. F. Morrison, Biochem. J. 58, 685 (1954).
JR. A. Peters [Biochem. J. 79, 261 (1961)] showed that NaCl and KCI inhibit the
reaction slightly.
[7] Isocitrate Dchydrogenase (TPN-Specific) from Pig Heart

[EC 1.1.1.42 threo-D~-Isocitrate:NADP oxidoreductase (decarboxylating)]
B y W. W. CLELAND,V. W. THOMPSON,and R. E. BARDEN
Mg++ or Mn++
threo-Ds-Isocitrate -b T P N + , * a-ketoglutarate
Keq -- 4.0 M, pH 6.8
-b HCO3- q- T P N H q- H+
The preparation from pig heart of TPN-specific isocitric dehy-

drogenase of very high specific activity (~12,000 units/mg) has been
described previously (Vol. V [89]). This report describes a simple pro-
cedure for isolating TPN-specific isocitric dehydrogenase which has a
specific activity of approximately 5000 units/mg.
Assay Method
Enzymatic activity is measured by recording the increase in absorb-
ance at 340 m~. The following assay mixture is employed: 1.0 ml of a
solution containing 1 m M E D T A , 0.3 m M dithiothreitol acid, and 100
m M Tris-HC1 buffer, pH 7.4; 20 m M MnSO4, 0.2 ml; 1.5 m M T P N , 0.2
ml; 80 m M threo-D~Ls-isocitrate, 0.05 ml; enzyme solution, 0.03-0.1 ml;
and water to a total volume of 3.0 ml. The reactions are carried out at
25 ° in silica cuvettes with a 1 cm light path. Under these conditions a unit
of enzyme has been defined as the amount of enzyme that will give a
change of 0.01 OD per minute (Vol. I [116]). Protein concentration is
[7] ISOCITRATE DEHYDROGENASE (TPN-SPECIFIC) 31
estimated from the optical density at 280 m~, assuming I mg/ml gives 1.0
OD28o/cm.
All solutions that come into contact with the enzyme (including
ammonium sulfate solutions) contain 0.3 m M dithiothreitol and all
operations are performed at 0-4 ° .
Tissue. Fresh pig hearts obtained at a slaughterhouse are placed
on ice for transportation to the laboratory. As quickly as possible the
ventricular muscle is freed of fat and vesicles, weighed, and sliced into
1-cm pieces. After the meat is ground thoroughly in a meat grinder, 4 ml
of a solution containing 0 . 8 8 M sucrose, 1 m M E D T A , and 0.3 m M
dithiothreitol are added for each gram of tissue, and the mixture is
homogenized with a loose-fitting pestle in a Potter-Elvehjem apparatus.
Differential Centrifugation. The homogenate is centrifuged at 1000
g for 10 minutes and the sediment is discarded. The supernatant is
centrifuged at 40,000 g for 11/~ hours and again the pellet is discarded.
First Ammonium Sulfate Cut. The supernatant from the 40,000 g
centrifugation is brought to 1.5 M (33% saturation) ammonium sulfate
by slowly adding the appropriate volume of 3.75 M ammonium sulfate. 1
After stirring for 10 minutes, sufficient solid ammonium sulfate is added
to bring the solution to 50% saturation. This solution is stirred for 15
minutes and centrifuged at 10,000 g for 10 minutes. The pellet is dis-
carded and the supernatant is brought to 80% saturation by the addition
of solid ammonium sulfate. After 2 hours of stirring, the solution is
centrifuged at 15,000 g for 15 minutes. 2 The supernatant is discarded, and
the pellet is dissolved in 33% saturated ammonium sulfate.
Second Ammonium Sulfate Cut. Protein which is insoluble in the
33% saturated solution after 15 minutes of stirring is removed by
centrifugation at 10,000 g for 10 minutes. The protein solution is then
brought to 70% saturation by the addition of solid ammonium sulfate.
After centrifugation for 10 minutes at 10,000 g, the pellet is dissolved in
2 . 0 M ammonium sulfate, in which the enzyme is stable for several
months at 0o. 3
'Ammonium sulfate, 1.5 M, was assumed to be 33% saturated. The amount of solid
ammonium sulfate added at each step was calculated on the basis of the informa-
tion contained in Vol. I [10], p. 76.
~The supernatant should be immediately assayed for enzymatic activity. If a
significant amount of activity is still in solution, stir the supernatant at 0 ° for
another 1~-1 hour and recentrifuge.
'A cloudy suspension is sometimes obtained; in this case, a small amount of 5 mM
phosphate, 15 mM KC1, 1 mM EDTA, 0.3 mM dithiothreitol, pH 7.0, is added
to dissolve the protein completely before it is added to Sephadex G-25.
Carboxymethyl-Cellulose Chromatography. Prior to chromatography,

the enzyme solution is desalted on a Sephadex G-25 column. The solvent
used contains 15 mM KC1, 5 mM phosphate, 1 mM EDTA, 0.3 mM
dithiothreitol, pH 7.0. The desalted protein solution is immediately
added to the CM-ceIlulose column (2 X 12 cm), which has been previ-
ously equilibrated with the buffer described above. Additional buffer is
added, and the eluant fractions arc assayed for absorbance at 280 m#.
When the 0D2so returns to the "background" of the buffer, the KCI con-
centration in the buffer is increased to 0.1 M to elute the enzymatically
active protein. Of the activity initially present, 60-85% is isolated along
with 2-3% of the protein. The specific activity can be increased from 170-
200 units/mg to 5000-6000 units/mg by this single step. The active
fractions are supplemented with bovine serum albumin to 2 mg/ml and
s~ored at 4 °.
The purification is summarized in the accompanying table.
PURIFICATION OF ISOCITRATEDEHYDROGENASE (TPN)
Total Specific
Volume Protein activity activity Yield
Step (ml) (rag) (units)" (units/mg) (%)
I. 40,000 g supernatant 185 1400 105,700 75 100

II. 50% saturation with 334 1180 99,240 84 94
ammonium sulfate,
supernatant
III. 80% saturation with 43.5 380 74,100 195 70
ammonium sulfate,
redissolved pellet
IV. 70% saturation with 12.7 300 64,400 215 61
ammonium sulfate,
redissolved pellet
V. CM-cellulose b 12 2.8 12,000 4300 37c
chromatography
" An aliquot of each fraction was desalted on a small Sephadex G-25 column before
the enzymatic activity was measured. A unit of enzyme is the amount that will give
a change of 0.01 OD per minute.
b A 97 mg portion of protein was chromatographed; fractions containing protein of
specific activity greater than 1800 units/mg were pooled. The specific activity of
the peak fraction was 5850 units/rag.
c Calculated by assuming that all of the 300 nag of protein was chromatographed
with the same efficiency as indicated for a 97 mg portion.
Properties
Purity. T P N - i s o c i t r a t e d e h y d r o g e n a s e isolated b y the purification
procedure described has a specific a c t i v i t y of a p p r o x i m a t e l y 5000 u n i t s /
[7] ISOCITRATE DEHYDROGENASE (TPN-SPECIFIC) 33
mg of protein. Disc gel electrophoresis4 of the enzyme gave an RI of 0.4

for the active band. The TPN-isoeitrate dehydrogenase was located with
specific stains on the basis of its enzymatic activity (Vol. VI [127], p.
969) .5
Kinetic Properties. Kinetic studiesGperformed with the enzyme in the
presence of 2 mM Mg ÷÷ and 33 mM imidazole, pH 6.8, have yielded the
following approximate K~'s: TPN, 0.1 y ; isocitrate, 0.5 ~M; TPNH, 1
~ / ; bicarbonate, 10 raM; a-ketoglutarate, 25 p2//. The mechanism is
sequential, and the ratio of maximum velocities in the forward and reverse
directions is 4. The dissociation constants for TPN and TPNH, measured
by competitive product inhibition versus each other, are both 0.3 ~M.
a-Ketoglutarate shows uncompetitive substrate inhibition, with a K~ of
480 p~/; isocitrate also shows substrate inhibition at high levels.
Specificity. The purified enzyme shows no DPN- or TPN-linked
dehydrogenase activity on malate, ethanol, lactate, glycerol, glutamate,
glycerol 3-phosphate, or glucose 6-phosphate, although some DPN-linked
dehydrogenase activities are present before the CM-cellulose chroma-
tography. Isocitrate is not oxidized by DPN. The purified enzyme is
free of aconitase.
Stability. TPN-isocitric dehydrogenase is quite unstable at low ionic
strength.
'B. J. Davis, Ann. N.Y. Acad. 8ci. 121, 404 (1964).

s We used 0.1 M Tris-ttC1 at pH 7.4 rather than at pH 8.5 and replaced MnCh with
MgC12. Dithiothreitol cannot be used in this assay mixture because it reduces
p-nitro-blue tetrazolium in the abaence of enzyme.
aV. W. Thompson and W. W. Cleland, to be published.
[8] Isocitrate Dehydrogenase (DPN-Specific)

from Bovine Heart 1
[EC 1.1.1.41 lhreo-vs-Isocitrate:NADoxidoreductase (decarboxylating)]
By G. W. E. PLAUT
Mn++
lhreo-D~-Isocitrate + D P N + (ADP~ a-ketoglutarate + CO2 + D P N H + H +
(1)
The procedures described herein constitute a modification of those
given earlier (Vol. I [119]). The enzyme is activated by A D P and is
inhibited by a number of nucleotides, la,~
Assay M e t h o d
Principle. Enzyme activity is determined by following the forma-
tion of D P N H as measured by the increase in optical density at
340 n ~ .
Reagents
Tris-acetate buffer, p H 7.2, 100 micromoles
MnC12, 4.0 micromoles
ADP, 2.0 micromoles
1.0 #mole of D P N ÷
threo-D~Ls-Isocitrate enzyme and water, 16 micromoles, in a final
volume of 3.0 ml
Procedure. Silica cells of 1.0 cm light path containing all com-
ponents of the reaction mixture except enzyme are placed into the
thermostatted curvette compartment of a spectrophotometer and al-
lowed to reach temperature equilibrium at 25 ° . The reaction is initiated
by the addition of enzyme in a plastic mixing spoon. The change
in absorbance at 340 m~ is measured in a spectrophotometer fitted
'yith a suitable recorder.
Initial reaction rates are estimated from the density change with
time of the initial linear portion of the record obtained.
The experimental work from this laboratory presented here and the preparation of
this article have been assisted by grants from the National Institute of Arthritis
and Metabolic Diseases, National Institutes of Health, United States Public
Health Service.
" R. F. Chert and G. W. E. Plaut, Federation Proc. 21, 244 (1962).
~R. F. Chen and G. W. E. Plaut, Biochemistry 2, 1023 (1963).
[8] ISOCITRATE DEHYDROGENASE (DI'N-SPECIF1C) 35
Units. One unit of enzyme is defined as that amount in 3.0 ml of

reaction mixture which causes a change of 0.01 in optical density at 340
m~ per minute at 25 ° in a cell of 1.0 cm light path. ~ Specific activity is
expressed as units per milligram of protein. Protein is determined by
the method of W a r b u r g and Christian (Vol. I I I 173]).
Purification Procedure ~
All manipulations are carried out at 2o-5 ° unless specified other-
wise.
PreparatioT~ of Aceto~e Powder. The procedure described previously
(Plaut and Sung, Vol. I [119]) is used except that the supernatant
fluid after low speed centrifugation of the sucrose-phosphate homog-
enate of bovine heart is acidified to p H 5.5 instead of p H 5.8-5.9
before sedimentation of particles enriched in mitochondria. This modifi-
cation results in greater recovery of enzyme activity, but lower specific
activity in the initial extract (step 1, below). About 230 g of acetone
powder is recovered from 30 kg of bovine heart.
Step 1. Extraction. Acetone powder (232 g) is mixed with 4.0
liters of 0 . 1 0 M potassium phosphate buffer at p H 7.2 in a Waring
blendor. The homogenate is centrifuged at 1000 g for 20 minutes.
The residue is treated with an additional 1600 ml of buffer and
centrifuged. The supernatant solutions are combined.
Step 2. Ammonium Sul]ate Fractionatio~ and Heat Step. Solid
a m m o n i u m sulfate is added to the extract while the reaction is main-
tained at p H 7.0-7.4 with solid Na~CO3 (approximately 0.1% of the
weight of a m m o n i u m sulfate added).
The precipitate formed between 30% and 50% saturation is mixed
with 500 ml of 30% saturated a m m o n i u m sulfate solution in a Potter-
E1vehjem homogenizer. The suspension is heated in a water bath at 50 °
for 15 minutes, cooled rapidly in an ice bath, and centrifuged. The
residue is discarded.
Step 3. Ammonium Sul]ate FractionatioT~. ~ The supernatant fluid
from step 2 is treated with saturated a m m o n i u m sulfate solution.
The fraction precipitating between 35% and 45% saturation is collected
'Therefore, 208 units is equivalent to the formation of 1 micromole of DPNH per
minute under the assay conditions above.
~Ammonium sulfate solutions used are adjusted to pH 7.0 with concentrated
ammonium hydroxide. Percent saturation is calculated on the basis of solubility
at 25 ° . The actual ammonium ion content at various steps in the purification
should be determined (e.g., by the method of Johnson').
M. J. Johnson, in "Manometric Techniques and Related Methods for the Study
of Tissue Metabolism" (W. W. Umbreit, R. H. Burris, and J. F. Stauffer, eds.),
2nd ed., p. 161. Burgess, Minneapolis, Minnesota, 1949.
by eentrifugation and suspended in 60 ml of 3 0 3 saturated ammonium

sulfate solution.
Step ~. Dialysis. To a 20-ml aliquot of the enzyme suspension from
step 3 is added 8 ml of saturated ammonium sulfate solution. The
residue recovered after centrifugation is dissolved in 27 ml of 5 mM
potassium phosphate buffer at pH 7.2, containing 1 mM ATP, e and is
dialyzed with high speed stirring for 2 hours against 2 liters of 5 mM
potassium phosphate at pH 7.2. The solution is clarified by centrifuga-
tion, and its ammonium ion content is diluted with the above buffer to
20 mM.
Step 5. Chromatography on DEAE-Cellulose. Four equal portions
(about 100 mg of protein in each) from the previous step are placed on
four 2.2 X 12 cm columns of DEAE-cellulose 7 (previously equilibrated
with 5 mM potassium phosphate at pH 7.2). A linear gradient consisting
of 150 ml of 5 mM potassium phosphate at pH 7.2 in the mixing chamber
and 150 ml of the buffer containing 0.2M NaCl in the reservoir is
applied to the column. A uniform flow rate of 2.2 ml per minute is
maintained with a constant-volume delivery pump. Fractions (15 ml) are
collected. The contents of tubes containing the major share of the
enzyme activity are pooled and treated with an equal volume of satu-
rated ammonium sulfate solution to precipitate the protein. The sus-
pension is centrifuged and the enzyme is stored as the precipitate
beneath the supernatant solution. The protein is dialyzed against buffer
as in step 4 (but without ATP), immediately before the next step.
Step 6. Hydroxylapatite Chromatography. The dialyzed enzyme
solution is placed on a 2.2 X 12 cm column of hydroxylapatite, s It is
eluted from the column at a flow rate of 1.5 ml per minute with a
linear gradient system consisting of 150 ml of 5 mM potassium phosphate
at pH 7.2 in the mixing chamber and 150 ml of 0.2 M potassium phos-
phate at pH 7.2 in the reservoir. The fractions (5.0 ml) with the highest
specific activity (3410-5520 units per milligram of protein) are pooled.
The solution is brought to 6 0 ~ saturation with saturated ammonium
sulfate. The precipitated protein is brought down by centrifugation and
is stored in the centrifuge tube beneath the supernatant fluid. Portions
of the residue are dissolved in buffer and dialyzed when needed.
An example of the purification procedure is given in Table I.
' The preparation contains sufficient ATPase at this stage of purification to convert
ATP to ADP. The latter stabilizes the enzyme.
DEAE-cellulose from the Brown Company, Berlin, New Hampshire, 0.9 meq/g
purified by the procedure of Kaziro et al. [Y. Kaziro, R. C. Warner, and J.-Y.
Chen, J. Biol. Chem. 236, 1917 (1961)].
aHydroxylapatite is prepared by the method of Levin (Vol. V [2]).
[8] ISOCITRA.TE DEHYDROGENASE (DPN-SPECIFIC) 37
TABLE I
PURIFICATION OF ISOCITRATE DEHYDROGENASE (DPN-LINKED)
FROM BOVINE HEART a'~
Total Activity Specific

Volume Activity activity yield activity
Step (ml) (units/ml) (units) (%) (units/mg)
1. Crude extract 5170 44 217,000 100 6.2

2. Ammonium sulfate and 525 528 277,200 128 75
heat treatment
3. Ammonium sulfate 62 4,500 279,000 129 134
fractionation
4. Dialysisc 37 2,280 84,800 100 200
5. DEAE-cellulose 14 3,200 45,080 53 1070
chromatography~
6. Hydroxylapatite 1.7 19,500 33,200 39 4500
chromatography c
" Data from R. F. Chen and G. W. E. Plaut [Biochemistry2, 1023 (1963)].
b Acetone powder, 232 g.
c One-third of the protein from step 3 was purified in steps 4 to 6. The activity yield
has been adjusted for the change in amounts.
The procedure described above gives enzyme preparations of high

specific activity, usually in reasonable yield. However, dialysis (step 4)
can lead to a substantial loss in activity since the enzyme is markedly
unstable in solutions of low ionic strength. An alternate procedure has
been developed in which the dialysis step is deleted and chromatography
on DEAE-cellulose (step 5) is replaced by another hydroxylapatite
chromatography step. This method of purification gives a final product of
somewhat lower specific activity, but with a more consistent recovery
of total activity.
Alternate Purification Method 9

Steps 1-3. The initial stage of the procedure corresponds to steps 1-3
of Table I, except that 135 g of acetone powder were used in the example
cited in Table I I and the volumes and amounts of reagents in the
fractionation steps were adjusted accordingly.
Step 4. First Hydroxylapatite Chromatography. The residue from
the preceding step is dissolved in a minimal volume of 5 m M potassium
phosphate at pH 7.2 and placed on a 2.2 X 12 cm column of hydroxyl-
apatite equilibrated previously with the buffer. The column is washed
with 100 ml of 10% saturated ammonium sulfate solution. The effluent
containing mainly inert protein is discarded. Then 30% saturated am-
R. F, Chen, A. J. Giorgio, and G. W. E. Plaut, unpublished observations, 1963.
TABLE II
ALTERNATE METHOD OF ENZYME PURIFICATION a'b
Total Activity Specific

Volume Activity activity yield activity
Step (ml) (units/ml) (units) (%) (units/mg)
1. Crude extract 2220 88 195,000 100 18.2

2. Ammonium sulfate and 200 980 196,000 100 194
heat treatment
3. Ammonium sulfate 50 3,400 170,000 87 440
fractionation
4. First hydroxylapatite 150 1,080 162,000 83 1060
chromatography
5. Second hydroxylapatite 4.9 23,000 113,000 58 3200
chromatography and
ammonium sulfate
precipitation
" Data from R. F. Chen, A. J. Giorgio, and G. W. E. Plaut, unpublished observa-
tion. 1963.
bAcetone powder, 135 g.
monium sulfate solution is applied to the column at a flow rate of 1 ml

per minute and the activity is recovered in 150-175 ml of effluent.
Step 5. Second Hydroxylapatite Chromatography. The solution con-
taining the activity from the previous step is brought to 50% saturation
with saturated ammonium sulfate solution. The precipitate is dissolved
in a minimal volume of 10 m M potassium phosphate at p H 7.2 and is
applied to a 2.2 X 12 cm column of hydroxylapatite previously equili-
brated with 10% saturated ammonium sulfate. Elution of enzyme activity
is accomplished by a gradient consisting of 150 ml of 10% saturated
ammonium sulfate in the mixing chamber and 150 ml of a solution of
10% saturated ammonium sulfate containing 0.2 M potassium phosphate
at p H 7.2 in the reservoir. Fractions containing the activity are combined
and brought to 50% saturation with saturated ammonium sulfate solu-
tion and centrifuged. The precipitated enzyme is stored in the centrifuge
tube beneath the supernatant fluid. In the fractionation cited in Table
II the residue was dissolved in 4.0 ml of 0.1 M potassium phosphate at
pH 7.2 before assay.
Propertiesla, 2, 10
Stability. The enzyme is markedly unstable at low ionic strength.
Thus, 50% of the activity is lost in 24 hours at 2 ° in a medium of ionic
~°R. F. Chen, D. M. Brown, and G. W. E. Plaut, Bioehemi.stry 3, 552 (1964).
[8] ISOCITRATE DEHYDROGENASE (DPN-SPECIFIC) 39
strength of 0.1. However, even under these conditions 90% of th(~

activity is retained after 4 days in solutions containing 5 mM ADP.
The enzyme is quite stable in 30% saturated ammonium sulfate
solution. It can be stored in this medium for at least 4 weeks at 2 ° with-
out significant loss of activity. The enzyme is less stable in solution,~
more than 40% saturated in ammonium sulfate.
Sedimentation Properties) ° Highly purified preparations of the en-
zyme have been found to contain a major active component (about 85%
of the total protein) in the ultracentrifuge, with a sedimentation constant
(S~o,,,,) of 10.3 S. On the basis of the sedimentation constant it has been
estimated that the molecular weight of the protein is around 300,000.
ADP modifies markedly the sedimentation of the protein. Thus, the
addition of 0.1 to 1 mM ADP results in the disappearance of the 10 S
component and the appearance of peak(s) at 19 to 26 S. The effect of
ADP is not due to covalent binding since ADP can be removed from
the enzyme by dialysis or gel filtration through Sephadex, leading to
reappearance of the component sedimenting at 10 S. The aggregation
caused by ADP occurs in the absence of added divalent metal ions.
Activators. ADP activates by increasing the affinity of the enzyme
for the substrate threo-Ds-isocitrate and certain divalent metal ions,
Mn ÷*, Mg +÷ (Vol. I [119]), or Co++,~° required for activity.
Values of K,~ in the absence and presence of ADP for isocitrate,
Mg ÷~, and Mn ÷÷ are shown in Table III. V.... is not changed by the
addition of ADP.
TABLE III
Km FOR threo-Ds-IsoCITRATE, ~{n ++, AND Mg ++ WITH AND WITHOUT A D P ~
Conditions ~ Km (mill)
Isocitrate
pH 6.5, no A D P 0.36
pH 6.5, 0.67 i n ] l ADP 0.10
pH 7.2, no A D P 1.5
pH 7.2, 0.67 m M ADP 0.14
Mn++
pH 7.2, no ADP 0.21
pH 7.2, 0.67 m M ADP 0.027
Mg++
pH 7.2, no A D P 1.8
pH 7.2, 0.67 m M ADP 0.18
D a t a from R. F. Chen and G. W. E. Plaut [Biochemistry 2, 1023 (1963)], and R. F.

Chen, D. M. Brown, and G. W. E. Plaut [Biochemistry 3, 554 (1964)].
b Conditions as described under Assay Method except for variations indicated.
The effect of ADP is highly specific; of a large number of nucleotides

tested only ADP and d-ADP are effective. In contrast to the enzyme
from yeast (Kornberg, Vol. I [118]) 5'-AMP is inactive. ADP exerts
its maximal effect at a concentration of around 1.3 mM, activity declines
at higher concentrations of the nucleotide.
pH Optimum. Original studies of the enzyme showed that optimal
activity occurred with a rather sharp peak at pH 6.5 in cacodylate
buffer when the threo-Ds-isocitrate concentration was 1.3 mM (Vol. I
[119]). The pH optimum shifts to pH 7.2 at this isocitrate concentra-
tion in the presence of 0.67 mM ADP, with a considerable broadening of
the pH-dependent activity peak. A similar change of pH optimum is
obtained in the absence of ADP by increasing the isocitrate concentra-
tion (13 mM). It can be seen in Table III that the apparent affinity of
the enzyme for isocitrate is greater at pH 6.5 than at pH 7.2, provided
ADP is absent.
Inhibitors. The enzyme from porcine heart is inhibited about 50% by
10 mM NaCN; at this concentration KCNS, NAN3, and Na2Mo04 are
inactive. 11 p-Hydroxymercuribenzoate inhibits the enzyme from porcine
heart. 11 p-Chloromercuriphenylsulfonate, 6.7 pjl/, inhibits the enzyme
from bovine heart completely when isocitrate is added to the reaction
mixture last; a combination of isocitrate and Mn ++ affords partial protec-
tion against p-chloromercuribenzenesulfonate inhibition when added to
the enzyme before the mercurial. The substrate, threo-D~-isocitrate, and
not the threo-b8 isomer, is responsible for the protection against inhibition
by p-chloromercuribenzenesulfonate. The same percent protection by Mn ÷÷
and isocitrate is obtained whether or not ADP is present in the assay
mixture. 1° In the protection experiments above, DPN ÷ was added last to
start the reactions. Hence, it seems likely that the combination of Mn +÷
and isocitrate can bind to the enzyme in the absence of ADP and/or
DPN ÷ (but, cf. Klingenberg et al.~2).
Inhibition competitive with DPN ÷ (K~0.78 mM at pH 7.2) is ob-
tained with DPNH (K,, 39 #M), ATP (K~, 0.15 mM), and ADPR
(Ki, 61 piP/). Acetyl pyridine-DPNH is about 70% as inhibitory as an
equivalent concentration of DPNH. TPNH alone is not an inhibitor;
however, it potentiates the inhibition by DPNH. ~ A number of other
nucleotides (IDP, GDP, ITP, UTP, 2'-AMP, T-AMP, 5'-AMP) inhibit;
however, when compared to ATP and ADPR, rather large amounts are
i~G. W. E. Plaut, in "The Enzymes" (P. D. Boyer, H. Lardy, and K. Myrblick,eds.),

~ M. Klingenberg, H. Goebell, and G. Wenske, Biochem. Z. 341, 199 (1965).
[8] ISOCITRATE DEHYDROGENASE (DPN-SPECIFIC) 41
required, thus raising the possibility that the inhibition is partly due to
chelation of Mn *÷.
Substrate Specificity. threo-Ds-Isocitrate appears so far to be the only
substrate; the other isomers of isocitrate, c/s-aconitate, citrate, D- and
L-hydroxyglutarate, malate, DL-fl-hydroxybutyrate, and glutaconate are
inactive. In contrast to the enzyme from yeast, 13 citrate neither activates
nor inhibits the preparation from bovine hearty No substrate inhibition
was observed with the purified enzyme either in the absence or presence
of 0.67 mM ADP at pH 7.2 when threo-DsL~-isocitrate concentrations up
to 26 mM were used. 1~ This appears to contrast with the reported
inhibition by 4 mM isocitrate of the DPN-linked dehydrogenase activity
of extracts from rat heart mitochondria.1~
Under comparable conditions acetyl pyridine-DPN + and thionicotina-
mide-DPN ÷ have 50% and 7%, respectively, of the activity of fl-DPN ~.
a-DPN ÷, TPN ÷, deamino-DPN ÷, pyridine aldehyde-DPN ÷, and NMN *
are inactiveY
Stereochemistry o/ HydrogeT~ Transfer. IG The hydrogen atom from
the a-position of threo-D,-isocitrate is transferred to the a-side of the
nicotinamide ring of DPN. threo-D~-Isocitrate-fl-T is oxidized by DPN*
to labeled a-ketoglutarate, without label in DPNH. The stereochemistry
of the hydrogen transfer in the oxidative decarboxylation of substrate
thus appears to be similar in DPN- and TPN-linked isocitrate dehydro-
genase reactions. ~6-2°
Occurrence. In animal tissues DPN-isocitrate dehydrogenase activity
has been found in extracts of acetone powders of mitochondria or
washed residues of heart from a variety of species, pigeon breast
nmscle, rat kidney, rat liver, and human placenta. However, the DPN
and TPN enzyme were not separated except in the cases of heart,
pigeon breast muscle, and human placenta. 2~,22
In the older work, mentioned above, ADP was not present in the
enzyme assay mixtures. This nucleotide was added in subsequent studies
in which the presence of the DPN-enzyme was reported to occur in
~J. A. Hathaway and D. E. Atkinson, J. Biol. Chem. 238, 2875 (1963).
~ R. F. Chert and G. W. E. Plaut, unpublished observation, 1963.
~H. Goebell and M. Klingenberg, Biochem. Z. 340, 441 (1964).
R. F. Chen and G. W. E. Plaut, Biochemistry 2, 752 (1963).
~7G. E. Lienhard and I. A. Rose, Biochemistry 3, 185 (1964).
~*S. Englard and S. P. Colowick, J. Biol. Chem. 226, 1047 (1957).
S. Englard, J. Biol. Chem. 235, 1510 (1960).
~°S. Englard and I. Listowsky, Biochem. Biophys. Res. Commun. 12, 356 (1963).
G. W. E. Plaut and S.-C. Sung. J. Biol. Chem. 207, 305 (1954).
2, S.-C. Sung and C. H. Hsu, Formosan Med. Assoc. 56, 103 (1957).
42 R E A C T I O N S ON T t t E CYCLE [9]
phosphate buffer-EDTA-reduced glutathione extracts of fresh mito-

chondria from the following sources: rat skeletal muscle, heart, kidney,
brain, and liver, pigeon heart, and locust flight muscle. 1~ Mitochondria
from locust flight muscle appear to contain a particularly high activity
of the enzyme, and extensive studies on the interrelationship of the
concentrations of DPN ÷, ADP, and isocitrate as affected by pH have
been made with crude extracts from this source? ~ The level of the DPN-
enzyme activity has also been compared to that of the TPN-enzyme in
extracts of acetone powders of mitochondria of brain, kidney, heart,
spleen, lung, and liver from various species, as well as in Ehrlich ascites
tumor. 2a
The determination of the level of DPN-enzyme of tissue extracts in
the presence of ADP may, however, present certain problems because
such preparations may still contain adenylate kinase leading to the
formation from ADP of ATP. ATP may phosphorylate DPN to TPN
in the presence of DPN kinase, followed by subsequent reduction of
TPN ÷ by isocitrate in the presence of the TPN-linked enzyme.24 Such
interference by other enzymes becomes difficult to eliminate in tissues,
such as liver, where the level of DPN-isocitrate dehydrogenase activity
appears to be lowJ 5,21,2s
The separation of the DPN-linked and TPN-linked isocitrate dehy-
drogenases from rabbit and porcine liver has been accomplished.25
~3A. M. Stein, J. H. Stein, and S. K. Kirkman, Biochemistry 6, 1370 (1967).
=4p. V. Vignais and P. M. Vignais, Biochim. Biophys. Acla 47, 515 (1961).
T. Aogaichi and G. W. E. Plaut, Biochem. Biophys. Res. Commun. 28, 628 (1967).
[9] I s o c i t r a t e D e h y d r o g e n a s e ( N A D - S p e c i f i c )
f r o m N e u r o s p o r a crassa
[EC 1.1.1.41 threo-D~-Isocitrate:NAD oxidoreduetase (decarboxylating)]
By R. A. COOKand B. D. SA~WAL
Mg++ (AMP)
threo-D.-Isocitrate T NAD +
a-ketoglutarate -k CO2 + NADH nu H +
Assay Method
Principle. The enzyme is assayed routinely by measuring the initial
rate of increase of absorbance at 340 m~ caused by the reduction of
NAD +. In crude extracts, the enzyme activity is measured by coupling
42 R E A C T I O N S ON T t t E CYCLE [9]
phosphate buffer-EDTA-reduced glutathione extracts of fresh mito-

chondria from the following sources: rat skeletal muscle, heart, kidney,
brain, and liver, pigeon heart, and locust flight muscle. 1~ Mitochondria
from locust flight muscle appear to contain a particularly high activity
of the enzyme, and extensive studies on the interrelationship of the
concentrations of DPN ÷, ADP, and isocitrate as affected by pH have
been made with crude extracts from this source? ~ The level of the DPN-
enzyme activity has also been compared to that of the TPN-enzyme in
extracts of acetone powders of mitochondria of brain, kidney, heart,
spleen, lung, and liver from various species, as well as in Ehrlich ascites
tumor. 2a
The determination of the level of DPN-enzyme of tissue extracts in
the presence of ADP may, however, present certain problems because
such preparations may still contain adenylate kinase leading to the
formation from ADP of ATP. ATP may phosphorylate DPN to TPN
in the presence of DPN kinase, followed by subsequent reduction of
TPN ÷ by isocitrate in the presence of the TPN-linked enzyme.24 Such
interference by other enzymes becomes difficult to eliminate in tissues,
such as liver, where the level of DPN-isocitrate dehydrogenase activity
appears to be lowJ 5,21,2s
The separation of the DPN-linked and TPN-linked isocitrate dehy-
drogenases from rabbit and porcine liver has been accomplished.25
~3A. M. Stein, J. H. Stein, and S. K. Kirkman, Biochemistry 6, 1370 (1967).
=4p. V. Vignais and P. M. Vignais, Biochim. Biophys. Acla 47, 515 (1961).
T. Aogaichi and G. W. E. Plaut, Biochem. Biophys. Res. Commun. 28, 628 (1967).
f r o m N e u r o s p o r a crassa
[EC 1.1.1.41 threo-D~-Isocitrate:NAD oxidoreduetase (decarboxylating)]
By R. A. COOKand B. D. SA~WAL
Mg++ (AMP)
threo-D.-Isocitrate T NAD +
a-ketoglutarate -k CO2 + NADH nu H +
Assay Method
Principle. The enzyme is assayed routinely by measuring the initial
rate of increase of absorbance at 340 m~ caused by the reduction of
NAD +. In crude extracts, the enzyme activity is measured by coupling
[9] ISOCITRATE DEHYDROGENASE FROM N'. crassa 43
the reaction to the reduction of dichlorophenol-indophenol with dia-

phorase.
Reagents
Tris-acetate, 0.2 M, pH 7.6
NAD ÷, 15 mM (10 mg/ml), prepared fresh
AMP, 20 raM, pH 7.6, prepared fresh
MgC12"6 H20, 0.1 M
Trisodium threo-DsLs-isocitrate, 25 mM, pH 7.6
Dichlorophenol-indophenol, 5.3 mM
Diaphorase (NADH:lipoamide oxidoreductase), from heart
muscle 1
Procedure. In crude extracts, containing NAD + oxidase activity, the

coupled assay is used. The following compounds are added to a 1 cm
light path cuvette: Tris-acetate buffer, 2.4 ml; dichlorophenol-indo-
phenol, 0.1 ml; NAD, 0.1 ml; MgCl~, 0.1 ml; AMP, 0.04 ml; isocitrate,
0.1 ml; an excess of diaphorase, a suitably diluted enzyme preparation,
and H~O to give a final volume of 3.0 ml. Isocitrate is omitted from
the control mixture. The reaction is started by the addition of the en-
zyme. The decrease in absorbancy is measured at 600 mtL over a
2-minute period.
With purified preparations of the enzyme, the assay mixture contains
buffer, 2.5 ml; NAD, 0.1 ml; AMP, 0.04 ml; MgCl~, 0.1 ml; isocitrate,
0.1 ml; and enzyme plus water to give a final volume of 3.0 ml. The
initial rate of increase of absorbance is measured at 340 m~ for at least
2 minutes. Isocitrate is omitted from the control mixture.
Units. One unit of enzyme is defined as the amount causing an
increase in absorbance of 0.001 per minute at 340 m~. Specific activity
is expressed as units per milligram of protein. Protein is determined by
the colorimetric method of Lowry et al. 2 With highly purified enzyme
preparations, protein is determined by absorbance measurements at 260
m~ and 280 m~. a
All procedures are carried out at 0-4 °, unless otherwise stated, and
all centrifugations are performed at 17,000 g for 15 minutes.
Organism and Growth Conditions. Wild-type strain STA4 of Neuro-
1V. Massey, Biochim. Biophys. Acta 37, 314 (1960).

O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193,
265 (1951).
' O. Warburg and W. Christian, Biochem. Z. 310, 304 (1941).
spora crassa is used. Conidia from 4-day-old slant cultures are washed
and dispensed into 100 ml of Vogel's 4 Medium N contained in 500-ml
Erlenmeyer flasks. The medium contains 0.5% sucrose plus 10 mM
citrate as energy source. The flasks are shaken vigorously by rotary
action at 28 ° for 24 hours. Mycelia from several flasks are used to
inoculate a carboy containing 10 liters of the same medium. The culture
is agitated by forced aeration and incubated for 48 hours at 28 ° . Cells
are harvested on two layers of cheesecloth, washed with deionized water,
and lyophilized. The yield of the cells is approximately 100-200 g wet
weight. Freeze-dried cell powder can be stored indefinitely at --20 ° if
kept in tightly sealed containers.
Step 1. Extraction. The enzyme is extracted by suspending finely
ground lyophilized cells in 0.2 M Tris-acetate, pH 7.6, containing 0.1 mM
EDTA and 0.1 mM dithiothreitoV (1 g of powder in 15 ml of buffer)
for 1 hour with mechanical stirring. The cell debris is removed by
centrifugation. The pH of the supernatant solution is adjusted to 5.0
with 2 0 ~ acetic acid, and the precipitate is discarded after centrifuga-
tion.
Step 2. Ethanol Fractionation2 Ethanol (precooled to --20 °) is
added gradually with stirring to the supernatant solution to give a final
concentration of 9%. After the preparation has stood for 15 minutes
at --10 °, the precipitate is removed by eentrifugation. Ethanol is then
added gradually to this supernatant solution to give a final concentration
of 2 0 ~ , and the solution is allowed to remain at --10 ° for 1 hour. The
precipitate is recovered by centrifugation and dissolved in 0.1 M Tris-
acetate buffer, pH 7.6, containing 0.1 mM EDTA and 0.1 m M dithio-
threitol. The final volume should be one-tenth the volume of the
original extract.
Step 3. Re]ractionation with Ethanol. T The pH of the suspension from
step 2 is adjusted to 5.0 with 20% acetic acid, and the precipitate is
removed by centrifugation. Ethanol is added to the supernatant solution
to a final concentration of 9% and allowed to stand for 15 minutes at
--10% The precipitate is again removed by centrifugation. More ethanol
is added to the supernatant solution to give a final concentration of 15%,
and the solution is maintained at --10 ° for 2 hours. The resulting pre-
cipitate is recovered by centrifugation and dissolved in a small volume of
20 mM phosphate buffer, pH 6.5, containing 0.1 mM EDTA.
Step 4. Ultracentri]ugation. The enzyme preparation from step 3 is
"H. J. Vogel, Microbiol. Genet. Bull. 13, 42 (1956).
' W. W. Cleland, Biochemistry 3, 480 (1964).
6B. D. Sanwal, M. W. Zink, and C. S. Stachow, J. Biol. Chem. 239, 1597 (lI~4).
~B. D. Sanwal and C. S. Stachow, Biochim. Biophys. Acta 96, 28 (1965).
[9] ISOCITRATE DEHYDROGENASE FROM N . crassa 45
centrifuged at 40,000 rpm in 4.5-ml lots for 1 hour in a swinging-bucket

rotor (SW 39L) of a Spinco Model L ultracentrifuge. The gelatinous
precipitate containing nucleoproteins is discarded. The enzyme prepa-
ration may be stored at --20 ° at this stage.
Step 5. DEAE-Cellulose Chromatography. The enzyme solution is
dialyzed for 6 hours against frequent changes of cold 50 mM phosphate
buffer, pH 6.5, containing 0.1 mM EDTA and 0.1 mM dithiothreitol. The
enzyme is relatively unstable at this stage and with prolonged dialysis
the activity is considerably reduced. The enzyme is chromatographed on
a DEAE-cellulose column (2.5 cm X 45 cm) equilibrated previously
with 50 mM phosphate buffer containing E D T A and dithiothreitol. The
SUMMARY OF PURIFICATION PROCEDURE a OF

LYOPHILIZED Neurospora crassa
Total Specific
protein activity Recovery
Fraction Total units (mg) (units/mg) (%)
1. Extraction
Crude extract 102,000 1470 69.5 100
After pH 5 precipitation 99,000 915 108 97
2. Ethanol fractionation
9% EtOH supernatant 108,000 717 150 102
9-20% fraction 90,000 161 560 88
3. Refractionation
After pH 5 precipitation 90,000 108 834 88
9% EtOH supernatant 81,000 88.5 915 80
9-15% fraction 62,000 36.4 1700 61
4. Ultracentrifugation 66,000 34.0 1940 64
5. DEAE chromatography 49,500 2.6 19000 48
For 10 g.
enzyme is eluted by applying a linear gradient of EDTA. The mixing

chamber contains 50 mM phosphate and 0.1 mM EDTA, pH 6.5, and
the reservoir contains only 70 mM EDTA, pH 6.5. Successive 5.0-ml
fractions are collected at 4 ° . The tubes containing enzyme activity are
pooled, and the enzyme is precipitated by adding solid (NH4)2SO, to
80% saturation. The enzyme is recovered by centrifugation and dissolved
in a small amount of 20 mM phosphate buffer, pH 6.5 containing 1 mM
EDTA. The enzyme is routinely stored for at least 3-4 months at --20 °
with little loss of activity.
Purity. The enzyme prepared by the above procedure is not homoge-
neous but yields one heavy and several light bands on disc electro-
phoresis in polyacrylamide gel. The heavy band can be selectively
stained by the nitro-blue tetrazolium-phenazine methosulfate method, s

The preparation lacks most of the interfering enzymes, such as the
NADP-specific isocitrate dehydrogenase, a-ketoglutarate dehydrogenase
complex, NAD- and NADP-specific glutamic dehydrogenase, aconitase,
NAD- and NADP-oxidase and isocitritase.
The purification procedure is summarized in the table.
Properties
NAD-specific isocitrate dehydrogenase serves a regulatory function
in vivo, and belongs to a class of enzymes which have been termed
"allosteric. ''9 These enzymes are characterized by the presence of an allo-
steric site which normally binds effectors that may be totally unrelated
to the substrate. The allosteric nature of isocitrate dehydrogenase is
reflected in the marked deviations of the initial velocity data from normal
Michaelis-Menten kinetics when isocitrate is used as the variable
substrate2
Activators and Inhibitors. The enzyme is activated at pH 7.6 by
citrate, which binds to the allosteric site. This activation is specific, since
anions and related compounds from the citric acid cycle such as malate,
fumarate, malonate, oxalate, tartrate, and acetate have no effect on
enzyme activity. 7 The substrate, isocitrate, also activates the enzyme by
binding to the allosteric site.
5'-AMP activates isocitrate dehydrogenase apparently by increasing
the affinity of both the allosteric and active sites. This effect is specific;
5'-GMP, 5'-IMP, 5P-UMP, 5'-CMP, ADP, and ATP tested at a concen-
tration of 1.6 mM do not substitute for AMP. The requirement for
adenylic acid in the activation of the enzyme is dispensable in the
presence of high concentrations of citrate or isocitrate at pH 7.6.
a-Ketoglutarate and L-glutamate inhibit isocitrate dehydrogenase activ-
ity at pH 7.6, possibly by binding to the allosteric site.
At pH 6.5 where the allosteric site is inoperative, the activators
citrate and isocitrate, and the inhibitors, a-ketoglutarate and glutamate,
have no effect on enzyme activity.
Ef]ect o] pH. The enzyme tested with saturating isocitrate (8 mM)
shows a pH optimum of 7.4-7.8 in Tris-acetate or phosphate buffer. At
pH 6.5, the allosteric site is "inoperative."
Enzyme Mechanism. NAD-specific isocitrate dehydrogenase is a
protein of molecular weight approximately 105,000 as indicated by
sucrose density gradient centrifugation. 1°
8C. L. Markert and F. Moller, Proc. Natl. Acad. Sci. U.S. 45, 753 (1959).
9j. Monod, J. P. Changeux, and F. Jacob, J. Mol. Biol. 6, 306 (1963).
1oR. A. Cook and B. D. Sanwal, unpublished results.
[10] ISOCITRATE DEHYDROGENASE (NAD-SPECIFIC) 47
In the presence of AMP, the reaction mechanism is most probably

ordered 11 with NAD * binding first, followed by isocitrate. The release of
products then occurs in the order: C02 a-ketoglutarate, and NADH~. In
the absence of AMP, the mechanism appears to become random 1~ (i.e.,
the steps for the addition of substrates to the enzyme become partially
rate limiting).
At pH 7.6 where the allosteric site is operative, sigmoid curves of
isocitrate vs velocity are obtained due to the sequential binding of two
isocitrate molecules. Binding of one molecule of isocitrate at the allosteric
site seems to be essential for the binding of a second molecule of isocitrate
at the active site (total allosterism)21
At pH 6.5 where the allosteric site seems to be "inoperative," normal
Michaelis-Menten kinetics are observed.
Kinetic Constants. At pH 7.6, in the presence of 2.2 mM citrate, the
Km for NAD is 0.33 mM and the K,~ for isocitrate is 0.15 mM. At pH
6.5, the Km for isocitrate is 0.26 mM and the Km for NAD is 0.26 raM.
11B. D. Sanwal, C. S. Stachow, and R. A. Cook, Biochemistry 4, 410 (1965).
~2B. D. Sanwal and R. A. Cook, Biochemistry 5, 886 (1966).
from Pea Mitochondria
[EC 1.1.1.41 threo-Ds-Isocitrate:NAD oxidoreductase (decarboxylating)]
By G. F. Cox
Mn++
Ds-Isocitrate q- NAD + , a-ketoglutarate q- COs -b NADH -b H + (1)
Assay Method
Principle. The method is based on the measurement of the increase in
optical density at 340 rn~ due to the production of NADH from NAD +
with the consequent oxidation of Ds-isocitrate. The assay is performed at
pH 7.6, which is optimal for the reaction.
Reagents
Trisodium D~L,-isocitrate, 120 mM (60 mM with respect to D~-
isocitrate)
Manganese sulfate, 30 mM
NAD +, 20 mM
Enzyme, diluted with a solution containing 50 mM N-2-hydroxy-
In the presence of AMP, the reaction mechanism is most probably

ordered 11 with NAD * binding first, followed by isocitrate. The release of
products then occurs in the order: C02 a-ketoglutarate, and NADH~. In
the absence of AMP, the mechanism appears to become random 1~ (i.e.,
the steps for the addition of substrates to the enzyme become partially
rate limiting).
At pH 7.6 where the allosteric site is operative, sigmoid curves of
isocitrate vs velocity are obtained due to the sequential binding of two
isocitrate molecules. Binding of one molecule of isocitrate at the allosteric
site seems to be essential for the binding of a second molecule of isocitrate
at the active site (total allosterism)21
At pH 6.5 where the allosteric site seems to be "inoperative," normal
Michaelis-Menten kinetics are observed.
Kinetic Constants. At pH 7.6, in the presence of 2.2 mM citrate, the
Km for NAD is 0.33 mM and the K,~ for isocitrate is 0.15 mM. At pH
6.5, the Km for isocitrate is 0.26 mM and the Km for NAD is 0.26 raM.
11B. D. Sanwal, C. S. Stachow, and R. A. Cook, Biochemistry 4, 410 (1965).
~2B. D. Sanwal and R. A. Cook, Biochemistry 5, 886 (1966).
from Pea Mitochondria
[EC 1.1.1.41 threo-Ds-Isocitrate:NAD oxidoreductase (decarboxylating)]
By G. F. Cox
Mn++
Ds-Isocitrate q- NAD + , a-ketoglutarate q- COs -b NADH -b H + (1)
Assay Method
Principle. The method is based on the measurement of the increase in
optical density at 340 rn~ due to the production of NADH from NAD +
with the consequent oxidation of Ds-isocitrate. The assay is performed at
pH 7.6, which is optimal for the reaction.
Reagents
Trisodium D~L,-isocitrate, 120 mM (60 mM with respect to D~-
isocitrate)
Manganese sulfate, 30 mM
NAD +, 20 mM
Enzyme, diluted with a solution containing 50 mM N-2-hydroxy-
ethylpiperazine-N'-2-ethanesulfonic acid (HEPES) pH 7.6, 5 M

glycerol to contain 7.5-30 units of activity per milliliter (see
definition of unit of activity below)
HEPES-NaOH buffer, 50 mM, pH 7.6
Procedure. The assays are performed using a spectrophotometer with

a temperature-controlled cell housing at 25 °. Into a 1 cm light path silica
cuvette are placed 2.6 ml of buffer, 0.1 ml of manganese sulfate, 0.1 ml
of trisodium n~L~-isocitrate, and 0.1 ml of NAD *. All reagents are dis-
solved in HEPES-NaOH pH 7.6. The reaction is started by the addition
of 0.1 ml of enzyme solution, and the rate of change of E at 340 m/~ is
measured.
Units. One unit of enzyme activity is defined as the production of
10 millimicromoles of N A D H per minute.
Specific activity is expressed as the units of enzyme activity per
milligram of protein.
Protein concentration is measured throughout by the spectrophoto-
metric method of Warburg and Christian. 1
Application of Assay to Crude Mitochondrial Extracts. If the crude
extract is passed through a column of Bio-Gel P-10 which has been pre-
viously equilibrated in a solution containing 50 mM HEPES, pH 7.6,
5 M glycerol, the enzyme can be satisfactorily studied under the assay
conditions described without interference from other enzyme reactions.
Peas (var. Alaska) are soaked overnight in running water and planted
thickly in well-moistened Vermiculite. The peas are incubated for 10
days at 25 ° in a dark room. At this stage the etiolated shoots are about
10 cm in length.
Grind 300 g of shoots in 50 g quantities, each with 50 ml of 0.5 M
sucrose, 0.1 M phosphate buffer pH 7.6, with a pestle and mortar. Strain
the ground tissue through nylon gauze and squeeze gently; the total
volume of filtrate is approximately 600 ml. All operations are at 0%
The extract is centrifuged at 1000 g for 5 minutes. The supernatant
is centrifuged for a further 15 minutes a~ 20,000 g to give a firm pellet
which is predominantly mitochondrial. The supernatant from this centrif-
ugation contains approximately 95% of the NADP-specific isocitrate
dehydrogenase activity. The NAD-specifie isocitrate dehydrogenase is
absent from this fraction. Washing of the pellets does not give significant
increases in initial specific activity and is therefore omitted.
The pellets are resuspended in 15 ml of a solution containing 0.1 M
IO. Warburg and W. Christian, Biochem. Z. 310, 384 (1942).
sodium bicarbonate and 5 M glycerol. A French press, cooled to 0 °, is

used to disintegrate the mitochondria, applying 3000 psi pressure by
hydraulic press. The extract is collected in a cooled vessel and then
centrifuged at 100,000 g for 20 minutes. The clear supcrnatant (volume
at)proximately 20 ml) is termed the crude extract.
Step I. A solution containing 0.1 M sodium bicarbonate and 5 M
glycerol is added to the crude extract until a protein concentration of 10
mg/ml is obtained; 1 g of alumina C), containing 8% solids is added per
100 mg of protein present, with the aim of absorbing 80-90% of the
original activity, and the gel is thoroughly dispersed. The suspension is
centrifuged at 5000 g for 5 minutes, and the supernatant is assayed to
establish the extent of the adsorption. If a satisfactory amount of the
enzyme has been adsorbed the supernatant is discarded. The pellet is
dispersed in 6 ml of a solution containing 50 m21I sodium bicarbonate,
0.2 M trisodium citrate, 5 M glycerol. The gel is dispersed thoroughly,
and allowed to stand for 15 minutes. Again the suspension is centrifuged
at 5000 g for 5 minutes. The supernatant is retained, and the pellet is
subjected to identical treatment using 3 ml of elution medium.
Step 2. The combined fractions are run through a desalting column
30 cm X 2 cm of Bio-Gel P-10 which has been equilibrated in a solution
containing 50 mM sodium bicarbonate and 5 M glycerol. The active
fractions are combined.
Step 3. A column of DEAE-cellulose 13 cm X 1.5 cm is prepared using
DEAE-cellulose equilibrated in 50 mM sodium bicarbonate, 5 M glycerol.
The column is packed under pressure provided by a small hand pump,
to give a flow rate of 60 ml per hour. The eluate from the desalting
column is pumped onto the column, again using the hand pump. It is
important to work with speed throughout this step as evidence suggests
that the enzyme is unstable on the DEAE-cellulose. The first wash is 20
ml of a solution containing 0.1 M sodium bicarbonate, 40 mM trisodium
citrate, and 5 M glycerol. This operation removes most of the remaining
pigmented material and entirely removes the NADP-specific isocitrate
dehydrogenase from the column. The column is then washed with a
solution containing 0.1 21I sodium bicarbonate, 60 mM trisodium citrate,
and 5 M glycerol; the eluate is collection in 1-ml fractions. This treat-
ment removes NAD-spccific isocitrate dehydrogenase from the column.
Step 4. The active fractions are combined (usually 3-4 ml) and passed
through a desalting column of Bio-Gel P-10 (15 cm X 2 cm) equilibrated
with a solution of 50 mM HEPES and 5 M glycerol, pH 7.6. The active
fractions are stored at - 1 5 °.
The purification procedure is summarized in the table.
The preparation obtained above is entirely free of the following
PURIFICATION OF ISOCITRATE DEHYDROGENASE (NAD-SPECIFIC)
FROM PEA MITOCHONDRIA
Total Specific
activity Protein activity Yield Purification
Fraction (units)a (mg/ml) (units)a (%) (fold)
Crude extract 1100 17.4 3.0 100 0

First C, eluate 578 4.3 22.5 52 7.5
Second C~ eluate 130 3.1 14.0 12.0 4.7
DEAE-cellulose
fractions
(a) 40 mM citrate
fractions 1-20 0 -- 0 0 0
(b) 60 mM citrate
fraction 24 23 0.24 96 2.0 32
fraction 25 47 0.26 180 4.5 60
fraction 26 33 0.24 138 3.0 46
fraction 27 20 0.15 133 2.0 44
Combined DEAE- 123 0.90 137 11.5 46
cellulose fractions
Nos. 24-27
One unit of enzyme activity = production of 10 millimicromoles of NADH per
minute. Specific activity is expressed as units of enzyme activity per milligram
of protein.
enzymes: (a) NADP-specific isocitrate dehydrogenase, (b) NAD-specific
glutamic dehydrogenase, (c) aconitase, (d) malic dehydrogenase.
A shortened purification can be carried out using the DEAE-cellulose
step alone. After being passed through Bio-Gel P-10, this preparation is
termed the "partially purified" enzyme; it has been used extensively for
the characterization of the enzyme.
Properties
Reaction with Substrate. Both the crude and the partially purified
preparations give sigmoid plots when the initial rates are plotted against
substrate concentration. The gradient of the plot of
log ~ X 100 against l o g S X 100
(the Hill plot) is variable between 2.0 and 3.0.

Activators. Citrate activates the enzyme at low concentrations. In the
presence of 1 m M citrate, " n o r m a l " Miehaelis-Menten kinetics are ob-
served and the gradient of the Hill plot falls to 1.0.
Stability oJ the Enzyme. In the absence of glycerol the enzyme is
very unstable. Even low concentrations of glycerol aid the stability, but
5 M has been found to be the most suitable concentration. When the

crude enzyme is stored at --15 ° in a solution containing 50 mM HEPES
at pH 7.6 and 5 M glycerol, it shows little loss of activity over a period
of 14 days. The purified enzyme is less stable under these conditions, but
20% of the original activity remains after 14 days.
pH Optimum ]or the Reaction. The pH optimum for the reaction is
7.6 in HEPES. The plot of initial rate against substrate concentration
becomes less sigmoid at pH values lower than this, and more sigmoid at
pH values above.
Metal Ion Requirement. The enzyme shows an absolute requirement
for certain divalent metal ions. Manganese, magnesium, and zinc ions
all activate the enzyme. Zinc inhibits the enzyme at higher concentra-
tions. The metal ion concentrations at half-maximal velocity are as
follows: manganese, 18 ~M; magnesium 57 ~M; and zinc, 50 ~M.
NAD ÷ Requirement. Absolute specificity is shown for NAD ÷ as the
coenzyme. The plot of initial rate against NAD ÷ concentration obeys
"normal" Miehaelis-Menten kinetics with both the crude and purified
preparations. The Michaelis constant for NAD ÷ is 0.23 mM.
NADH Effect. Both the crude and the purified enzyme are competi-
tively inhibited by NADH, and the K~ as calculated from the results with
the purified enzyme is 0.19 mM. The inhibition of the crude enzyme is
confused by the presence of an NADH oxidasc in the system which results
in sigmoid plots of rate against NAD ÷ concentration in the presence of
NADH.
Reversibility of the Reaction. The reaction appears to be irreversible
in the assay conditions used. No oxidation of NADH occurred using
buffers over the range of pH 6.0 to 8.5.
Importance o] --SH Groups. When the enzyme is prepared in the
manner described, no requirement for cysteine can be demonstrated. 2
Iodoacetate (1 mM) has no effect at saturating substrate concentrations,
but p-hydroiymercuribenzoate (66 /~M) inhibits the enzyme by 40%
under the same conditions.
Inhibitors. Monovalent anions inhibit the enzyme competitively and
increase the sigmoid nature of the rate/substrate plots. A typical series
are the halogens, in which there is increasing inhibition with increasing
ionic size. As a result of this property considerable buffer effects were
observed when using Tris-HCl. HEPES does not appear to have this
disadvantage.
~D. D. Davies, J. Exptl. Botany 6, 212 (1955).

[11] ,~-Ketoglutarate Dehydrogenase from Pig Heart

By D. R. SANADI
Assay Methods
Principles. a-Ketoglutarate dehydrogenase is an integrated, multi-
enzyme complex present in mitochondria. 1 It catalyzes the oxidative
decarboxylation of a-ketoglutarate (KG) to succinyl-CoA 2 as shown in
Eq. (1).
KG -[- CoASH -}- NAD + --~ succinyl-CoA -I- C02 -~ NADH T H + (1)
The reaction rate may be measured with purified preparations of. the
enzyme by the absorbance increase produced by the reduction of NAD.
In crude preparations, NAD reduction is not a suitable measure of the
activity, mainly because of the presence of compounds that cause re-
oxidation of NADH. The oxidation of KG by ferricyanide [Eq. (2)],
measured by a manometric procedure, 1 is applicable at all stages of puri-
fication, including the homogenate of heart muscle.
KG ~- 2 Fe(CN)68- --~ succinate -~ CO2 ~ 2 Fe(CN)s 4- -{- 2 H + (2)
NAD Reduction Assay
Reagents
Phosphate buffer, 0.2 M, pH 7.2
CoASH, 1 mM
Cysteine.HC1, 0.1 M, adjusted to pH 7.0-7.5
NAD, 10 mM, pH 7.2
KG, 3 raM, pH 7.2
Procedure. The following components are mixed in a test tube: phos-
phate, 1.0 ml; CoASH, 0.15 ml; cysteine, 0.1 ml; NAD, 0.1 ml; KG, 0.5
ml; and sufficient distilled water to bring the total volume, including the
enzyme to be added last, to 3.0 ml. The solution is incubated at 30 ° for
2 minutes and then transferred to the cuvette with a 1 cm light path. The
cuvette chamber maintained at 30 °. The reaction is initiated by the
addition of 0.02 to 0.1 ml of enzyme preparation; the formation of NADH
is measured by the absorbance increase at 340 m~. The reference cuvette
contains water.
ID. R. Sanadi, J. W. Littlefield, and R. M. Bock, J. Biol. Chem. 197, 851 (1952).
=D. R. Sanadi and J. W. Littlefield, J. Biol. Chem. 201, 103 (1953).
[11] o~-KETOGLUTARATE DEHYDROGENASE FROM PIG HEART 53
The original* assay medium contained only 0.1 mM NAD, which

presumably gives only 6 8 3 of the maximal velocity2 Increasing NAD
to 0.3 mM increases the activity to 87•o of the maximum velocity.
Manometric Assay using Ferricyanide

Reagents
Sodium bicarbonate, 1 M
Diphosphothiamine, 2 mg/ml
MgC12, 0.1 M
Bovine serum albumin, 30 mg/ml, pH 6.9
Potassium ferricyanide, 0.5 M
KG, 0.1 M, pH 7.2
Procedure. The main compartment of the Warburg vessel receives
bicarbonate, 0.4 ml; diphosphothiamine, 0.1 ml; MgCl~, 0.2 ml; albumin,
1.0 ml; KG, 0.5 ml; and enzyme preparation and distilled water to make
a final volume of 2.9 ml. Ferricyanide, 0.1 ml, is placed in the side arm.
The vessels are gassed for 5 minutes with C02 and equilibrated in a
water bath at 37 ° for another 5 minutes. The stopcocks are closed and
the ferricyanide is tipped in. The C02 evolved in the first 10 minutes is
proportional to the enzyme concentration.
Additional Methods. KG oxidation by ferricyanide can be followed
spectrophotometrically by measuring the decrease in absorbance at 410
n~. 3 2,6-Dichlorophenol-indophenol may be used as the electron acceptor
instead of ferricyanide, in which case the absorbance decrease at 600
m~ is measured. These spectrophotometric methods are satisfactory after
the active mitochondrial fraction is separated from the homogenate. The
use of cyanide to inhibit cytochrome oxidase is essential in measuring the
activity of particulate preparations. It must be recognized also that the
reaction rate with the artificial acceptors decreases with time, and
meaningful assays are obtained only during the first 30 seconds. This
difficulty has been largely overcome in the manometric assay by using
bovine serum albumin as a protective agent. The albumin, however, has
no protective effect on the spectrophotometric assays of short duration
using the artificial acceptors.
Diced pig heart is homogenized in 150-g batches with 450 ml of 30
mM phosphate buffer, pH 7.2, in a Waring blendor for 2 minutes. A con-
venient amount of tissue to start with is 1.5 kg, which is the amount used
sV. Massey, Biochim. Biophys. Acta 38, 447 (1960).
in the present description. The entire procedure is carried out at 0-4 ° .
The homogenate is centrifuged at 2000 g for 30 minutes. The cloudy
supernatant liquid is adjusted to pH 5.4 with acetic acid and centrifuged
again for 20 minutes. The precipitate is suspended with a glass homog-
enizer in 300 ml of water and recentrifuged at 4000 g for l0 minutes.
The washed precipitate is again suspended in 100 ml water, the pH of
the suspension is adjusted to 7.0 with 1 N NaOH, and the volume is
brought to 250 ml with distilled water. The suspension is frozen at - - 2 0 °
and thawed at least twice over a period of 24 hours or more. The protein
coagulated by this treatment is removed by centrifugation at 18,000 g
for 20 minutes.
Ammonium acetate is added to the slightly pink supernatant solution
in the amount of 38.5 g/100 ml. After an equilibration period of at least
10 minutes, the mixture is centrifuged for 20 minutes at 18,000 g. The
supernatant liquid is collected, and the same amount of ammonium
acetate is again dissolved in it. After 10 minutes, the suspension is centri-
fuged as above. The yellowish precipitate is dissolved in 10 ml of 10 mM
sodium bicarbonate or 20 mM phosphate buffer, pH 7.2, and dialyzed
against the same buffer for 6-8 hours. At this stage of the preparation,
approximately 10% of the activity of the homogenate is recovered with
350-fold purification. The activity of the preparation in the CoA-linked
assay is generally 4-5 micromoles of NAD reduced per minute per milli-
gram of protein.
Additional 2- to 3-fold increases in specific activity have been
reported by adsorption of the ammonium acetate fraction on a column of
calcium phosphate gel2 Separation of some lipoyl dehydrogenase, together
with some colorless protein, has been observed during this fractionation.
The activity of the complex treated in this manner is reported to be
approximately 15 micromoles of NAD reduced per minute per milli-
gram of protein.
Hirashima, Hayakawa and Koike 3~ have recently described another
procedure for the purification of pig heart a-ketoglutarate dehydrogenase.
The preparation was homogeneous in electrophoretic and sedimentation
analysis. Its activity, assayed under Massey's conditions, 3 was 2.4-4.8
/~moles NAD reduced per minute per mg protein, which is in agreement
with the value reported by Sanadi, Littlefield, and Bock. 1
Properties
Both the ammonium acetate fraction and the calcium phosphate gel
preparation show a major broad peak, comprising over 90% of the pro-
tein in the ultracentrifuge. 1,8 The S2o,w extrapolated to zero protein con-
3, M. Hirashima, T. H a y a k a w a and M. Koike, J. Biol. Che.m. 242, 902 (1967).
[12] DEHYDROGENASE FROM E. coli
a-KETOGLUTARATE 55
centration was 36 S.3~ The absorption spectrum shows maxima at 455 m~

and around 360 m~, and a shoulder in the region of 480 m~. Diphospho-
thiamine, lipoic acid, and flavin are present as tightly bound cofactors in
the amounts of 3.1, 4.5, and 4.1 micromoles, respectively, per gram of
protein. Dissociation of the KG dehydrogenase complex occurs in the
presence of 2.5 M urea into at least two component fractions; these can
recombine to form a complex which has the same sedimentation constant
as the original complex2 The lipoyl dehydrogenase component can be
isolated also from a trypsin digest of the complex. 4
The Km values for KG, NAD, and CoA with the KG dehydrogenase
complex are 13 ~M, 4.5 ~ I , and ,~0.1 ~M, respectively? Hirashima et al. 4
report a value of 0.11 mM for the Km with KG.
The KG dehydrogenase activity is highly sensitive to arsenite and
Cd +*, which probably bind the dithiol grouping in the lipoic acid and the
lipoyldehydrogenase.5 The inhibition can be reversed by dithiol com-
pounds like 2,3-dimercaptopropanol, but not by monothiol compounds.
Tshikawa and co-workers~ have reported recently the isolation of an
a-ketoglutarate dehydrogenase complex from bovine kidney mitochondria.
Its properties appear to be similar to those of the heart enzyme. It can be
resolved into dihydrolipoyl transsuccinylase and flavoprotein fractions.
'R. L. Searls and D. R. Sanadi, J. Biol. Chem. 235, 2485 (1960).
D. R. Sanadi, M. Langley, and F. White, J. Biol. Chem. 234, 183 (1959).
~E. Ishikawa, R. M. Oliver, and L. J. Reed, Proc. Natl. Acid. Sci. U~S. 56, 534 (1966).
[12] ~ - K e t o g l u t a r a t e D e h y d r o g e n a s e C o m p l e x
from Escherichia coli
By LESTER J. REED and BARID B. MUKHERJEE
a-Ketoglutarate -{- CoA ~- DPN + --~
succinyl-CoA ~- COs -b DPNH -~ H + (1)
Enzyme systems that catalyze reaction (1) have been isolated from
pig heart muscle,1-3 E s c h e r i c h i a coli, 4 and beef kidney mitochondria~ as
multicnzyme complexes with molecular weights of several million. The
I D. R. Sanadi, J. W. Littlefield, and R. M. Bock, J. Biol. Chem. 197, 851 (1952).
3M. Hirashima, T. Hayakawa, and M. Koike, J. Biol. Chem. 242, 902 (1967).
M. Koike, L. J. Reed, and W. R. Carroll, J. Biol. Chem. 235, 1924 (1960).
~E. Ishikawa, R. M. Oliver, and L. J. Reed, Proc. Natl. Acad. Sci. U.S. 56, 534
(1966).
a-KETOGLUTARATE 55
centration was 36 S.3~ The absorption spectrum shows maxima at 455 m~

and around 360 m~, and a shoulder in the region of 480 m~. Diphospho-
thiamine, lipoic acid, and flavin are present as tightly bound cofactors in
the amounts of 3.1, 4.5, and 4.1 micromoles, respectively, per gram of
protein. Dissociation of the KG dehydrogenase complex occurs in the
presence of 2.5 M urea into at least two component fractions; these can
recombine to form a complex which has the same sedimentation constant
as the original complex2 The lipoyl dehydrogenase component can be
isolated also from a trypsin digest of the complex. 4
The Km values for KG, NAD, and CoA with the KG dehydrogenase
complex are 13 ~M, 4.5 ~ I , and ,~0.1 ~M, respectively? Hirashima et al. 4
report a value of 0.11 mM for the Km with KG.
The KG dehydrogenase activity is highly sensitive to arsenite and
Cd +*, which probably bind the dithiol grouping in the lipoic acid and the
lipoyldehydrogenase.5 The inhibition can be reversed by dithiol com-
pounds like 2,3-dimercaptopropanol, but not by monothiol compounds.
Tshikawa and co-workers~ have reported recently the isolation of an
a-ketoglutarate dehydrogenase complex from bovine kidney mitochondria.
Its properties appear to be similar to those of the heart enzyme. It can be
resolved into dihydrolipoyl transsuccinylase and flavoprotein fractions.
'R. L. Searls and D. R. Sanadi, J. Biol. Chem. 235, 2485 (1960).
D. R. Sanadi, M. Langley, and F. White, J. Biol. Chem. 234, 183 (1959).
~E. Ishikawa, R. M. Oliver, and L. J. Reed, Proc. Natl. Acid. Sci. U~S. 56, 534 (1966).
[12] ~ - K e t o g l u t a r a t e D e h y d r o g e n a s e C o m p l e x
from Escherichia coli
By LESTER J. REED and BARID B. MUKHERJEE
a-Ketoglutarate -{- CoA ~- DPN + --~
succinyl-CoA ~- COs -b DPNH -~ H + (1)
Enzyme systems that catalyze reaction (1) have been isolated from
pig heart muscle,1-3 E s c h e r i c h i a coli, 4 and beef kidney mitochondria~ as
multicnzyme complexes with molecular weights of several million. The
I D. R. Sanadi, J. W. Littlefield, and R. M. Bock, J. Biol. Chem. 197, 851 (1952).
3M. Hirashima, T. Hayakawa, and M. Koike, J. Biol. Chem. 242, 902 (1967).
M. Koike, L. J. Reed, and W. R. Carroll, J. Biol. Chem. 235, 1924 (1960).
~E. Ishikawa, R. M. Oliver, and L. J. Reed, Proc. Natl. Acad. Sci. U.S. 56, 534
(1966).
E. coli a-ketoglutarate dehydrogenase complex has been separated into

three enzymes, a-ketoglutarate dehydrogenase (E1),6 dihydrolipoyl trans-
succinylase (Ez), ~ and dihydrolipoyl dehydrogenase (E3), and the com-
plex has been reconstituted from the isolated enzymes.7 Various data
indicate that the oxidative decarboxylation of a-ketoglutarate repre-
sented by Eq. (1) proceeds via the sequence shown in Eqs. (2-6).
HO~C(CH~)~COCO2H ~- TPP-E,--~
[HO2C(CH2)2CH(OH)-TPP]-E, + C02 (2)
[HO2C(CHQ2CH(OH)-TPP]-EI + [LipS~]-~--~
[HO2C(CH2)2CO-SLipSH]-F_a + TPP-E, (3)
[HO~C(CH~)2CO-SLipSH]-F~ ~- HSCoA-~
[Lip(SH)2]-F_a + HO2C(CH2)2CO-SCoA (4)
[Lip(SH)2]-F~ T E r F A D ~ [LipS2]-E2 + reduced E3-FAD (5)
Reduced E r F A D -~ DPN + --* E r F A D + DPNH + H + (6)
In its functional form the lipoyl moiety [LipS2] is bound in amide link-
age to the c-amino group of a lysine residue2 The purification and prop-
erties of the E. coli a-ketoglutarate dehydrogenase complex are described
below.
Assay Methods
There is as yet no sensitive and convenient assay for the a-keto-
glutarate dehydrogenase complex that m a y be used at all levels of
purity of the enzyme complex. The D P N reduction assay described below
is the only reliable assay for the intact complex, but it is not suitable
for crude preparations that contain D P N H oxidase. Assays for the
three component enzymes of the a-ketoglutarate dehydrogenase complex
have been described, but these assays do not distinguish between the
free and combined enzymes. To the extent that the enzymes are associated
in the a-ketoglutarate dehydrogenase complex these assays may be
used to follow the purification of the complex. Thus the ferricyanide
reduction assay described below provides an estimate of the activity of
e It appears that enzyme (ED catalyzes both the decarboxylation of a-ketoglutarate
and the reductive succinylation of the protein-bound lipoyl moiety [Eqs. (2) and
(3)], whereas enzyme (ED catalyzes only the transsuccinylation [Eq. (4)]. There-
fore, it seems appropri~tte to designate (E,) a-ketoglutarate dehydrogenase and
(E~) dihydrolipoyl transsuccinylase (succinyltransferase)rather than a-ketoglutarate
decarboxylase and lipoyl rcductase-transsuccinylase, respectively [footnote (7)].
~B. B. Mukherjee, J. Matthews, D. L. Homey, and L. J. Reed, J. Biol. Chem. 240,
PC2268 (1965).
S H. Nawa, W. T. Brady, M. Koike, and L. J. Reed, J. Am. Chem. Soc. 82, 896
(1960).
ot-KETOGLUTARATE 57
the a-ketoglutarate dehydrogenase component (E~) of the complex. As-

says for dihydrolipoyl transsuccinylase (Ez) ~ and for dihydrolipoyl
dehydrogenase (E3) TM have been described in previous volumes of this
series.
Ferricyanide Reduction
a-Ketoglutarate + 2 Fe(CN)6 -~ + H~O --~
succinate + CO2 + 2 F e ( C N ) c 4 + 2 H + (7)
Principle. The assay is based on colorimetric determination of ferro-
cyanide (as Prussian blue) produced by oxidative decarboxylation of
a-ketoglutarate with ferricyanide as electron acceptor [Eq. (7)]. This
reaction is catalyzed by the a-ketoglutarate dehydrogenase component
of the a-ketoglutarate dehydrogenase complex.
Procedure. The procedure is identical with that described for pyru-
vate dehydrogenase in a previous volume of this series/° with the ex-
ception that potassium a-ketoglutarate is substituted for potassium
pyruvate.
Units. One unit is the amount of enzyme required to produce 2
micromoles of ferrocyanide per hour under the conditions described.
Specific activity is expressed as units per milligram of protein. Protein is
determined by the biuret method, ~1 with crystalline bovine serum albumin
as the standard.
Other Methods o] Assay. The rate of C02 evolution [Eq. (7) ] can be
used to estimate a-ketoglutarate dehydrogenase activity? ~
DPN Reduction
Principle. The assay is based on spectrophotometric determination of
the rate of formation of D P N H [Eq. (1)].
Reagents
Potassium phosphate buffer, 0.5 M, pH 8.0
Magnesium chloride, 10 mM
DPN, 0.1 M
Cysteine hydrochloride, 30 mM neutralized before use
° E. Knight, Jr. and I. C. GunsMus, Vol. V [90]. Dihydrolipoamide is the preferred

substrate in this assay [cf. footnote (10)]. Succinate-grown E. coli cells provide
a rich source of succinate thiokinase (C. D. Upper, Ph.D. Dissertation, University
of Illinois, 1964).
:oL. d. Reed and C. R. Willms, Vol. IX [50].
X~A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949).
~2L. P. Hager, Ph.D. Dissertation, University of Illinois, 1953.
Thiamine pyrophosphate, 20 mM
CoA, 3 mM prepared before use
Potassium a-ketoglutarate, 0.1 M
Procedure. To a cuvette with a 1 em light path add phosphate buffer~

0.1 ml; magnesium chloride, 0.1 ml; DPN, 0.02 ml; cysteine, 0.1 ml;
thiamine pyrophosphate, 0.01 ml; water and enzyme to make a total vol.
ume of 0.97 ml. The reaction is initiated at 25 ° by the simultaneous addi-
tion of 0.02 ml of CoA and 0.01 ml of potassium a-ketoglutarate. The
blank cell contains phosphate buffer, 0.1 ml; magnesium chloride, 0.1 ml;
DPN, 0.02 ml; and water, 0.78 ml. The increase in absorbance at 340 m#
is followed with a recording spectrophotometer. An increase of 0.03-0.15
during the initial phase of the reaction (about 1 minute) is a linear
function of protein concentration.
One unit is defined as the amount of enzyme that catalyzes an initial
rate of formation of 1 micromole of D P N H per minute. Specific activity
is expressed as units per milligram of protein. 13
Other Methods o] Assay. 3-Acetyl diphosphopyridine nuc]eotide
(AcPyDPN) has been substituted for D P N in an attempt to circumvent
destruction or reoxidation of D P N H in crude E. coli extracts) 4
In the procedure described in a previous volume of this series TM both
the a-ketoglutarate and pyruvate dehydrogenase complexes are purified
together, and are separated in the final step by isoelectric precipitation.
Several modifications have been incorporated into the procedure, which
is described in detail.
Reagents
Protamine solution, 2%, pH 6.2, prepared before use and kept at
room temperature. Protamine sulfate (Nutritional Biochemicals
Corporation) is suspended in water, the pH is adjusted to 6.2
with 10% KOH, and the mixture is centrifuged to remove in-
soluble material.
Potassium phosphate buffer, 20 mM, pH 7.0
Potassium phosphate buffer, 50 raM, pH 7.0
Acetic acid, 1% (v/v)
Sodium acetate, 10 mM
,3 The specific activity of highly purified preparations of the a-ketoglutarate dehydro-

genase complex in this assay is approximately 15 units/mg protein.
~ C. R. Amarasingham and B. D. Davis, J. Biol. Chem. 240, 3664 (1965).
o~-KETOGLUTARATE 59
Yeast ribonucleic acid solution, 1%, pH 6.2. Yeast ribonucleic acid

(Nutritional Biochemicals Corporation) is suspended in water,
the pH is adjusted to 6.2 with 10% KOH, and the mixture is
centrifuged to remove insoluble material.
Growth o] Cells. E. coli, Crookes strain, is grown in the medium

described by Hager (see also Korkesl~), with the exception that sodium
pyruvate TM is substituted for glucose. ~7 The cells can be grown conve-
niently in 45-liter batches of medium in a Biogen continuous culture
apparatus. The conditions are 5 hours of growth at 35 °, a chamber speed
of 200 rpm, and an air pressure of 10 psi. The cells are harvested with
a refrigerated Sharples centrifuge and are stored at --20 ° until needed.
The yield of cell paste is 6-7 g per liter of medium.
Preparation of Cell-Free Extract. All subsequent operations are
carried out at 0--5°. The thawed cells are suspended in 20 mM phosphate
buffer, pH 7.0, by means of a Waring blendor, at a concentration of
20 g of cell paste per 50 ml of buffer; 70-ml portions of the suspension
are treated for 7 minutes in a 10-kc sonic oscillator (Raytheon). Cell
debris is removed by centrifugation for 30 minutes at 53,700 g (20,000
rpm) in the No. 21 rotor of a Spinco Model L ultracentrifuge.
Fractionation with Protamine. The cell-free extract is diluted with
20 mM phosphate buffer, pH 7.0, to a protein concentration of ap-
proximately 20 mg/ml. Then 900 ml of diluted extract is adjusted to pH
6.15 with 1% acetic acid, and 135 ml (0.15 volume) of 2% protamine
solution, pH 6.2, is added dropwise with stirring. The mixture is stirred for
an additional 15 minutes and then centrifuged for 30 minutes at maxi-
mum speed in the No. 845 rotor of an International Model PR-2 centri-
fuge. The gelatinous, white precipitate is discarded, and 27 ml (0.03
volume) of 2% protamine solution is stirred into the supernatant fluid.
The mixture is stirred for 15 minutes and the precipitate is collected
by centrifugation. Two 27-ml portions (0.03 volume) of 2% protamine
,5 S. Korkes, Vol. I [77].
~'~Crystalline sodium pyruvate, type II, dimer-free, was obtained from Sigma
Chemical Company. The sodium pyruvate solution is filter-sterilized and is added
to the medium to give a final concentration of 60 g per liter.
" U. Henning, G. Dennert, R. Hertel, and W. S. Shipp reported [Cold Spring Harbor
Syrup. Quant. Biol. 31, 227 (1966)] t h a t pyruvate is a nutritional inducer for the pyr-
uvate dehydrogenase complex. We have confirmed this finding and have observed
that extracts of aerobic pyruvate-grown cells contain three to five times as much
pyruvate dehydrogenase complex as extracts of aerobic glucose-grown cells. Both
extracts contain essentially the same amount of a-ketoglutarate dehydrogenase
complex. The results of Amarasingham and Davis (footnote 14) indicate that
acetic acid is a nutritional inducer for the a-ketoglutarate dehydrogenase complex
and t h a t a-ketoglutarate may be the direct physiological inducer.
6(3 REACTIONS ON THE CYCLE [12]
solution are added successively to the supernatant fluid as described

above, and the precipitates are collected by centrifugation. Aliquots of
the supernatant fluids from the four successive additions of 2 ~ prot-
amine solution are assayed for a-ketoglutarate dehydrogenase activity
(ferricyanide reduction assay) and for pyruvate dismutation activity. 1°
Usually, the third or fourth addition, or both, of 2% protamine solution
results in precipitation of over 9 0 ~ of the a-ketoglutarate and pyruvate
dehydrogenase complexes.
The grayish yellow precipitate is suspended in 125 ml of 0.1 M
phosphate buffer, pH 7.0, by means of a large glass homogenizer
equipped with a motor-driven Teflon pestle. The suspension is stirred for
45 minutes and then centrifuged for 20 minutes at 47,000 g (20,000 rpm)
in the No. 30 rotor of a Spinco Model L ultracentrifuge. The precipitate
is discarded, and to the yellow solution is added 0.05 ml of 1 ~ yeast
ribonucleic acid solution, pH 6.2, for every 10 mg of protein. TM The solu-
tion is diluted with an equal volume of distilled water and allowed to
stand overnight in an ice bath. The solution is centrifuged for 20 min-
utes at 47,000 g, and the precipitate is discarded.
Ultracentri]ugation. The clear, yellow solution from the preceding
step, designated protamine eluate, is diluted with 50 mM phosphate
buffer, pH 7.0, to a protein concentration of 5 mg/ml. The solution is
centrifuged for 2½ hours at 144,000 g (40,000 rpm) in the No. 40 rotor
of a Spinco Model L ultracentrifuge. The supernatant fluid is separated
from the gelatinous yellow pellet and is discarded. To each centrifuge
tube is added sufficient 0.05 M phosphate buffer, pH 7.0, to cover the
yellow pellet (about 1 ml), and the tubes are shaken occasionally during
a period of several hours to facilitate solution of the pellets. The solu-
tions are combined and are clarified, if necessary, by ccntrifugation for
20 minutes at 36,000 g (20,000 rpm). The solution contains both the
a-ketoglutarate and pyruvate dehydrogenase complexes and variable,
but small, amounts of lower molecular weight contaminant(s).
Isoelectric Precipitation. To the clear, yellow solution from the
preceding step is added 0.01 ml of 1% yeast ribonucleic acid solution, pH
6.2, for every 10 mg of protein. TM The solution is diluted with 10 mM
sodium acetate to a protein concentration of 5 mg/ml. If the solution is
cloudy it is centrifuged for 5 minutes at 14,000 g (13,000 rpm) in an
International Model HR-1 centrifuge, and the gray precipitate is dis-
carded. The pH of the solution is carefully adjusted to 5.7 by dropwise
~Ribonucleie acid is added to remove protamine. Otherwise, variable amounts of

the a-ketoglutarate and pyruvate dehydrogenase complexes may precipitate when
the solution is diluted prior to ultracentrifugation. Also, a sharp separation of the
two complexes may not be achieved in the isoelectric precipitation stop if protaminc
is present.
[12] a-KETOGLUTARATE DEHYDROGENASE FROM E. coli 61
addition, with stirring, of 1% acetic acid. The mixture is stirred for an

additional 5 minutes, and the yellow precipitate is collected by centrif-
ugation. This precipitate contains the a-ketoglutarate dehydrogenase
complex. The pH of the supernatant fluid is lowered to 5.0, and the
yellow precipitate is collected by centrifugation. This precipitate con-
tains the pyruvate dehydrogenase complex (approximately 280 mg of
protein). The two precipitates are dissolved separately in 20 mM phos-
phate buffer, pH 7.0. The solutions are clarified, if necessary, by centrif-
ugation for 20 minutes at 36,000 g (20,000 rpm). The preparations are
stored in the frozen state, preferably at --90 ° , and retain full activity for
several months. A summary of the purification is given in the table.
PURIFICATION PROCEDURE a
Volume Protein Specific Recovery

Fraction (ml) (mg) activity ~ (%)
Diluted extract 900 19,200 3.7 (100)

Protamine eluate 125 1,230 37 64
Ultracentrifuge pellet 30 579 65 53
Precipitate, pH 6.0-5.7 5 161 180 41
Three hundred grams of cell paste.
b Micromoles/2 of ferrocyanide produced per hour per milligram of protein.
C o m m e n t s . Usually, the preparations of the a-ketoglutarate and
pyruvate dehydrogenase complexes contain less than 5% of impurity, as
revealed by ultracentrifugal analysis. If larger amounts of impurity
are present the ultracentrifugation or isoelectric precipitation steps, or
both, are repeated. Alternatively, the preparations are fractionated with
solid ammonium sulfate. The a-ketoglutarate dehydrogenase complex
precipitates between 0.25 and 0.32 saturation, and the pyruvate dehydro-
genase complex precipitates between 0.40 and 0.48 saturation.
Properties
Specificity.The enzyme complex is specific for D P N . It exhibits little,
if any, activity toward pyruvate.
Physical Constants2 The electrophoretic mobility of different prepa-
rations of the a-ketoglutarate dehydrogenase complex varied from --5.1
to --4.9 X 10 -0 cm ~ volt-I sec-I in 50 m M potassium phosphate buffer, p H
6.9. The enzyme complex exhibits a sedimentation coefficient (S°o.w) of 40
S, a diffusion coefficient (D~o,w) of 1.51 X 10 -7 cm 2 sec-I (protein concen-
tration, 3.2 mg/ml), and a partial specific volume (~) of 0.731 ml/g. TM
Based on these data, the calculated molecular weight is 2.4 X 10 ". The
frictional ratio (]/]o) is 1.6.
~'D. W. Kupke, Federation Ib'oc. 25, 990 (1966).
[13] Succinate Thiokinase from Pig Heart

[EC 6.2.1.4 Suecinate: CoA ligase (GDP)]
By SUNGMAN CHA
Succinyl-CoA + GDP + Pi ~ succinate + CoA + GTP
Assay Methods 1
Method I
Principle. The reaction in the reverse direction is assayed by measure-
ment of the increase in absorbance at 235 m~ which is due to the forma-
tion of the thioester bond of succinyl-CoA.
Reagents
Tris-succinate, 0.1 M, pH 7.4 [0.1 M in terms of succinate, approxi-
mately 0.22M in terms of Tris(hydroxymethyl)aminomethane]
MgCl~, 0.1 M
GTP, 1 mM
CoA, 1 mM
Procedure. To a 1-ml cuvette with a 1 cm light path, add 0.5 ml of
Tris-succinate, 0.1 ml each of MgCI2, GTP, and CoA, and enough water
to make the final volume 1.0 ml (0.2 ml if the volume of the enzyme solu-
tion to be used is less than 50 ~1). Alternatively, large amounts of all
reagents and water, except CoA, may be mixed, divided into small
aliquots, and kept frozen almost indefinitely. After having been warmed
to 30 °, 0.9 ml of this mixture, is used. The reaction is initiated by the
addition of enzyme solution. The change in absorbance at 235 m~ is
measured by a spectrophotometer.
Method II
Principle. The reaction may also be assayed in the reverse direction
by coupling to the pyruvate kinase and lactate dehydrogenase system
according to the following equations.
GTP + CoA -J- succinate ~ GDP + succinyl-CoA + P~ (1)
GDP -~- phosphoenolpyruvate ~ pyruvate -t- GTP (2)
Pyruvate -~- NADH + H + ~ lactate + NAD + (3)
IS. Cha and R. E. Parks, Jr., J. Biol. Chem. 239, 1961 (1964).
[13] SUCCINATE THIOKINASE FROM PIG HEART 63
The rate of formation of GDP by reaction (1) is determined by the

absorbance decrease at 340 m~ due to the disappearance of NADH.
Reagents
MgCl~, 0.2 M in 2.0 111 KC1
Phosphoenolpyruvate, 31 mM (10 mg/ml, trisodium salt, Calbio-
chem)
NADH, 4 mM
Tris-succinate, 0.1 M, pH 7.4
GTP, 1 mM
CoA, 1 mM
A mixture of pyruvate kinase (Sigma, type II, rabbit muscle, 100
~ f units/mg), and lactate dehydrogenase (Sigma, type III, beef
heart muscle, 300-600 ~M units/rag), 2 mg/ml each in Tris-
acetate buffer, 0.1 M, pH 7.4
Procedure. To a 1 ml euvette with a 1 cm light path, add 0.5 ml of

Tris-succinate, an appropriate amount of water to make the final
volume 1 ml, 0.1 ml each of GTP and CoA, 0.05 ml each of MgCI~-KC1,
phosphoenolpyruvate, and NADH, and 0.05 ml of the pyruvate kinase-
lactate dehydrogenase mixture, in that order. The reaction is initiated by
the addition of suceinic thiokinase solution, and the absorbance decrease
at 340 mu at 30 ° is recorded.
Methods I a*~d I I U~its. One unit of enzyme is the amount of
enzyme catalyzing the conversion of 1 micromole of substrate per min-
ute. The conversion factor of absorbance-change to concentration is
4.0 absorbance units per micromole in Method I, and 6.22 absorbance
units per micromole in Method II. Specific activity is expressed as units
of enzyme per milligram of protein. Protein in concentrated solutions is
determined by the biuret procedure. 2 When interfering materials, such
as ammonium sulfate and Tris buffer, are present, the biuret procedure is
performed after the protein precipitated with 10% trichloroacetic acid.
Protein in dilute solutions is estimated by the absorbance at 280 m~,
employing the factor 0.9 absorbance unit per milligram per milliliter.
Comments on Assay Methods. Method I is the simplest and most
convenient. However, the linearity between observed rates and amount
of enzyme does not hold when the rate is greater than approximately
0.04 absorbance unit per minute, probably due to the instability of
succinyl-CoA, the product being measured. Furthermore, the absorbance
2A. G. Gornall, C. J. Bardawill, and M. M. David. J. Biol. Chem. 177, 751 (1949) ;
see also Vol. I I I [73].
64 REACTIONS ON TIIE CYCLE [13]
¢.
~J
~9
¢,i ¢5 ,~ ,d
of the reaction mixture at 235 m~ is quite high (approximately 1.3).

Therefore, without a spectrophotometer designed to work at high
absorbances {e.g., Gilford), it is difficult to assay an enzyme preparation
with low activity. Most of these difficulties are overcome by the use
of Method II, which may be adapted to situations where measurement
of succinyl-CoA is not feasible, as in the presence of arsenate or
hydroxylamine. The reaction in the forward direction may be assayed
either by the direct spectrophotometric measurement of the disappear-
ance of succinyl-CoA in the presence of GDP, Pi, and Mg+÷,~ or by
coupling to the a-ketoglutarate dehydrogenase reaction2 ,~ The direct
measurement of succinyl-CoA disappearance suffers from the high initial
absorbance, the necessity of synthesizing the substrate, and the insta-
bility of the compound. The hydroxamate method of Kaufman 5 may be
adapted to mammalian enzyme by replacing ATP with either GTP or
ITP, 4 but hydroxylamine in the reaction mixture is a strong inhibitor of
the enzyme,1 and there are few advantages of this method over the
spectrophotometric assays. One unit of enzyme as defined above cor-
responds to approximately four Kaufman units (micromoles of succino-
hydroxamate formed in 30 minutes at 37 ° in the presence of GTP).
Purification Procedure 6
The enzyme from pig heart may be purified about 800-fold over the
crude extract in a two-stage procedure. The following procedure, using
fresh, nonfrozen pig hearts is carried out at 0-5 °. If the purification
started with two batches, the products may be pooled at the end of step
2, and treated as a double-size preparation following the further steps
described below without modifications. Results of a typical first-stage
preparation are presented in the table.
First-Stage Purification
Step 1. Preparation o] Crude Extract. Four or five fresh pig hearts
are freed of fat and connective tissue, ground with a meat grinder, and
washed with cold distilled water in a big container (4-10 liters) until the
washings are almost colorless. The washed mince is transferred to a
cheesecloth bag, and as much water as possible is pressed out by hand.
Six hundred grams of the washed tissue are homogenized with three vol-
H. Hift, L. Ouellot, J, W. Littlefield, and D, R. Sanadi, J. Biol. Clwm. 204, 565
(1953).
*R. Mazumder, D. R. Sanadi, and V. W. Rodwell, J. Biol. Chem. 235, 2546 (1960).
sS. Kaufman, C. Gilvarg, C. Cori, and S. Ochoa, J. Biol. Chem. 203, 869 (1953) :
see also Vol. I [120].
*S. Cha, C.-J. M. Cha, and R. E. Parks, Jr., J. Biol. Chem. 242, 2577 (1967).
umes of 10 m M K2HP04 in a high-speed Waring blendor for 3 minutes.

The homogenate is centrifuged for 30 minutes at 10,000 g, and the resi-
due is discarded. Four to five hours are required for this step, during
which one should start packing the two columns for the next step. How-
ever, if necessary, the crude extract m a y be kept overnight without a
significant loss of activity.
Step 2. Calcium Phosphate Gel-Cellulose Column Chromatography. 7,s
The crude extract is divided into two equal volumes, each of which is
applied to the prepared gel-cellulose columns. After the extract is com-
pletely loaded, each column is washed with 100-200 ml of l0 m M potas-
sium phosphate buffer, p H 7.4. The eluate up to this point m a y be
discarded. The enzyme is eluted with a linear gradient of potassium
phosphate buffer, pH 7.4, from 10 m M to 0 . 3 M in a total volume of 2
liters. Fractions of 100 ml are collected. The bulk of the succinic thio-
kinase emerges in three to four fractions, almost free of heme proteins,
and the highest activity is usually found in fraction 11 or 12. The time
required for this step is 5-10 hours, depending on the flow rates of the
columns. Quite often, a small amount of the enzyme (about 10% of the
total and usually discarded) emerges in another peak at around fraction
5 with heme proteins, perhaps because of the presence of channels in the
column. I f the amount in this peak is too large to be discarded, as m a y
occasionally be the case, this portion m a y be pooled separately, and
The calcium phosphate gel is prepared by the method of K. K. Tsuboi and P. B.

Hudson [J. Biol. Chem. 224, 879 (1957)]. To 1 liter of 0.5 M Na2HPO,, add 30 ml
of concentrated ammonium hydroxide, followed by 7.5 liters of 0.1 M CaCh, while
stirring vigorously. Allow the gel to settle overnight, siphon off the supernatant,
and add distilled water to the original volume. Repeat this washing procedure
until the supernatant fluid is free of chloride ions. After as much supernatant fluid
as possible is siphoned off, the dry weight per unit volume is determined before use.
The gel may be stored at room temperature or in a refrigerator for a long time.
Commercially available hydroxylapatite gel may also be used.
Two identical columns are employed. For each column, 130 g of cellulose powder
(Whatman, Column Chromedia, coarse fibers, obtainable from Reeve Angel & Co.)
are suspended in 1 liter of 30 mM Tris-acetate buffer, pit 8.0; then an appropriate
volume of calcium phosphate gel containing 5 g by dry weight is added to the
suspension, mixed well, and kept at 2-5 ° overnight or longer. The whole suspen-
sion is packed into a large glass column (5 × 50 cm) with glass wool at the
bottom. The packing is facilitated by means of gentle suction produced by
attaching a 4- to 5-foot-long tubing filled with water to the bottom of the column.
The unsettled portion of the suspension is stirred frequently to prevent channeling
and a slow flow rate due to a gross separation of the gel and the cellulose. When
the gel and the cellulose have settled completely, to minimize channeling 10 g of
cellulose preswollen by an overnight soak in about 200 ml of 30 mM Tris-acetate
buffer, is overlaid on top of the column. The column is washed with 100-200 ml
of the same buffer before use.
similar material from several preparations may be accumulated as am-

monium sulfate suspension (as in step 3), then step 2 may be repeated
after dialysis.
Step 3. Ammonium Sulfate Fractionation. To each liter of the pooled
eluate from the previous step, 445 g of solid ammonium sulfate is added
(70~c saturation at 0 °) and dissolved.9 After the sample has stood 1 hour
or longer (usually overnight), the precipitate is collected by centrifuga-
tion at 10,000 g for 30 minutes. It is dissolved in the smallest practical
volume of 50 mM Tris-aeetate buffer, pH 8.0, and dialyzed thoroughly
against the same buffer with three to four changes of 4 liters each,
usually overnight. Immediately before the next step the enzyme solution
is dialyzed against 4 liters of 0.03 M Tris-aeetate buffer, pH 8.0, for 2
hours.
Step 4. DEAE-Cellulose Column Chromatography. The enzyme solu-
tion from the previous step is loaded on to a DEAE-eellulose column
(acetate form, 2.5 X 20 cm) which has been equilibrated with 0.03M
Tris-acetate buffer, pH 8.0. The column is washed with 50 ml of the same
buffer. A linear gradient of Tris-acetate buffer, pH 8.0, 30 mM to 0.3 M
in a total volume of 500 ml is then applied, and 10-ml fractions are
collected. Here again, the enzyme emerges in two peaks, one at about
tube No. 9, and the other somewhat between tube Nos. 15 and 20, pos-
sibly representing different forms of the enzyme. The first peak usually
contains a small amount of enzyme with a low specific activity. It
takes about 4-6 hours for this step, depending on the flow rate of the
column which can be improved greatly by the exhaustive removal of fines
from the DEAE-cellulose. The enzyme is rather unstable at the end
of this step. Therefore, the next step should be carried out as soon as
possible. If necessary, the procedure may be interrupted overnight after
the enzyme is loaded on the next column (step 5).
Step 5. Second Calcium Phosphate Gel-Cellulose Column Chroma-
tography. Fifteen grams of cellulose powder and 400 mg of calcium
phosphate gel (dry weight), suspended in 200 ml of 30 mM Tris-acetatc
buffer, pH 8.0, are packed into a glass column of 2.5 cm diameter, with
a fritted glass disk. The column is washed with 50-200 ml of the same
buffer. After the enzyme solution from the previous step is loaded, the
enzyme is eluted by a linear gradient of potassium phosphate buffer,
while the concentration of Tris-acetate buffer and the pH are held
constant. This is achieved by starting the gradient with 250 ml of 50 mM
Tris-acetate buffer, pH 7.4, in the mixing chamber, and 250 ml of a
A m m o n i u m sulfate fractionation may be carried out in two steps, i.e., by dis-
carding 45% precipitate. However, this additional procedure has little effect on
the overall result.
pH 7.4 buffer solution, 0.25M potassium phosphate in 50 mM Tris-

acetate in the reservoir. The fractions containing the bulk of the enzyme
are pooled, and the enzyme is stored in a refrigerator as precipitate in
80% saturated ammonium sulfate.
Second-Stage Purification
Step 6. Sephadex G-IO0 Gel Filtration. Several (up to 10) batches of
the first-stage purification product in 80% saturated ammonium sulfate
are pooled and centrifuged. The precipitate is dissolved in a very small
volume (less than 2 ml) of 50 mM Tris-acetate buffer, pH 8.0. De-
natured proteins, if any, are removed by centrifugation. The resulting
clear solution of enzyme is loaded on a Sephadex G-100 column,
1.9 X 60 cm, equilibrated in the same buffer. The enzyme is eluted with
the same buffer, and 2-ml fractions are collected. The fractions con-
taining the bulk (70-80%) of the enzyme are combined and subjected
to the next step. Proteins emerge in two overlapping peaks. The second
protein peak, usually smaller than the first, coincides with the enzymatic
peak (around tube No. 35). The eluates corresponding to the front end
of the enzymatic peak are pooled separately, and similar materials
from several preparations may be accumulated as an ammonium sulfate
suspension; then the second stage may be repeated.
Step 7. Second DEAE-Cellulose Column Chromatography. The en-
zyme solution from the previous step is loaded on the column without
further treatment, and step 4 is repeated. As in the first stage, the
next step should follow immediately after this.
Step 8. Third Calcium Phosphate-Gel Cellulose Column Chroma-
tography. The procedure is the same as in step 5.
Step 9. Second Sephadex G-IO0 Gel Filtration. The procedure is the
same as in step 6. The elution pattern of this step shows relatively
constant specific activity of about 110 ~ units per milligram of
protein, at the center of the peak, and about 10% recovery of the enzyme
from the crude extract may be achieved.
Properties
Stability and pH Optimum. e The optimal pH for activity is reported
to be 7.4 for the enzyme from pig kidney cortex. 4 The enzyme is quite
stable at a pH above 5.5 up to 9.2 (the highest tested), and is inactivated
rapidly at a pH below 5. The enzyme loses a considerable portion of its
activity (as much as 50% at a time) through freezing and thawing,
but not through lyophilization. It is also unstable m a dilute buffer,
e.g., Tris-acetate buffer at concentrations less than 0.05 M. A solution of
the enzyme in 0.25 M Tris buffer, pH 7.4 to 8.0, may be kept for weeks
without an appreciable loss of activity. A suspension in 80% saturated

ammonium sulfate at 0 °, or lyophilized powder at --20 °, may be kept
for years.
Substrate Specificity and Kinetic Parameters. The enzyme from pig
heart, as well as that from other animals belonging to different phyla,
namely Chordata (Mammalia, Amphibia, Pisces), Arthropoda (Crusta-
cea, Insecta), Annelida, and Brachiopoda, is specific for guanine nucleo-
tides, and ATP does not serve as a substrate. 1°, 1~ Some analogs of GDP
and GTP such as ITP, 8-azaGDP, 8-azaGTP, and 6-thioGTP have been
shown to replace the guanine nucleotides. Itaconic acid 12 and dephospho-
CoA 6 are the only known substitutes for succinic acid and CoA, respec-
tively. Apparent Michaelis constants for various substrates 6,~3 are:
succinyl-CoA, 20--60 #M; GDP, 2-8 p2~/; 8-azaGDP, 30-110 p21//; phos-
phate, 150-480 p_M; CoA, 20 tLM; GTP, 5-10 #M; azaGTP, 70-210
~M; 6-thioGTP, 50 vM; succinate, 400-700 vii/.
Molecular Properties2 The highest specific activity ever observed
with the enzyme from pig heart is 120 tLM units/mg. The enzyme ap-
pears to have a molecular weight of about 70,000, and at least two,
probably four, active centers per molecule.
Catalytic Activities and Reaction Mechanism2 ,14 The enzyme also
catalyzes the following reactions in the presence of Mg++: the arsenolysis
of succinyl-CoA, the arsenolysis of GTP in the presence of CoA, the
formation of GDP from GTP in the presence of suceinate, GDP-GTP
exchange, P~-GTP exchange in the presence of CoA, succinate-succinyl-
is not completely understood; however, the following seems to be the
major pathway:
E + succinyl-CoA ~ E ~ CoA + succinate

E ~ CoA -b P~ ,~- E ~ P -t- CoA
E~-~P+GDP~E+GTP
~°D. R. Sanadi, D. M. Gibson, P. Ayengar, and M. Jacob, J. Biol. Chem. 218, 505
(1955).
11It. A. Leonard, J. P. Green, and S. Cha, unpublished works (1966).
12S.-F. Wang, J. Adler, and It. A. Lardy, J. Biol. Chem. 236, 26 (1961).
13S. Cha and R. E. Parks, Jr., J. Biol. Chem. 239, 1968 (1964).
,4 S. Cha, C.-J. M. Cha, and R. E. Parks, Jr., J. Biol. Chem. 242, 2582 (1967).
[14] Succinyl Coenzyme A Synthetase f r o m E s c h e r i c h i a coli

[EC 6.2.1.5 Succinate:CoA ligase (ADP)]
By W. A. BRIDGER,R. F. RAMALEY,and P. D. BOYER
Mg++
Succinyl-CoA q- P~ ~ ADP ~ " succinate + CoA + ATP ~ (1)
Assay Method
Principle. The rate of synthesis of succinyl-CoA is determined by
measuring the increase in absorbance at 230 m~ accompanying thioester
bond formation.
Reagents
Assay mixture:
Tris-HC1, 50 mM, pH 7.2
KC1, 0.1 M
MgC12, 10 m M
Sodium succinate, 10 m M
ATP, 0.4 m M
CoA, 10 m M
Enzyme, 0.00][-0.03 unit (0.03 to 1/~g of pure enzyme)
Unit. A unit is defined as that amount of enzyme catalyzing the
formation of 1 micromole of succinyl-CoA per minute at 25 ° under assay
conditions as given.
Procedure. The procedure given is based on a modification s of that
used in Gunsalus' laboratory. 3 One ml of assay mixture and 10 ~1 of 10
m M CoA are mixed in a silica cuvette (1 cm light path) in the
thermostatted cell compartment of a recording spectrophotometer. The
same mixture is placed in the blank cuvette. After temperature equili-
bration, 5-50 ~1 of solution containing the amount of enzyme indicated
is added to the sample cuvette; the solutions are mixed quickly, and the
rate of increase of absorbance at 230 m# is recorded. Calculation of the
rate of formation of succinyl-CoA is based on the value of 4.5 X 103 for
the ZXcM4 at 230 m~ accompanying the reaction.
1The abbreviations used are: P~, inorganic orthophosphate; CoA, coenzyme A;
ADP and ATP, adenosine-5'-di- and triphosphate.
2R. F. Ramaley, W. A. Bridger, R. W. Moyer, and P. D. Boyer, J. Biol. Chem.,
242, 4287 (1967).
3j. Gibson, C. D. Upper, and I. C. Gunsalus, J. Biol. Chem., 242, 2474 (1967).
' E. R. Stadtman, Vol. III, p. 931.
[14] SUCCINYL-COA
SYNTHETASE FROM E. coli 71
The enzyme also may be assayed by measuring succinyl hydroxamate

formation after carrying out the reaction in the presence of hydroxyl-
amine2 This assay is unsatisfactory for most purposes, however, since it
is less sensitive, requires much higher substrate concentrations, and the
progress of the reaction cannot be recorded.
Growth of Cells. E. coli (Crookes' strain) are grown from nutrient
agar cultures by inoculation of two culture tubes containing 7 ml of 1%
tryptone, 1% yeast extract, 0.5% K2HP04, and 0.3% glucose. These
cultures, after 6-8 hours' growth at 37 °, are used to inoculate 13 liters
of medium (0.0005% yeast extract, 0.001% F e S Q . 7 H20, 0.04%
MgS04" 7 H,.,O, 0.3% NH,C1, 2.2% sodium succinate hexahydrate, 20 mM
KH~PO~, 20 mM K~HPO~) at 37 ° contained in a New Brunswick Micro-
ferm Fermentor. The culture is grown overnight at medium aeration (4
liters of air per minute) and low agitation (50 rpm), and the following
morning the aeration is increased to over 16 liters per minute and the
agitation is made maximal (170 rpm). An antifoaming agent (Dow
Corning Antifoam A) is used as necessary, and 85% phosphoric acid is
added to keep the pH between 7 and 8 during growth. The cells are
grown until they reach an optical density (measured on a 1:10 dilution
at 660 mt~) of 13 to 15, after which they are maintained in continuous
culture by pumping in fresh medium and pumping out the cell suspen-
sion at a rate (approximately 5 liters per hour) such that the optical
density deos not fall below 10.
The cell suspension from the fermentor is chilled quickly by pump-
ing it through a coil of tubing immersed in ice water and is kept at 2 °
in large refrigerated stainless steel tanks. The cells are harvested by
periodic centrifugation with a Centrico Westphalia or Sharples con-
tinuous-flow centrifuge. Approximately l0 g of wet packed cells is
obtained per liter of medium. The packed cells are resuspended in 20 mM
MgC12-20 mM potassium phosphate, pH 7.2 (1 liter of buffer per kilo-
gram of packed cells) by means of a blender controlled by a variable
transformer. The suspension may be frozen in 800 ml portions until used.
Sonic Extraction. An 800 ml portion of the cell suspension is sonicated
for three 10-minute intervals with a Bronson sonicator using tap 7. The
suspension is stirred and kept immersed in an ethanol-ice bath during
this treatment. Then 400 ml of 20 mM magnesium chloride-20 mM
potassium phosphate, pH 7.2, is stirred in and the cellular debris and un-
broken cells are removed by centrifugation for 45 minutes at 12,000 q.
~F. Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945).

The supernatant is decanted and the residue is resuspended in 400 ml of

the same buffer, sonicated for an additional 30 minute total, centrifuged
as before, and the supernatant is combined with that of the first sonica-
tion. The pH of the extract is adjusted to 7.2 with concentrated am-
monium hydroxide.
Acetone Fractionation. Five grams of ammonium sulfate are added
per 100 ml of extract and the pH is readjusted to 7.2 with NH4OH. The
mixture is stirred in a --20 ° cold room and 70 ml of acetone, chilled b y
passage through a coil of tubing immersed in a dry ice-acetone slurry, is
slowly added per 100 ml of original extract. The mixture is stirred for an
additional 30 minutes after all the acetone is added, and then centrifuged
for 30 minutes at 12,000 g in a rotor and centrifuge previously chilled
to --20 °. The residue is discarded and to the supernatant fluid is added
30 ml of cold acetone per 100 ml of original extract. The mixtureis
stirred and centrifuged as before, the supernatant is discarded, and the
precipitate is allowed to drain overnight at --20 ° .
Ammonium Sulfate Fractionation. The precipitate is extracted with
1100 ml of ammonium sulfate solution (300 g ammonium sulfate added
to 1 liter of water and adjusted to pH 7.2 with ammonium hydroxide)
by the use of a blendor controlled with a variable transformer. The
suspension is stirred for 15-30 minutes, then centrifuged for 30 minutes
at 12,000 g. The residue is discarded and to the extract is added 10 g of
ammonium sulfate per 100 ml of solution. The solution is maintained at
pH 7.2. The precipitate is collected by centrifugation for 30 minutes at
12,000 g and dissolved in a minimal volume (25-50 ml) of 50 mM
potassium phosphate-50 mM potassium chloride, pH 7.2.
Ammonium Sulfate Removal and Heat Treatment. The enzyme solu-
tion is passed through a 3.8 X 40 cm column of Sephadex G-50 (medium)
which had been equilibrated with 50 mM potassium phosphate 50 mM
potassium chloride, pH 7.2. The protein-containing fractions are pooled,
heated to 52 ° in a 62 ° water bath, and held at 52 ° for 5 minutes. The
solution is then chilled rapidly and centrifuged for 30 minutes at 27,000 g.
Chromatography on Diethylaminoethyl Sephadex (DEAE-S). The
enzyme solution is applied to a 5 X 50 em column of DEAE-S which has
been equilibrated with 50 mM potassium phosphate-50 mM potassium
chloride, pH 7.2, and the column is then washed with 1 liter of this buffer.
The enzyme is eluted by a linear gradient of 2 liters of the starting buffer
to 2 liters of 50 mM potassium phosphate-0.18 M potassium chloride, pH
7.2. The enzyme appears at the end of the gradient as a broad peak. The
fractions containing the major portion of the enzyme are pooled and re-
applied to a 4 X 170 column of DEAE-S which is eluted as above with
the addition of 2 liters of 50 mM potassium phosphate-0.18 M potassium
[14] SUCCINYL-COA SYNTHETASE FROM E. coli 73
chloride after the gradient elution has been completed. Ammonium sulfate
(50 g per ml pooled eluatc) is added, and the p H is adjusted to 7.2 with
N H 4 O H ; the enzyme m a y be stored at - - 2 0 ° until used.
C h r o m a t o g r a p h y on C a l c i u m P h o s p h a t e Gel. The preparation after
D E A E - S c h r o m a t o g r a p h y still contains traces of impurities, most of
which can be removed by chromatography on calcium phosphate gel.
The enzyme is collected by centrifugation for 20 minutes at 27,000 g,
resuspended in a minimal volume of 50 m M Tris-HCl-0.10 M KCI, p H 7.2,
and passed through a 1 X 50 cm Scphadex G-50 column previously
equilibrated with this buffer. The enzyme is then placed on a 2 X 30 cm
column of calcium phosphate geV and elutcd with a 4 liter linear gradient
PURIFICATION OF SUCCINYL-CoA SYNTHETASE ~
Total
Volume activity Protein Specific
Fraction (ml) (units) b (rag) activity
l. Sonic extract 800 10,500 11,000 09

2. 35-50% acetone precipitate -- 7,000 3,900 18
3. Ammonium sulfate precipitate 1100 7,300 3,200 2.3
4. After first DEAE-Sephadex 300 7,600 240 31 1
chromatography
5. After second DEAE-Sephadex 350 7,000 220 31 6
chromatography
6. After calcium phosphate gel 250 6,500 200 32.1
chromatography
° The enzyme was purified from 400 g of E. coli (Crookes strain) cell paste. Protein
was determined by the method of Lowry et al. [O. H. Lowry, N. J. Rosebrough,
A. L. Farr, and R. J. Randall, J. Biol. Chem. 19S, 265 (1951)] for steps 1-3 and
from A~0 as described herein for steps 4-6.
b A unit is the amount of enzyme required to catalyze the formation of 1 micromole of
succinyl-CoA per minute at 25° under the assay conditions.
of potassium phosphate from 0.0 to 0.10 M, p H 7.2, containing 50 m M
T r i s - H C l and 0.1 M KC1 throughout the gradient. The enzyme is precipi-
tated as before with 5 g of a m m o n i u m sulfate per 100 ml of pooled eluant,
adjusted to pH. 7.2, and stored at 2-4 °.
The purified enzyme m a y be stored in this way for m a n y months with
no change in activity, or m a y be stored frozen in small batches dissolved
in 0.1 M Tris-HC1-0.1 M KC1, p H 7.2. No loss of activity accompanies
repeated freezing and thawing.
The results of a purification of enzyme from 400 g of E . coli are
shown in the table. The enzyme purified as described shows high ap-
Prepared by the method of H. W. Siegelman, G. A. Wicczorek, and B. C. Turner,
Anal. Biochem. 13, 402 (1965).
parent homogeneity in the analytical ultracentrifuge or by disc electro-

phoresis. Remaining, however, is a trace ATPase (turnover number about
2 per minute per mole of succinyl-CoA synthase at 37°), perhaps a
property of succinyl-CoA synthase. In spite of the apparent high
purity, the enzyme as isolated may have a specific activity ranging from
21 to 47 units per milligram. This variation could result from partial
inactivation before the final steps of purification or from unknown effects
of culture conditions.
Properties
Specificity. Succinyl-CoA synthetase from bacterial and plant sources
has been found to be specific for A D P and A T P 7,s whereas the enzyme
from mammalian sources will use either G T P and G D P or I T P and I D P 2
Dephospho-CoA (CoA lacking the 3'-phosphoryl residue on the ribose
portion) is a substrate but has a higher Km than CoA. 1° Pantetheine,
while not a substrate in the overall reaction, has been shown 11 to
catalyze the CoA-dependent Pi ~ A T P exchange (see below).
Molecular Weight. The molecular weight has been determined to be
141,000 --+4000 by sedimentation equilibrium. 2 T r e a t m e n t with 1 m M
p-mercuribenzoate results in a marked lowering of the sedimentation co-
efficient and gives rise to separable phosphorylated and nonphosphorylated
components. 12
Ultraviolet Absorption. A solution at p H 7.2 containing 1 mg of
succinyl-CoA synthetase per milliliter has an absorbance of 0.51 at
280 m/~.2 The ratio of absorbance at 280 m~ to that at 260 m/~ is 1.75. ~
Estimation o] Phosphoenzyme Form. Succinyl-CoA synthase may
be converted to a form containing 3-phosphohistidine by exposure to
A T P and Mg ~ or to succinyl-CoA, P~, and Mg ÷÷. A T P will phosphorylate
up to one histidine residue per mole of enzyme, with an apparent - - A F of
2000 calories, but more than one phosphoryl group per mole is obtained
by reaction with succinyl-CoA and p~.2 The estimation of phospho-
enzyme is made by a phenol extraction procedure, following exposure
of the enzyme to ~2P-labeled substrate. The reaction is carried out con-
veniently in a 1-2 ml volume in a 12-ml centrifuge tube. The reaction
is stopped with an excess of E D T A , and 2 ml of liquefied phenol (ad-
7S. Kaufman and S. G. A. Alivisatos, J. Biol. Chem. 216, 141 (1955).
SR. A. Smith, I. R. Frank, and I. C. Gunsalus, Federation Proc. 16, 251 (1957).
9D. R. Sanadi, D. M. Gibson, P. Ayengar, and M. Jacob, J. Biol. Chem. 218, 505
(1956)..
1oR. H. Moyer and R. A. Smith, Biochem. Biophys. Res. Commun. 22, 603 (1966).
~IR. W. Moyer, R. F. Ramaley, L. G. Butler, and P. D. Boyer, J. Biol. Chem.
242, 4299 (1967).
1.-W. A. Bridger, unpublished experiments.
[15] 3-KETOACID COA-TRANSFERASE 75
]usted to pH 7 just before use) is added. To the tube is then added 4

ml of wash solution (8% w/v phenol, 10 mM Pi, 10 mM EDTA, pH 7)
and the solution is mixed with a vortex mixer for 30 seconds. After a
brief centrifugation in a clinical centrifuge at top speed, the upper
layer is carefully removed by aspiration. The extraction is repeated
until the upper layer contains no radioactivity {usually 5-8 extractions),
and the phenol layer is then quantitatively transferred to a planchet,
dried under heat lamps, and counted.
Other Reactions. Succinyl-CoA synthetase will catalyze a variety of
exchange reactions among its substrates. Exchange between ADP and
ATP has been observed 13 in harmony with formation of the phos-
phorylated enzyme. Pi ~ ATP exchange is absolutely dependent on the
presence of CoA but greatly stimulated by succinate. 2,~ Succinate
succinyl-CoA exchange has also been observed. 1~
The latter two exchanges suggest the existence of a form of enzyme
containing tightly bound CoA. This has led to the observation that
incubation of the enzyme with Mg ++ and succinyl-CoA results in the
formation of an isolable "high-energy" form which is apparently free of
all substrates except CoA. 11 Incubation of this enzyme form with P~,
ADP, and Mg ++ leads to the production of an equivalent amount of ATP.
Another possible intermediate has been postulated by Nishimura and
Meister, ~4 who showed that incubation of the enzyme with CoA and
14C-labeled succinyl-CoA led to the incorporation of label into succinate,
whereas incubation with ADP and 32P-labeled succinyl phosphate gave
radioactive ATP. These data suggest the participation of succinyl phos-
phate as an intermediate.
~3S. Kaufman, J. Biol. Chem. 216, 153 (1955).
~*J. S. Nishimura and A. Meister, Biochemistry 4, 1457 (1965).
[15] 3-Ketoacid CoA-Transferase

[EC 2.8.3.5 Succinyl-CoA:3-oxoacid CoA-transferase]
B y Louis B. HERSH and WM. P. JENCKS
Succinyl-CoA q- acetoacetate ~ acetoacetyl-CoA -{- succinate
Assay M e t h o d
Principle. T h e formation of acetoacetyl-CoA, as its magnesium-chel-
ated enolate ion, is followed spectrophotometrically at 310 m/~.I
ij. R. Stern, M, J. Coon, A. del Campillo, and M. C. Schneider, J. Biol. Chem. 221,
15 (1956).
]usted to pH 7 just before use) is added. To the tube is then added 4

ml of wash solution (8% w/v phenol, 10 mM Pi, 10 mM EDTA, pH 7)
and the solution is mixed with a vortex mixer for 30 seconds. After a
brief centrifugation in a clinical centrifuge at top speed, the upper
layer is carefully removed by aspiration. The extraction is repeated
until the upper layer contains no radioactivity {usually 5-8 extractions),
and the phenol layer is then quantitatively transferred to a planchet,
dried under heat lamps, and counted.
Other Reactions. Succinyl-CoA synthetase will catalyze a variety of
exchange reactions among its substrates. Exchange between ADP and
ATP has been observed 13 in harmony with formation of the phos-
phorylated enzyme. Pi ~ ATP exchange is absolutely dependent on the
presence of CoA but greatly stimulated by succinate. 2,~ Succinate
succinyl-CoA exchange has also been observed. 1~
The latter two exchanges suggest the existence of a form of enzyme
containing tightly bound CoA. This has led to the observation that
incubation of the enzyme with Mg ++ and succinyl-CoA results in the
formation of an isolable "high-energy" form which is apparently free of
all substrates except CoA. 11 Incubation of this enzyme form with P~,
ADP, and Mg ++ leads to the production of an equivalent amount of ATP.
Another possible intermediate has been postulated by Nishimura and
Meister, ~4 who showed that incubation of the enzyme with CoA and
14C-labeled succinyl-CoA led to the incorporation of label into succinate,
whereas incubation with ADP and 32P-labeled succinyl phosphate gave
radioactive ATP. These data suggest the participation of succinyl phos-
phate as an intermediate.
~3S. Kaufman, J. Biol. Chem. 216, 153 (1955).
~*J. S. Nishimura and A. Meister, Biochemistry 4, 1457 (1965).
[15] 3-Ketoacid CoA-Transferase

[EC 2.8.3.5 Succinyl-CoA:3-oxoacid CoA-transferase]
B y Louis B. HERSH and WM. P. JENCKS
Succinyl-CoA q- acetoacetate ~ acetoacetyl-CoA -{- succinate
Assay M e t h o d
Principle. T h e formation of acetoacetyl-CoA, as its magnesium-chel-
ated enolate ion, is followed spectrophotometrically at 310 m/~.I
ij. R. Stern, M, J. Coon, A. del Campillo, and M. C. Schneider, J. Biol. Chem. 221,
15 (1956).
Reagents
Tris-HC1 buffer, 20 mM, p H 8.10 ___ 0.02 at 25 ° containing 15 m M

magnesium chloride
Sodium acetoacetatc, 1.0 M (prepared according to the method of
Seeley 2)
Succinyl-CoA, 1.8 m M (prepared according to the method of
Simon and Shemin 3)
P r o c e d u r e . In a 3.5 ml quartz cuvette of 1.0 cm light path are placed 1.0
ml of buffer, 0.20 ml of sodium acetoacetate, 0.40 ml of succinyl-CoA,
enzyme, and water to a final volume of 3.0 ml. The reaction mixture
(excluding enzyme) is temperature equilibrated at 25 ° , and the reaction
is initiated by the addition of enzyme. Readings of the absorbance at 310
m~ are recorded every 15 seconds for 2 minutes. The reaction is usually
linear for the first 60-90 seconds, and this rate is used to calculate
enzyme activity.
Interfering enzymes preclude the use of this assay system until after
the third purification step. However, Stern has described alternative
assay systems that can be used with crude enzyme fractions. 1 A lag
PURIFICATION O F CoA-TRANSFERASE
Total protein Total Specific

Step (rag) units b activityb
1. Extraction t Taken from the procedure -- --

2. Ammonium sulfate of Stern et al.o -- --
(35-65%)
3. Heat and acid 19,700 78,000 4.0
4. Ammonium sulfate 9,200 80,000 8.7
(50-70%)
5. DEAE, pH 7 273 79,000 290.0
6. DEAE, pH 8 33 39,500 1200.0
a See text footnote 1.
b See text explanation of units.
period followed by a linear increase in absorbance at 310 m# m a y occur
with the heat- and acid-treated enzyme in the direct assay. In these
cases the linear portion of the rate curve is used to calculate enzyme
activity.
U n i t s . One unit of activity as defined by Stern e t al. 1 is t h a t amount
of enzyme required to cause an absorbance change of 0.01 per minute.
Since the molar extinction coefficient of acetoacetyl-CoA is 1.19 X 10s
*See Vol. I [105].
SE. J. Simon and D. Shemin, J. Am. Chem. 8oc. 75, 2520 (1953).
under the conditions of the assay,1 an absorbanee change of 0.01 cor-

responds to the formation of 2.5 X 10-3 micromoles of acetoacetyl-CoA.
Four hundred Stern units corresponds to the formation of 1 micromole
of substrate per minute (one international unit). Specific activity is
defined as the number of Stern units per milligram of protein.
Protein is determined by the method of Warburg and Christian. 4
This method of protein measurement gives a value 17% less than the
more accurate microbiuret method 5 when highly purified enzyme is used. ';
The specific activity of the purest preparation (see table) is 1040 units/
mg, based on the microbiuret method.
The following purification procedure is a modification of the method
of Stern et alJ All operations are performed at 4 °, unless otherwise noted,
and all determinations of pH are carried out at this temperature with
a pH meter calibrated at 4 °. All dialysis tubing is boiled in neutral I mM
EDTA prior to use. A summary of the procedure is shown in the table.
Step I. Extra.ction. Fifty pig hearts obtained immediately after death
are packed in ice. The hearts are cleaned of fat, blood clots, and connec-
tive tissue, and are then diced and passed twice through an electric
mincer. The minced hearts are washed five times with distilled water at
4°; each wash contains five times the weight of mince. The washed mince
is filtered through a large table-top Biichner filter and dried as much as
possible by suction. The mince is extracted as follows: (a) Into a 5-1iter
Waring blendor is placed a given weight of mince and 1,5 volumes of 50
mM potassium phosphate buffer, pH 7.4, containing 0.2M potassium
chloride (i.e., for 850 g of mince 1275 ml of potassium phosphate buffer
is added). (b) The mince is blended for 5 minutes at two-thirds maximal
speed. (c) Buffer, 1.5 volumes (1275 ml) is added and the mixture is
blended for an additional 5 minutes at one-third maximal speed.
The mince is centrifuged at 13,000 g for 20 minutes. The supernatant
is passed through 10 layers of cheesecloth, and the precipitate is dis-
carded.
Step 2. Ammonium Sul]ate Fractionation. The centrifuged extract is
brought to 35% saturation with ammonium sulfate by the addition of
245 g of ammonium sulfate per liter of extract. The solution is stirred for
1 hour and is then centrifuged at 13,000 g for 15 minutes. The precipitate
is discarded.
The supernatant solution is brought to 6 5 ~ saturation with am-
Warburg and Christian cited by E. Layne, Vol. III [73].
a R. F. Itzhaki and D. M. Gill, Anal. Biochem. 9, 401 (1964).
oL. B. Hersh and W. P. Jencks, J. Biol. Chem. 242, 3481 (1967).
monium sulfate by the addition of 210 g of ammonium sulfate for each

liter of extract. After stirring for 1 hour, the solution is centrifuged at
13,000 g for 15 minutes. The supernatant is discarded. The precipitate is
dissoh'ed in a volume of 17 mM potassium l~hosphate buffer, pH 7.4,
equal to one-tenth the starting volume. The dissolved precipitate is
dialyzed against 40 volumes of 17 mM potassium phosphate buffer, pH
7.4, for 12 hours.
Step 3. Heat and Acid Treatmer~t. The 65% ammonium sulfate frac-
tion is diluted to a protein concentration of 10 mg/ml with 17 mM potas-
sium phosphate buffer, pH 7.4. Three hundred (300) ml or 1 liter of this
solution in a beaker is placed in a water bath at 55 °. The temperature of
the solution is allowed to reach 50 ° in 9-14 minutes, during which time
the solution is stirred with a glass rod. The solution is maintained at 50 °
for 6 minutes with stirring, after which it is quickly cooled in ice. When
the temperature of the enzyme solution has reached 5 ° , it is centrifuged
at 13,000 g for 20 minutes. The precipitate is discarded.
The supernatant solution is adjusted to pH 5.8 by the addition of
0.1 M acetic acid and is then centrifuged at 13,000 g for 5 minutes. The
supernatant is quickly adjusted to pH 7 by the addition of 1.0 M potas-
sium bicarbonate. The solution is frozen.
Step $. Ammonium Sul]ate Fractionation. The heat- and acid-treated
enzyme (7.0 liters) is thawed and adjusted to pH 7.4 with 1.0M sodium
hydroxide. Ammonium sulfate is added to the solution to 5 0 ~ satura-
tion (313 g of ammonium sulfate/liter of solution). The solution is
stirred for 1 hour and then centrifuged at 13,000 g for 20 minutes. The
precipitate is discarded.
The supernatant is brought to approximately 70% saturation with
ammonium sulfate by the addition of 160 g of ammonium sulfate per
liter of the original solution. The solution is stirred for 1-2 hours and is
then centrifuged at 13,000 g for 45 minutes. The precipitate is dissolved
in a minimum volume (approximately 200 ml) of 5 mM potassium
phosphate buffer, pH 7, containing 50 mM potassium chloride and is
dialyzed against 14 liters of 5 mM potassium phosphate buffer, pH 7,
containing 50 mM potassium chloride for 12 hours and then against 3
changes of 9 liters of 5 mM potassium phosphate buffer, pH 7 for 8
hours each.
Step 5. DEAE Chromatography at pH 7. The dialyzed 50-70%
ammonium sulfate fraction (235 ml, about 10 g of protein) is placed on
a DEAE column (8 cm >( 80 cm) previously equilibrated at pH 7 with
5 mM potassium phosphate buffer. The column is washed with 5 mM
potassium phosphate buffer, pH 7, until the nonadsorbed protein has
[15] 3-KF~TOACID COA-TRANSFERASE 79
moved two-thirds of the length of the column; the movement of non-

adsorbed protein can be followed by its reddish brown color.
A linear gradient, consisting of 6 liters of 5 mM potassium phosphate
buffer, pH 7, containing 20 mM potassium chloride in the mixing chamber
and 6 liters of 5 mM potassium phosphate buffer, pH 7, containing 0.25 M
potassium chloride in the reservoir, is applied to ttle column. Fractions of
20 ml each are collected at this point. The enzyme is eluted at about 50
mM potassium chloride.
The fractions containing enzyme {approximately 1 liter) are pooled
aud put in dialysis tubing, which is placed in a plastic tray and covered
with Carbowax 4000 (Union Carbide). The solution is concentrated to
about 100 ml overnight at 4 ° by this procedure. The concentrated solu-
tion is placed in fresh dialysis tubing and is dialyzed against: (a) 8
liters of 5 mM Tris-HC1 buffer, pH 8, containing 50 mM potassium
chloride for 12 hours; (b) 6 liters of 5 mM Tris-HC1 buffer, pH 8 con-
raining 20 mM potassium chloride for 5 hours; and (c) three changes of
6 liters of 5 mM Tris-HC1 buffer, pH 8 for 6 hours each.
Step 6. DEAE Chromatography at pH 8. The dialyzed DEAE pH 7
fraction (105 ml, about 270 mg of protein) is applied to a DEAE
column (4 cm X 32 cm) previously equilibrated with 5 n~]I Tris-HC1
buffer, pH 8. The column is washed with one column volume of Tris-HC1
buffer (400 ml) after which a linear gradient consisting of 3 liters of 5
mM Tris-HC1 buffer, pH 8, in the mixing chamber and 3 liters of 5 mM
Tris-HC1 buffer, pH 8, containing 0.2M potassium chloride in the
reservoir is applied to the column. Fractions of 15 ml each are collected
at a flow rate of 3 ml/minute. The fractions containing enzyme are pooled
(approximately 300 ml), placed in dialysis tubing, and concentrated by
dialysis against Ficoll (Pharmacia). The concentration technique em-
ployed is the same as that used with the DEAE pH 7 fractions except
that Ficoll is used in place of Carbowax. The concentrated enzyme (5
ml) is dialyzed against three changes of 20 mM potassium phosphate
buffer, pH 7.4.
Step 6 does not give reproducible results and often results in poor
recovery of enzyme activity. Adsorption on alumina C~, gel may be used
as a further purification step: The enzyme is adsorbed from 5 mM
potassium phosphate buffer, pH 7.0, onto 6.6 times its weight of gel:
Inactive protein is removed by repeated washing with 0.1 M potassium
phosphate buffer, pH 7.0, after which the enzyme is eluted by repeated
washing with 0.25M buffer, concentrated, and dialyzed as described
above.
~See Vol. I [11].
80 REACTIONS ON THE CYCLE [lS]
The purified enzyme can be divided into small aliquots and stored
frozen.
In some cases thc enzyme was prepared in large quantities through
steps 1 and 2 at the New England Enzyme Center.
Purity. The enzyme has never been completely purified; however,
Stern et al. 1 have estimated that the pure enzyme has a specific activity
of 1300 units/mg. 1 The best preparations of CoA-transferase are approxi-
mately 92% pure.
Distribution. The enzyme has been reported in dog skeletal muscle,s
dog heart, s and pig kidney,1 as well as pig heart.
Properties
Sedimentation Behavior and Molecular Weight. A sedimentation
coefficient (S2o,w) of 5.5 has been obtained for an enzyme solution con-
taining 7 mg of protein/ml in 20 mM potassium phosphate buffer, pH
7.6,e while a value of 5.08 has been reported using a protein concentra-
tion of 12 mg/ml in the same buffer.~ A tentative molecular weight of
78,000 has been estimated for the enzyme by Sephadex chromatography;6
however this value awaits verification by techniques which take account
of possible molecular asymmetry of the protein.
Stability. The purified enzyme gradually loses activity when stored
at --20 ° at a protein concentration of 1.4 mg/ml in 20 mM potassium
phosphate buffer, pH 7.6.8 After 9 months at --20 ° the specific activity
of the enzyme decreased from 1200 to 642, and after 12 months the
specific activity decreased to 485.
Activators and Inhibitors. There are no known activators of the
enzyme. It has been reported that the enzyme is not inhibited by 1 mM
potassium EDTA or 10 mM iodoacetate. 1 Activity is inhibited by salts
and is sensitive only to the nature of the anion. The order of inhibitory
power of monovalent anions is SCN- ~ ClO( ~ I- ~ Br- ~ C1- ~ F-.
Divalent anions have relatively little effect. Chloride ion acts as a com-
petitive inhibitor with respect to the acid substrates succinate and aceto-
acetate. Incubation with acetoacetyl-CoA in the absence of other sub-
strates causes a loss of activity unless the enzyme has been rigorously
freed of metal ions2
Specificity. The enzyme has been reported to transfer coenzyme A
from succinyl-CoA to acetoacetate, fl-ketovalerate, fl-ketoisocaproate,
and p-ketocaproate, with relative activities of 100, 70, 57, and 32,1 as
well as to a-methylacetoacetate~ and malonic semialdehydeJ° Coenzyme
~G. K. K. Menon and J. R. Stern, J. Biol. Chem. 235, 3393 (1960).
o M. J. Coon, unpublished results. I
10G. K. K. Menon, J. R. Stern, F. P. Kupiecki, and M. J. Coon, Biochim. Biophys.
Acta 44, 602 (1960).
[16] SrCCINATE DEHYDROGENASE 81
A is transferred from acetoacetyl-CoA to succinate, 1 malonic semialde-

hyde, 1° and malonate, although malonate reacts 50 times slower than
succinate, s Succinyl-S-pantetheine, suceinyl-S-glutathione, and acetoace-
tyl-S-pantetheine are inactive as substrates for the enzyme. 1
Effect o] pH. With succinyl-CoA and acetoacetate as substrates the
enzymatic reaction rate increases with p H from pH 7.0 to pH 9.1.1 With
acetoacetyl-CoA and suecinate as substrates the reaction shows little
dependence on pH between pH 8.1 and pH 8.7. 6
Kinetics and Mechanism of Action. Reciprocal plots of rate against
substrate concentration at varying concentrations of the second substrate
exhibit parallel lines for the reaction in both directions, i.e., the reaction
follows "ping-pong ''11 kinetics? Product inhibition patterns and quanti-
tative studies of the acetoacetate-acetoacetyl-CoA and succinate-suc-
cinyl-CoA exchange reactions catalyzed by this enzyme suggest the two-
step mechanism shown in Eqs. (1) and (2).
E d- acetoacetyl-CoA ~- E • • acetoacetyl-CoA ~ E-CoA d- acetoacetate
(1)
E-CoA d- succinate ~ E • • succinyl-CoA ~ E ~ succinyl-CoA (2)
The separation of the overall reaction into its two component half
reactions and the isolation of an enzymatically active enzyme-coenzyme A
intermediate provide additional support for this mechanism. 6
l~W. W. Cleland, Biochim. Biophys. Acta 67, 104, 173, 188 (1963).
[16] Succinate Dehydrogenase

[EC 1.3.99.1 Succinate: (acceptor) oxidoreductase]
By C. VE~-~ER, D. V. DERVARTANIAN, and W. P. ZEYLEMAKER
Assay M e t h o d
In previous volumes of this series methods of assay were described for
the particle-bound I and the soluble enzyme, 2 as well as a method of
purification of the enzyme from beef heart mitochondria 2 and M~ro-
coccus l~ctilyticus3 Enzyme prepared according to several methods de-
scribed in the literature 2-4 cannot be used to reconstitute the suceinate
oxidase activity of a Keilin and Hartree 5 heart muscle preparation where
I W. D. Bonner, Vol. I [121].
2p. Bernath and T. P. Singer, ¥oi. ¥, p. 82.
s T. P. Singer, E. B. Kearney, and P. Bernath, J. Biol. Chem. o.9-3,599 (1956).
~T. Y. Wang, C. L. Tsou, and Y. L. Wang, Sci. Sinica Peking 5, 73 (1956).
*D. Keilin and E. F. Hartree, Proc. Roy. Soc. London B129, 277 (1940).
[16] SrCCINATE DEHYDROGENASE 81
A is transferred from acetoacetyl-CoA to succinate, 1 malonic semialde-

hyde, 1° and malonate, although malonate reacts 50 times slower than
succinate, s Succinyl-S-pantetheine, suceinyl-S-glutathione, and acetoace-
tyl-S-pantetheine are inactive as substrates for the enzyme. 1
Effect o] pH. With succinyl-CoA and acetoacetate as substrates the
enzymatic reaction rate increases with p H from pH 7.0 to pH 9.1.1 With
acetoacetyl-CoA and suecinate as substrates the reaction shows little
dependence on pH between pH 8.1 and pH 8.7. 6
Kinetics and Mechanism of Action. Reciprocal plots of rate against
substrate concentration at varying concentrations of the second substrate
exhibit parallel lines for the reaction in both directions, i.e., the reaction
follows "ping-pong ''11 kinetics? Product inhibition patterns and quanti-
tative studies of the acetoacetate-acetoacetyl-CoA and succinate-suc-
cinyl-CoA exchange reactions catalyzed by this enzyme suggest the two-
step mechanism shown in Eqs. (1) and (2).
E d- acetoacetyl-CoA ~- E • • acetoacetyl-CoA ~ E-CoA d- acetoacetate
(1)
E-CoA d- succinate ~ E • • succinyl-CoA ~ E ~ succinyl-CoA (2)
The separation of the overall reaction into its two component half
reactions and the isolation of an enzymatically active enzyme-coenzyme A
intermediate provide additional support for this mechanism. 6
l~W. W. Cleland, Biochim. Biophys. Acta 67, 104, 173, 188 (1963).
[16] Succinate Dehydrogenase

By C. VE~-~ER, D. V. DERVARTANIAN, and W. P. ZEYLEMAKER
Assay M e t h o d
In previous volumes of this series methods of assay were described for
the particle-bound I and the soluble enzyme, 2 as well as a method of
purification of the enzyme from beef heart mitochondria 2 and M~ro-
coccus l~ctilyticus3 Enzyme prepared according to several methods de-
scribed in the literature 2-4 cannot be used to reconstitute the suceinate
oxidase activity of a Keilin and Hartree 5 heart muscle preparation where
I W. D. Bonner, Vol. I [121].
2p. Bernath and T. P. Singer, ¥oi. ¥, p. 82.
s T. P. Singer, E. B. Kearney, and P. Bernath, J. Biol. Chem. o.9-3,599 (1956).
~T. Y. Wang, C. L. Tsou, and Y. L. Wang, Sci. Sinica Peking 5, 73 (1956).
*D. Keilin and E. F. Hartree, Proc. Roy. Soc. London B129, 277 (1940).
this activity had been destroyed by alkali treatment2 ,~ A preparation

which is capable of reconstituting succinate oxidase is described below. *,8
It is similar to the preparation of Keilin and King 6 and to a modification
of the method of Wang and co-workers.4 As outlined in the literature, 8,9
there is no relationship between activities measured with the artificial
electron acceptors 1.-0 and the more labile reconstruction activity. There-
fore, it has been suggested that reconstitution activity be used as an
indicator of the native state of the soluble enzyme.
Two methods are generally used for the determination of the activity
of soluble succinate dehydrogenase with artificial elcctron acceptors.
The first method, a manometric one with phenazine methyl sulfate
(PMS), can ' ~. modified for spectrophotometric use although the use of
a light-sensitive acceptor requires certain precautions. It has been
claimed, s that the rates obtained with the spectrophotometric modification
are not proportional with the enzyme concentration. However when a
suitable control lacking succinate is used the rates are closely propor-
tional. Nevertheless in our experience the second method, which uses
K3Fe(CN)e as acceptor, is more convenient as a routine assay.
The method of determination of the reconstitution activity of a
soluble, purified succinate dehydrogenase preparation is also described.
M a n o m e t r i c M e t h o d with Phenazine M e t h y l Sul]ate :,'°
Reagents
Succinate, 0.4M, pH 7.6
Bovine serum albumin in H20, 3% (w/v)
Cyanide, 30 mM neutralized
Phenazine methyl sulfate (PMS) in H20, 1 ~ (w/v), carefully
protected from light
Enzyme, in oxygen-free 30 mM phosphate buffer containing 0.1%
bovine serum albumin, diluted to give an uptake between 2 and
7 t~l of 02 per minute in the assay
Procedure. Add to tile main compartments of five Warburg vessels
phosphate buffer, 0.5 ml; succinate, 0.3 ml; bovine serum albumin, 0.!
ml; enzyme and H=0 to a final volume of 3 ml. Different amounts of
PMS are pipetted to the side arms of the vessels; recommended amounts
D. Keitin and T. E. King, Proc. Roy. Soc. Lo~don B152, 163 (1960).
7T. E. King, J. Biol. Chem. 236, 2342 (1961).
ST. E. King, J. Biol. C]~ern. 238, 4032 (1963).
oT. E. King, J. Biol. Chem. 238, 4037 (1963).
~°E. B. Kearney and T. P. Singer, J. Biol. Chem. 219, 963 (1956).
[16] SUCCINATE DEHYDROGENASE 83
are 0.2, 0.1, 0.07, 0.05, and 0.04 ml (concentration range, 2.2-0.43 raM).
Cyanide, 0.1 ml, is added last. Each vessel is connected immediately to
its manometer with the stopcock closed, then placed in the water bath at
38 ° (the pressure is released by opening the stopcock). After 7 minutes'
equilibration, the contents are tipped and the oxygen uptake is recorded
in the interval 2-7 minutes after tipping.
The activity is calculated from double reciprocal plots of activity
against dye concentration. In this determination one mole of succinate
reduces one mole of oxygen.
In the spectrophotometric adaptation of this method, all reagents are
used in the amounts described, as well as 0.1 ml of 0.15 mM 2,6-dichloro-
phenol-indophenol (~ ~-- 21 )< 103 M -l'sec -1 at 600 mt~). Add the reagents
to a cuvette thermostatted at 38 ° and start the reaction by adding
an amount of enzyme that gives a change in extinction of 0.02-0.1 per
minute measured as initial rate. A blank rate (all reagents except suc-
cinate) must be determined separately.
In this determination 1 mole of succinate reduces 1 mole of dye.
Spectrophotometric Method with KsFe(CN)~ (footnotes 8, 11)

This method is a modification of the method of Slater and Bonner. 1,12
Reagents
EDTA, 30 mM pH 7.6
KCN, 0.03 M, neutralized
Succinate, 0.4 M, pH 7.6
Bovine serum albumin in H20, 3% (w/v)
K3Fe(CN)6, 75 mM stored in a dark bottle
Enzyme in oxygen-free 30 mM phosphate buffer containing 0.1%
bovine serum albumin, diluted to give a change in extinction of
0.02-0.08 per minute, measured as initial rate
Procedure. Add to a spectrophotometer cuvette thermostatted at 25°:
H20 to a final volume of 2.9 ml; phosphate buffer, 1 ml; EDTA, 0.1 ml;
succinate, 0.3 ml; bovine serum albumin, 0.1 ml; and K3Fe(CN)6, 0.2 ml;
0.1 ml of KCN is added only when particle-bound enzyme is assayed.
After noting the extinction at 455 mt~ (c-----150M-~'cm-1), start the
reaction by addition of the enzyme, and follow the change in extinction
during the first 2 minutes. Initial rates are taken as a measure of activity.
A blank rate (all reagents except succinate) must be determined
separately.
11D. V. DerVartanian and C. Veeger, BiocMm. Biophys. Act(t 92, 233 (1964).
i~E. C. Slater and W. D. Bonner, Biochem. J. 52, 185 (1952).
In this determination 1 mole of succinate reduces 2 moles of
KaFe(CN)6. Concentrations of K~Fe (CN)e of 0.2-5 mM can be used for
the calculation of maximal velocities from double reciprocal plots. Con-
centrations of K~Fe(CN)6 above 5 mM are inhibitory. At low concen-
trations of K3Fe(CN)~ rates can be measured by following the reaction
at 420 m~ (c ~ 1.03 X l0 s M -l"cm-1).
Succinate Oxidase o] Heart Muscle Particles. Reconstitution of the

oxygen uptake of particles deprived of succinate oxidase activity.
Reagents
Succinate pH 7.6, 0.4 M
EDTA, 30 mM, neutralized
Cytochrome c 1% (w/v), in H20
Heart muscle preparation prepared as described below under A of
this section
Cytochrome c-deficient heart muscle preparation prepared as de-
scribed under purification procedure
Alkali-treated heart muscle preparation, prepared as described
under B of this section
Soluble succinate dehydrogenase purified up to the gel eluate stage
of the purification procedure.
A. PREPARATION OF THE HEART MUSCLE PREPARATION OF KEILIN AND
HARTREE2'7'13 The procedure is similar to the one described for prepa-
ration of starting material for the soluble enzyme, except that the meat
mince is washed only with tap water.
B . PREPARATION OF THE ALKALI-TREATED HEART MUSCLE PREPARA-
TION2 ,7 The pH of a heart muscle preparation as described under A,
protein concentration approximately 10 mg/ml, is adjusted to pH 9.3
with 1 N NaOH. The mixture is incubated for 90 minutes at a tempera-
ture of 38 °. After 90 minutes the mixture is cooled to room temperaturc
and adjusted to pH 7.6 by careful addition of 1 N HCI. This prepara-
tion can be stored for 3 days at 0 ° without significant loss in reconsti-
tution activity.
Procedure. a. SUCCINATE OXIDASE ACTIVITY. The succinate oxidase
activity is measured manometrically in Warburg flasks at 38 ° in a
system containing: H20 to a final volume of 3 ml; phosphate buffer,
1 ml; cytochrome c, 0.1 ml; EDTA, 0.1 ml. The enzyme preparation (in
main compartment), is either heart particles prepared as described
~ E . C. Slater, Biochem. J. 45, 1 (1949).
under A or B, or the cytochrome c-deficient heart muscle preparation

which is the starting material for the soluble enzyme; 0.3 ml of succinate
is added to the side arm. After temperature equilibration the reaction is
started by tipping the contents from the side arm and the oxygen uptake
is recorded.
Keilin and Hartree heart muscle preparation has a specific activity
of 0.7-1.4 micromoles of succinate oxidized per minute per milligram of
protein. The alkali-treated heart muscle particles have a residual suc-
cinate oxidase activity averaging below 5% of the original activity.
b. RECONSTITUTION OF SUCCINATE OXmASE ACTIVITY. The mixture of
H~O, phosphate buffer, eytochrome c, and EDTA in the amounts given
under procedure a is added to the main compartment of the Warburg
flasks, followed by the alkali-treated heart muscle preparation in a
concentration of about 1 mg/ml final volume, and 0.5-1 mg of soluble
succinate dehydrogenase; 0.3 ml succinate is added to the side arm.
After 7 minutes' temperature equilibrium at 38 °, the reaction is started
by tipping the contents from the side arm and the oxygen-uptake is
recorded.
Maximal restoration of suceinate oxidase activity is obtained with
varying ratios of soluble enzyme to heart muscle preparation depending
on pretreatment, purity and age of the preparation. Fully reconstituted
alkali-treated heart muscle preparation oxidizes 0.2-0.3 mieromole of
succinate per minute per milligram of heart muscle protein.
The procedure comprises 2 parts: (A) preparation of cytochrome
c-deficient heart muscle preparation; (B) preparation of the soluble
enzyme.
A few essential precautions have to be taken in part 2 of the
procedure to make sure that reproducible preparations are obtained in
terms of activity with artificial hydrogen acceptors and reconstitution
activity as well as kinetic and spectral properties. Glass-distilled H~O
should be used throughout the entire purification procedure, and all steps
should be carried out at 0-4 ° in the presence of 1 mM EDTA. It is very
important to eliminate as much oxygen as possible by flushing the solu-
tions with pyrogallol-purified N2. It is preferable to perform all purifica-
tion steps and centrifugations in closed tubes under N2.
The whole procedure from the butanol extraction through the second
(NH4)2SO~ precipitation takes less than 31/~ hours. Precipitation and
fractionation with (NH4)~S0, significantly increase the purity of the
enzyme. On the other hand, purification occurs at the expense of the re-
constitution activity, the loss of which is not reproducible.
In studies with the enzyme purified up to the gel eluate stage of the
procedure, it must be kept in mind that the enzyme may contain traces
of succinate.
A. Preparation of Cytochrome c-Deficient Heart Muscle Preparation

Twenty pig hearts are cleaned of fat and connective tissue and lninced
in a meat grinder. The mince is washed with 8-10 changes of 30 liters of
tap water, for 15 minutes each time, efficient mechanical stirring being
used, and then squeezed by hand through cheesecloth. The water of the
last change should be light yellow, not pink.
The next step is necessary to remove cytochrome c and other soluble
hemoproteins which are difficult to remove during the purification of the
soluble enzyme. This step is not included in the normal procedure for
the Keilin and Hartree heart muscle preparation. The mince is ex-
tracted overnight at 0 ° with 20 liters of 0.15 M phosphate buffer pH 7.6
containing 1 mM EDTA, squeezed through cheesecloth, and washed twice
with tap water.
Grind 800 g of wet mince, 400 ml of 20 mM phosphate pH 7.6 con-
taining 1 mM EDTA, and 600 g acid-washed and neutralized sand at 0 °
in a mechanical mortar (manufacturers: Pascall Engineering Company,
Ltd., Crawley, Sussex, England) until a homogeneous paste is obtained,
usually for 20 minutes. ~ Add 600 ml of the same cold phosphate buffer and
stir for another 5 minutes. The suspension is centrifuged for 15 minutes at
1000 g.
Carefully decant the supernatant and after pH adjustment to 5.7
by the addition of 1 N acetic acid, stir for 5 minutes and centrifuge for
15 minutes at 1500 g. Discard the supernatant, wash the precipitate with
cold water, and then centrifuge for an additional 15 minutes at 1500 g.
Homogenize the precipitate in 1-1.5 liters of 50 mM borate-50 mM
phosphate buffer, pH 8.0, final protein concentration 10-15 mg/ml. In
the case of a normal Keilin and Hartree heart muscle preparation, the
precipitate is homogenized in 0.1 M phosphate buffer pH 7.6.
These preparations can be stored for weeks at 0 °. The succinate
oxidase activity declines gradually, while the activity with artificial
hydrogen acceptors remains almost unaffected.
B. Preparation o] the Soluble Enzyme

All steps are carried out in the absence of 02.
Step 1. Solubilization and Gel Eluate. The heart muscle preparation
is made anaerobic by flushing it with purified N~. Add sodium succinate
to a concentration of 40 mM (the color changes to green) and allow the
preparation to stand 2-24 hours in a closed bottle at 0% Add to this
preparation one-fifth of its volume of n-butanol (of --20°), and stir o,"
shake the mixture for 30 minutes while N_o is bubbling through the
solution (at 0°). Centrifuge the mixture in closed transparent polyethyl-
ene tubes under N., for 20 minutes at 1500 g.
After centrifugation three layers are visible: a sediment, a clear
yellow-orange middle layer, and a very turbid upper layer. The middle
layer is carefully withdrawn by means of an adjustable suction system.
Volume is 800-1100 ml. Contamination with the two other layers must
be avoided as it leads to impure preparations. Adjust the pH of the
extract to 6.0 by the addition of 1 N acetic acid. Add calcium phosphate
gel (prepared according to Keilin and Hal~treeTM) to a final concentration
of 4 mg/ml. Stir the suspension for 5 minutes, then centrifuge it for 4
minutes at 1000 g.
Discard the supernatant, wash the gel once by stirring with deoxy-
genated water, and centrifuge the suspension at 1000 g for 5 minutes.
Discard the supernatant and add 150-200 ml of 80 mM phosphate buffer
pH 7.6 to the tubes. The tubes are made anaerobic, closed, shaken for
10 minutes, then centrifuged at 30,000 q for 5 minutes to remove any
butanol-denatured protein bound to the gel.
The dark brown gel eluate is collected and either used for further
purification or stored in sealed polyethylene tubes under liquid N_o, as
described in the next section.
Step 2. Fractio77ation with (NH,)~_SO,. Adjust the pH of the gel-
eluate to 7.2 with 1N acetic acid and add solid (NH,)~SO, to 65%
saturation (450 g per liter), in 5 minutes, while a stream of N2 is flushed
over the solution. Centrifuge the mixture for 10 minutes at 23,000 q.
Dissolve the precipitate in 30 ml of 0.1 M phosphate buffer pH 7.6. Add
12 ml of a saturated (at 20 °) solution of (NH,)oSO4 adjusted to pH 7.2
with concentrated NH,OH (0.3 saturation). The pH of the (NH4)~SO,
solution is measured in a 1 : 10 dilution. This addition takes ,5 minutes and
is performed under a stream of N... Centrifuge the mixture for 5 minutes
at 23,000 g and discard the precipitate.
Add approximately 20 ml of the saturated (NH,)_oS04 solution to the
supernatant (0.5 saturation) in the course of 5 minutes under a stream of
N2. Centrifuge the mixture at 30,000 g for 5 minutes, discard the super-
natant, and remove the last traces of (NH~)2SO, with filter paper under
a stream of N.,. Wash the sediment with anaerobic 0.1M phosphate
bufer pH 7.6 by carefully placing a few drops of the buffer on top of
the pellet and then removing them. Dissolve the precipitate in about 3
ml of 0.1 M phosphate buffer pH 7.6, amt place the solution in a poly-
ethylene tube covered with a self-sealing rubber stopper through which
"D. Keilin and E. F. tIartree, Proc. Roy. Soc. London B19.,4, 397 (1938).
a needle is inserted. The contents of the tube are subjected to 5-6

cycles of evacuation and refilling with N~, ending with the latter. With-
draw the needle and store the solution in liquid N2. Such frozen prepa-
rations are stable for at least 3 weeks. Before use, thaw the contents of
a tube at 0 ° and then centrifuge under N2 for 5 minutes at 30,000 g to
remove a slight turbidity.
The various steps are summarized in the table.
Properties
The amount of flavin has been determined to be 1 mole per 200,000-
250,000 g of protein at the second (NH,)2SO, fractionation step. In
comparison with the other preparations described 2.4 it is estimated that
the enzyme at the second (NH~)2S04 step is more than 70% pure. The
flavin (FAD) is covalently linked to the protein. 15,16 There are 8 atoms
of nonheme iron per mole of flavin,",~s which is twice as much as in
other preparations2.4 The enzyme contains 4-8 atoms of labile sulfide per
mole of flavin.1''18 The failure of the enzyme containing four nonheme
iron atoms to reconstitute oxygen uptake might be due to degradation of
the form containing 8 atoms. It also cannot be excluded that the enzyme
as isolated here is a mixture of a primary succinate dehydrogenase con-
taining four iron atoms and a labile nonheme iron protein essential for
the connection with the respiratory chain.
The enzyme is very unstable at room temperature even when kept
under N2. Under all conditions the reconstitution activity declines faster
than the activity with PMS and K~Fe(CN)6. The reeonstitution activity
is fairly stable upon storage under liquid N~; the activity with hydrogen
acceptors is also stable under these conditions. When stored at room tem-
perature the absorbance of the enzyme declines slowly over the entire
wavelength range. The process is slower under anaerobic conditions, but
there is no relation between decline in absorbance and inactivation."
About 22% activation is observed upon incubation at room tempera-
ture." This is small in comparison with values obtained with another
preparation, 3,~9 indicating that this preparation is fully activated. The
spectral changes observed with competitive inhibitors ~9-~ which were first
~T. Y. Wang, C. L. Tsou, and Y. L. Wang, Sc/. 8inica Peking 7, 65 (1958).
~*E. B. Kearney, J. Biol. Chem. 235, 865 (1960).
" T . E. King, Biochem. Biophys. Res. Commun. 16, 511 (1964).
= W. P. Zeylemaker, D. V. DerVartanian, and C. Veeger, Biochim. Biophys. Acta
09, 183 (10~5).
~E. B. Kearney, J. Biol. Chem. ~20, 363 (1957).
2°D. V. DerVartanian and C. Veeger, Biochim. Biophys. Acta 105, 424 (1965).
riD. V. DerVartanlan, W. P. Zeylemaker, and C. Veeger, Syrup. Flavins Flavo-
proteins 8, 183 (1906).
O O O
C e-
e- ., ¢D O
r~
0 '~ ¢~'~ O ~
~ a0 ~'~
O
,.,.~ ~ ~ ~ e, " ~
,00 REACTIONS ON Tim C'~'CLE [16]
attributed to activation, were found to be due to the formation of two

classes of enzyme-inhibitor complexesJ 1 Reduced glutathione and BAL
cause marked changes in the absorption spectrum of the enzyme. 2°
Besides succinatc, the enzyme also oxidizes L-ch]orosuccinate,
L-methyl succinate, D-malatc, and L-malatc. '-'~ D-Chlorosuccinate,
D-methyl succinate, malonate, methylene succinate, malcate, acetoace-
tare, and oxaloacetate are competitive inhibitors. The kinetically esti-
mated K~ values agree well with disassociation constants of spectrally
detectable enzyme-inhibitor complexes. ~,~° The catalytic center activity
of the enzyme at 25 ° extrapolated to infinite succinate and K3Fe(CN)6
concentrations is 3900 min -~. The same value is obtained at 25 ° at infinite
succinate, and PMS concentrations in the spectrophotometric PMS-
promoted reduction of 2,6-dichlorophenol indophenol. -~2The kinetic results
indicate t h a t the dissociation of fumarate from the reoxidizcd enzyme
is the rate-limiting step in the overall reaction. 2~ The catalytic center
activity of the reconstituted particles with a limiting amount of soluble
enzyme, at 37 ° and with a succinate concentration of 40 mM, has been
found to be 10,000 min -~ (footnote 9).
In contrast to data reported in the literature, 25-~7 the enzyme ex-
changes, upon reduction with succinate in D20, its protons with different
rates, ~8 which leads to the formation of ( - - ) - ( R ) - s u c c i n a t e acid-d1,
meso-succinate-d~., ( - - ) - ( R , R ) - s u c c i n a t c - d 2 , (--)-(R)-succinate-d3 and
succinate-d4. Furthermore the formation of ( ~ ) - ( S ) - d e u t e r a t e d suc-
cinates could be observed -~s in case succinate-d4 was exchanged in H20.
Application for Analytical Purposes

As described elsewhere, the purified enzyme is suitable for the
microdete~mination of succinate (see this volume [69]).
52W. P. Zcylemaker and C. Veeger, unpublished results.

~A. Guiditta and T. P. Singer, J. Biol. Chem. 234, 666 (1959).
T. E. King, R. L. Howard, D. F. Wilson, and J. C. R. Li, J. Biol. Chem. 237,
2941 (1962).
u O. Gawron, A. J. Glaid, and J. Francisco, Biochem. Biophys. Res. Commun. 9,
237 (1964).
Hj. Kahn and D. Rittenberg, Biochem. Biophys. Res. Commun. 27, 484 (1967).
sTM. Hiifner, L. M. Buckle)', and T. C. Hollocher, Bioc]~em. Biophys. Res. Com.mun.
28, 791 (1967).
ssj. R~tey, J. Seibl, D. Arigoni, J. W. Cornforth, G. Ryback, W. P. Zeylemaker, and
C. Veegcr, Nature 216, 5122 (1967).
[17] FUMARASE 91
[17] F u m a r a s e
[EC 4.2.1.2 L-Malatehydro-lyase]
B y ROBERT L. HILL and RALPH A. BRADSHAW
Fumarate + H20 ~ L-malate
Assay Method
Distinct differences in the chemical and physical properties of
fumarate and L-malate allow fumarase to be assayed in several ways. 1
The most convenient method is a continuous assay, 2 in which changes in
fumarate concentration are measured spectrophotometrically between
250 and 300 m~. The activity of fumarase is extremely sensitive to
temperature and to the concentration and type of anion in the assay
mixture. Each of these parameters must be controlled carefully for
accurate activity measurements.
Procedure2 An aliquot of enzyme is added directly to a cuvette of
1 cm light path containing 3 ml of 50 mM L-malate and 50 mM sodium
phosphate buffer, pH 7.3. The increase in absorbance at 250 m~ is
observed at 10-second intervals for at least 60 seconds. Although the
reaction remains linear between 0 and 1 optical density units (OD), the
most reproducible results are found in the range of 0-0.5 OD. Because
of the marked variation in activity with temperature, the temperature
of the assay mixture must be determined accurately with a calibrated
thermometer. Alternatively, the temperature of the assay mixture may be
held constant by temperature control of the cell housing of the spectro-
photometer.
Units. The number of units of activity for an aliquot of enzyme in 3 ml
of substrate is defined as the initial rate of change in optical density per
l0 seconds times 103. The observed activity is corrected to the activity
at 25 ° , if necessary, by assuming that the activity varies by 8% per
degree between 22 and 28% The number of units per milliliter of enzyme
solution is calculated from the size of the aliquot taken for assay. The
specific activity is defined as the total number of units per milliliter of
enzyme at 25 ° at a protein concentration of 1 mg per milliliter. The
protein concentration of samples of pure fumarase is calculated from the
extinction coefficient at 280 m~ (0.51 for a solution of 1 mg/ml).3
'V. Massey, Vol. I, p. 729.

3L. Kanarek and R. L. Hill, J. Biol. Chem. 239, 4202 (1964).
A sample calculation for determination of each of these parameters is

given below.
If 5 ~l of a solution of fumarase (10 ml total volume) gives a change
in optical density at 250 m~ equal to 0.029 per 10 seconds at 24 °, then
Number of units -- [(0.029)(10a)]/(0.92) = 31.5

Units per ml = (31.5)(200) = 6300, and
Total units -- (6300)(10) -- 63,000
If this solution has an optical density of 0.102 at 280 m~, then
Specific activity = [(6300)(0.510)]/(0.102) - 31,500
This specific activity corresponds to a turnover number of 77,000

molecules of malate per molecule of enzyme per minute at 25 °, pH 7.3.
The method for estimation of specific activity given here differs from
that of Frieden e t al.," who measured the concentration of the enzyme
solution at 250 m~ rather than 280 m#. Since enzyme solutions are often
slightly turbid, errors in absorbance measurements as the result of light
scattering are less at 280 m~ than at 250 m~. Thus estimation of enzyme
concentration at 280 m~ may be more accurate. For very precise measure-
ments, it has been found desirable to calculate the amount of light
scattering which results from the slight turbidity of the enzyme solutions.
The procedure described here s was developed from methods described
earlier by Massey 1 and by Frieden e t al. ~ This method gives a higher
yield than these earlier methods and requires only a short time to obtain
pure enzyme. As much as 100 mg of crystalline fumarase may be ob-
tained per kilogram of pig heart muscle, an increase of 5 to 6 times the
yield given by the other methods. The low yields given by earlier methods
appear to have resulted from the loss of 60-70~ of the fumarase content
of heart muscle when the tissue was washed with water prior to extrac-
tion of fumarase with buffer.'
The treatment of the tissue prior to extraction may have a marked
effect on the behavior of fumarase during its isolation. If fumarase is
prepared from muscle which has been frozen, somewhat different con-
centrations of ammonium sulfate are required to obtain the same yields
of enzyme in steps 2-5 in the following procedure. For this reason it
appears desirable to prepare fumarase from heart muscle that has not
been frozen prior to treatment.
' C. Frieden, R. M. Bock, and R. A. Alberty, J. Am. Chem. Soc. 76, 2482 (1954).
[17] FUMARASE 93
The following procedure can also be used with horse heart muscle to
give highly pure, crystalline fumarase. 5
Step 1. Extraction of Muscle. Fifty to 70 hearts can be processed in
this step in 1 day, but it is convenient to extract 5 hearts in a single
operation and pool extracts at the end of step 1. Five swine hearts (200-
300 g each), chilled in ice at the slaughterhouse, are trimmed of fat and
connective tissue and cut into small cubes about 2 cm on a side. The
diced tissue (about 750-950 g) is then homogenized in a Waring blendor
(1 gallon capacity) at room temperature with 2.25 liters of 10 mM
sodium phosphate buffer, pH 7.3. The hearts are blended for 1 minute
at a high speed, which is sufficient to thoroughly homogenize the mixture.
The speed of the blendor is then reduced and the blending is continued
until the temperature of the homogenate reaches 28 °. The time required
to achieve this temperature is dependent upon the initial temperature of
the tissue, but generally it is between 5 and 10 minutes. The resulting
homogenate is then adjusted to pH 5.2 with 1 M sodium acetate buffer,
pH 4.0. A volume of 40-60 ml is usually required. The homogenate is
then centrifuged at 4 ° for 30 minutes at 1340 g. The precipitate is
discarded, and the volume of the supernatant solution is measured.
Step 2. First Ammonium Sulfate Fractionation. The solution from step
1 is brought to 0.55 saturation with solid ammonium sulfate (351 g per
liter). After it has been stirred mechanically for 10 minutes, the solution
is stored at 4 ° for 2 days. Although fumarase may be recovered im-
mediately from the resulting precipitate, storage for 2 days gives higher
yields of enzyme, and most of the supernatant solution can be removed
conveniently by suction. The supernatant solution contains less than 5%
of the activity in the original extract. The ammonium sulfate precipitate
is collected from the remainder of the solution by centrifugation at
4100 g and then dissolved in 200 ml of 10 mM phosphate buffer, pH 7.3.
This solution is fractionated further as described in step 3. The specific
activity of fumarase at this stage is about 170; the yield is 95% (see
the table).
Step 3. Second Ammonium Sulfate Fractionation. The ammonium
sulfate concentration in the redissoh'ed precipitate from step 2 is esti-
mated by assuming that the volume increment over 200 ml is the result
of addition of this volume of 0.55 saturated ammonium sulfate solution.
The solution is then brought to 0.35 saturation (209 g per liter) by the
addition of the appropriate amount of solid salt. The mixture is stirred
mechanically for 10 minutes and then centrifuged at 4 ° for 30 minutes
at 4100 g. Long periods of settling are not required at this point. The
35% ammonium sulfate precipitate contains about 5% of the original
~L. Kanarek, personal communication (1967).
fumarase and may be discarded. The specific activity of the supernatant

solution is about 276 with an overall yield of 90% (see the table).
Step 4. Third Ammonium Sulfate Fractionation. The supernatant
solution from step 3 is brought to 0.55 saturation with ammonium sulfate
by addition of solid salt (129 g per liter). The mixture is stirred for 10
minutes and then centrifuged at 4100 g at 4 ° for 30 minutes. The super-
natant solution which contains about 0-2% of the original fumarase may
be discarded. The precipitate, when dissolved in phosphate buffer, has
a specific activity of 328 with a yield of 87% (see the table).
Step 5. First Crystallization. The precipitate from step 4 is dissolved
in 125 ml of 10 mM phosphate buffer, pH 7.3, at room temperature. This
solution occasionally contains insoluble material which is inactive and
may be removed by centrifugation. The solution is adjusted to pH 7.3
with 1 N sodium hydroxide and the ammonium sulfate concentration is
estimated as described in step 3. Phosphate buffer (10 mM, pH 7.3),
saturated with ammonium sulfate at room temperature, is then added to
bring the salt concentration to 0.45 saturation. A 15 ml aliquot of the
resulting solution is centrifuged, and the density of the supernatant
solution is measured. The ammonium sulfate concentration may be
calculated from the relationship,
Ammonium sulfate [(Observed density.~ ]

(% saturation) = [\ 0.240 ] -- 4.212 X 100
Additional buffer, saturated with ammonium sulfate, is then added to
bring the solution to 0.50 saturation. After 3 days at 5 ° a well-defined
crystalline sheen is evident. The suspension, which contains crystals and
amorphous precipitate, is allowed to warm slowly to room temperature
and sufficient 10 mM phosphate buffer, pH 7.3, is added to give a final
ammonium sulfate concentration of 0.35 saturation. The solution is then
stirred for 1 hour at room temperature and brought to 0.50 saturation
with saturated ammonium sulfate. This addition should be made slowly
with constant stirring. The solution is then stored for 3 days at 5°; in
this time additional crystals form. It is advantageous at this point to
harvest the crystals and prepare them for recrystallization as described
in step 6. Only a slight increase in the quantity of crystalline fumarase
is obtained by repeating this step. The specific activity of the crystals
at this stage is about 9000 with a 78% yield (see the table).
Step 6. Recrystallization. The suspension of crystals from step 5 is
centrifuged for 1 hour at 4080 g and the resulting supernatant solution
is discarded. The precipitate is suspended in 80 ml of 0.35 saturated
ammonium sulfate at room temperature, collected by centrifugation, and
then dissolved in 20 ml of 10 mM phosphate buffer, pH 7.3. After the
[17] FUMARASE !)5
crystals have been dissolved, the insoluble material m a y be removed by

centrifugation and discarded. The clear solution is allowed to w a r m to
room temperature and adjusted to p H 7.3 if necessary. A solution of
saturated a m m o n i u m sulfate is then added drop by drop, with stirring,
to bring the fired ammonium ~ulfatc concentration to 0.46 saturation.
The solution is then allowed to stand for 48 hours at 5 °. At this time the
solution is warmed to 25 ° and brought to 0.48 saturation with am-
monium sulfate and again is allowed to stand for 48 hours at 5 °. The
solution is brought to room temperature again and a m m o n i u m sulfate
concentration brought to 0.50 saturation, and allowed to stand for several
days at 5 °. At this point, the crystals m a y be harvested. A slightly
higher yield of fumarase can be obtained by adjusting the solution of
crystals in 0.50 saturated a m m o n i u m sulfate to 0.40 saturation with
10 m M phosphate buffer, p H 7.3. This solution is stirred mechanically
for 1 hour at 25 ° and brought to 0.46 saturation as described above. After
24-48 hours at 5 ° the solution is brought again to 0.50 saturation and
allowed to stand at 5 ° for 24-48 hours. By this procedure more than
95% of the fumarase originally present at the start of step 6 is recovered
in crystalline form. After washing the crystals with 0.15 saturated am-
monium sulfate, p H 7.3, and dissolving them in 10 m M phosphate, p H
7.3, the specific activity is 31,500 with a yield of 72% (see the table).
About 90 mg of crystals are obtained in this step. The crystals m a y be
stored at 5 ° in 0.504).55 saturated a m m o n i u m sulfate.
Purity. Fumarase prepared by this method reaches a constant specific
ISOLATION OF CRYSTALLINE FUMARASE FROM SWINE HEART MUSCLE
Protein Specific
concen- activity
Volume Units tration a (units/ Protein ~ Yield
Step and fraction (ml) (X 10-~) (mg/ml) rag) (rag) (%)
1. Muscle extract 2250 4.0 47 38 105,750 100

2. First ammonium sulfate
Supernatant solution 2650 0.27 34 3 90,100 7
Precipitate 280 3.7 77.5 170 21,700 95
3. Second ammonium sulfate
Supernatant solution 500 3.65 26.5 276 13,250 90
Precipitate -- O. 15 -- -- -- 4
4. Third ammonium sulfate
Supernatant solution -- 0.05 -- -- -- 2
Precipitate 175 3.49 60.6 328 10,605 87
5. First crystallization 200 3.14 1.75 9,000 350 78
6. Recrystallization 50 2.90 1.84 31,500 92 72
a Assuming an extinction coefficient of 0.510.
activity of 31,500 units per milligram after two recrystallizations. Re-

peated recrystallization does not increase the specific activity. The
enzyme behaves as a homogeneous protein on ultracentrifugation (sedi-
mentation velocity, and sedimentation equilibrium 6) and on starch gel
electrophoresis.
Stability. Crystalline fumarase has been observed to be stable for
periods as long as two years when stored in a crystalline suspension at
0.50-0.55 saturated ammonium sulfate at 5 ° . In solution, the stability of
the enzyme is affected markedly by various anions. In the presence of
10 m M phosphate, pH 7.3, sterilized, dilute solutions of fumarase have
been kep~ for 3 weeks at both 5 ° and 25 ° with no loss in activity. 7
Fumarase is quite unstable in the absence of phosphate, s Solutions of
the enzyme are partially inactivated by freezing and thawing.
Properties
Physical Properties. The S~o,~ of fumarase extrapolated to zero con-
centration, is 9.09 X 1013 seconds. The diffusion coefficient determined by
Cecil and 0gston 9 is 4.05 X 10 -7 cm 2 per second at a concentration of 7
mg/ml. The molecular weight calculated from this diffusion coefficient
and the $2o,~ of fumarase at 7 mg/ml (8.5 X 10-13 seconds) is 197,000.
Sedimentation equilibrium analysis of the native enzyme at 50 m M
potassium phosphate buffer, pH 7.3 containing 1 ~ sucrose and 1 m M
2-mercaptoethanol, gave a value for the weight average molecular weight
of 194,000___ 2000. 6 The partial specific volume, calculated from the
amino acid composition is 0.738 ml/g2
The isoelectric point of fumarase is dependent on the type of anions
present. At an ionic strength of 0.1, the isoelectric point in Tris-acetate
was calculated to be 7.35 ± 0.05. In 10 m M Tris-acetate containing
9 m M sodium chloride, the enzyme appears to be isoelectric at pH
6.6 ± 0.2. The isoelectric point in phosphate buffer at an ionic strength
of 0.21 was estimated to be about 5.3. TM The isoionic point of fumarase,
determined by measuring the pH of a solution that was deionized on a
mixed-bed, ion-exchange resin was shown to be 7.95 ± 0.05. 3 The fact
that this value is higher than the isoeleetric point in buffers containing
acetate, chloride, or phosphate ions suggests that binding of anions to
fumarase occurs in dilute buffer systems at a neutral pH.
' L. Kanarek, C. Marler, R. A. Bradshaw, R. E. Fellows, and R. L. Hill, J. Biol.

Chem. 239, 4207 (1964).
7R. A. Bradshaw and R. L. Hill, unpublished observations (1967).
8j. Teipel and R. L. Hill, unpublished observations (1967).
°R. Cecil and A. G. Ogston, Biochem. J. 57, 494 (1952).
1°N. Shavit, R. G. Wolfe, and R. A. Alberty, J. Biol. Chem. 233, 1382 (1958).
[17] FUMAR~.SE 97
The optical rotatory dispersion of fumarase has been measured with

a Rudolf spectropolarimeter, between 578 and 313 m~. ~ With aid of the
Moffitt equation, 11 in which a value of 212 m~ was assumed for Xo, the
parameters a~ and bo were found to be --81 and --311, respectively. If
bo may be considered to reflect the helical content of a protein, then
fumarase may be judged to contain about 50% of its amino acid residues
in helical form.
Specificity. The enzyme apparently possesses an absolute specificity
for the substrates fumarate and L-malate. A large variety of compounds
structurally related to fumarate or L-malate remain unaltered by the
enzyme.1
The E fleet o / p H and Buf]er Composition on Activity. The effect of
pH on the enzymatic activity of fumarase has been discussed in detail by
Massey 1 and by Alberty. 12 The enzyme shows a bell-shaped dependence
on pH, which suggests that fumarase possesses both an acidic and basic
functional group in its active site. The pKa of these groups indicates that
they may be 2 imidazole groups or an imidazole and a carboxyl group. 1~
The effect of buffer composition on enzymatic activity is complicated
by binding of anions. Monovalent anions such as iodide, bromide, and
chloride have been observed to inhibit, although polyvalent anions, such
as phosphate and sulfate, show activation in dilute concentrations.
At high concentrations these ions also inhibit. To avoid the complex
problems associated with these polyvalent anions, Alberty and co-
workers 1~,1~,15 have studied the kinetic properties of fumarase in Tris-
acetate and have consequently been able to elucidate many of the
features of the fumarase mechanism. It should be emphasized that the
presence of anions in solutions of the enzyme may have a large effect on
the activity and should be regulated closely at all times.
Equilibrium Constant. The equilibrium constant for the L-malate-
fumarate reaction has been shown to be
L-malate
K ~ = fumarate = 4.4 at 25°, 0.01 ionic strength
This value is independent of hydrogen ion concentration at pH's greater

than 6.5. At pH values less than 6.5 the equilibrium ratio varies with
charge as the result of the differences in the ionization constants of
11w. Moffitt, J. Chem. Phys. 25, 467 (1956).
~2R. A. Alberty, in "The Enzymes" (P. D. Boyer, H. Lardy, and K. Myrblick, eds.),
~ D. A. Brant, L. B. Barnett, and R. A. Alberty, J. Am. Chem. Soc. 85, 2204 (1963).
~'C. Frieden and R. A. Alberty, J. Biol. Chem. 212, 859 (1955).
uC. Frieden, R. G. Wolfe, and R. A. Alberty, J. Am. Chem. ~oc. 79, 1523 (1957).
fumaric and malic acid. 16 The AH of the reaction has been reported to be
between --3560 and --3960 calories per mole. 16-1s The free energy
accompanying the reaction at 25 ° has been calculated to be --880
calories per mole. 19
S u b u n i t S t r u c t u r e . Several lines of evidence suggest that fumarase
is composed of 4 identical polypeptide chains associated by noncovalent
forces2 The molecular weight of the native enzyme, 194,000, is reduced
to 48,500 in 8 M urea or 6 M guanidine hydrochloride.6 The addition of
0.1 M mercaptoethanol does not reduce this value further. Amino terminal
end-group analysis indicates that the enzyme possesses 3.6 residues of
alanine per molecule. Analysis of the soluble tryptic peptides by the
peptide mapping technique shows about one-fourth the number of
tryptic peptides anticipated from the total lysine plus arginine content
of the enzyme2
T h i o l Groups. Amino acid analysis shows that fumarase contains 12
residues of half-cysteine measured as cysteic acid2 p-Chloromercuri-
benzoate titration, in the presence of 2 M urea, reveals 12 residues of
thiol groups per molecule. These results indicate that fumarase is devoid
of disulfide bonds. Reaction of the native enzyme with thiol reagents
shows that the enzyme is irreversibly inactivated by a variety of sulf-
hydryl reagents. 2°,-'1 The rate of inactivation is markedly dependent
on the nature and concentration of the thiol reagent. In the presence
of a 50% excess of p-chloromercuribenzoate, 40-48 hours is required for
complete conversion of the thiol groups to the corresponding mercaptide
derivative. The rate of inactivation of the enzyme is directly proportional
to the number of thiol groups reacted. Furthermore, with the exception
of p-chloromercuribenzoate, all reagents which result in the formation
of a derivative with a formal positive or negative charge on the thiol
group cause dissociation of the enzyme into dimers. These results, along
with a number of other lines of evidence,21 suggest that the thiol groups
of fumarase are not associated with structures in the active site of the
enzyme but are buried in the interior of the molecule in hydrophobic
environments. As a result of this protected position of the thiol groups,
it is not necessary to maintain a dilute concentration of mercaptan in
solutions of this enzyme. Under conditions in which the molecule is
denatured, i.e., 8 M urea or 6 M guanidine hydrochloride, dilute con-
R. M. Bock and R. A. Alberty, J. Am. Chem. Soc. 75, 1921 (1953).

1~V. Massey, Biochem. J. 53, 72 (1953).
~SE. M. Scott and R. Powell, J. Am. Chem. Soc. 70, 1104 (1948).
I~K. Burton and M. A. Krebs, Biochem. J. 54, 94 (1953).
~°R. L. Hill and L. Kanarek, Brookhaven Symp. Biol. 17, 80 (1964).
•.1G. W. Robinson, R. A. Bradshaw, L. Kanarek, and R. L. Hill, J. Biol. Chem.
242, 2709 (1967).
[18] MITOCHONDRIAL L-MALA.TE DEHYDROGENASE OF BEEF HEART (~)9
centrations of mercaptoethanol or similar mercaptan compound must

be added to the solution to avoid oxidation of these residues.
Inhibitors
Competitive and Noncompetitive Inhibitors. Besides the complex
activation-inhibition behavior of "mions and sub~trates with fumarase, a
number of compounds have been demonstrated to act as competitive
inhibitors. 1 Adipate, succinate, glutarate, malonate, tartrate, mesaeonate,
maleate, D-malate, citrate, and trans-aeonitate behave in this manner.
Thiocyanate and acetylene dicarboxylate were the only compounds to
show noncompetitive inhibition. Interestingly enough, acetate, butyrate,
crotonate, L-aspartate, acetoacetate, and the mono and dimethyl esters
of fumarate do not appear to inhibit the enzyme, although Jacobson -~2
has reported that crotonate acts as a weak competitive inhibitor. Frieden 23
has reported m-tartrate to be the best competitive inhibitor investigated.
In addition to a large number of sulfhydryl reagents, which act as
irreversible inhibitors, iodoacetate 2. and ),-bromocrotonate~-~ also irre-
versibly inactivate the enzyme. Analysis of the site of modification of
these reagents indicates that histidine, methionine, and possibly lysine
are involved. These modifications, which are protected by substrate and
competitive inhibitor, may well involve active site residues.
"K. Jacobson, Enzymologia 16, 113 (1953).
2~C. Frieden, P h . D . Thesis, University of Wisconsin, Madison, Wisconsin, 1956.
'~ R. A. Bradshaw, G. M. Hass, and R. L. Hill, manuscript in preparation (1967).
~G. W. Robinson and R. L. Hill, unpublished observations (1967).
[18] Mitochondrial L-Malate Dehydrogenase of Beef Heart

[EC 1.1.1.37 L-Malate : NAD oxidoreductase]
By SASHA ENGLARD a n d LEWIS SIEGEL
L-Malate + NAD + ~ oxaloacetate + NADH + H +
Assay Method
Principle. Mitochondrial malate dehydrogenase activity is measured
spectrophotometrically by the increase in absorption at 340 m~ due to
NAD + reduction in presence of L-malate.
Reagents
Glycine-NaOH, 0.12 M, pH 10.0
L-Malic acid, 0.85 M neutralized to pH 7.5 with NaOH
NAD ÷, 37.5 mM, adjusted to pH 6.5
[18] MITOCHONDRIAL L-MALA.TE DEHYDROGENASE OF BEEF HEART (~)9
centrations of mercaptoethanol or similar mercaptan compound must

be added to the solution to avoid oxidation of these residues.
Inhibitors
Competitive and Noncompetitive Inhibitors. Besides the complex
activation-inhibition behavior of "mions and sub~trates with fumarase, a
number of compounds have been demonstrated to act as competitive
inhibitors. 1 Adipate, succinate, glutarate, malonate, tartrate, mesaeonate,
maleate, D-malate, citrate, and trans-aeonitate behave in this manner.
Thiocyanate and acetylene dicarboxylate were the only compounds to
show noncompetitive inhibition. Interestingly enough, acetate, butyrate,
crotonate, L-aspartate, acetoacetate, and the mono and dimethyl esters
of fumarate do not appear to inhibit the enzyme, although Jacobson -~2
has reported that crotonate acts as a weak competitive inhibitor. Frieden 23
has reported m-tartrate to be the best competitive inhibitor investigated.
In addition to a large number of sulfhydryl reagents, which act as
irreversible inhibitors, iodoacetate 2. and ),-bromocrotonate~-~ also irre-
versibly inactivate the enzyme. Analysis of the site of modification of
these reagents indicates that histidine, methionine, and possibly lysine
are involved. These modifications, which are protected by substrate and
competitive inhibitor, may well involve active site residues.
"K. Jacobson, Enzymologia 16, 113 (1953).
2~C. Frieden, P h . D . Thesis, University of Wisconsin, Madison, Wisconsin, 1956.
'~ R. A. Bradshaw, G. M. Hass, and R. L. Hill, manuscript in preparation (1967).
~G. W. Robinson and R. L. Hill, unpublished observations (1967).
[18] Mitochondrial L-Malate Dehydrogenase of Beef Heart

[EC 1.1.1.37 L-Malate : NAD oxidoreductase]
By SASHA ENGLARD a n d LEWIS SIEGEL
L-Malate + NAD + ~ oxaloacetate + NADH + H +
Assay Method
Principle. Mitochondrial malate dehydrogenase activity is measured
spectrophotometrically by the increase in absorption at 340 m~ due to
NAD + reduction in presence of L-malate.
Reagents
Glycine-NaOH, 0.12 M, pH 10.0
L-Malic acid, 0.85 M neutralized to pH 7.5 with NaOH
NAD ÷, 37.5 mM, adjusted to pH 6.5
Procedure. Into a 1 em light path cuvette, pipette 2.5 ml of glyeinc-
NaOH, 0.3 ml of L-malate, and 0.2 ml of NAD ÷. The reaction, carried out
at 28-30 °, is initiated by addition of 10-50 ~l of an enzyme solution
properly diluted with 0.1 M potassium phosphate buffer, pH 7.4. Dilu-
tions of enzyme are chosen to give a change in absorbance of 0.015-0.060
per minute. The rate of N A D H formation is conveniently followed at
340 m~ with a Gilford model 2000 multiple sample absorbance recorder.
Units. A unit of activity is defined as that amount of enzyme required
to convert 1 micromole of NAD ÷ to N A D H per minute as determined
from the initial rates of absorbancy change at 340 m~ under the assay
conditions just described. Specific activity is defined as the number of
units per milligram of protein per milliliter. Protein is measured by the
method of Lowry et al.} crystalline bovine serum albumin being used
as a standard.
Various methods and modifications of existing procedures have been
reported for the purification of mammalian heart muscle mitochondria]
malate dehydrogenases. 2-9 The procedure described here for the purifica-
tion of beef heart mitochondrial malate dehydrogenase I°,11 includes a
number of steps described initially by Straub 2 as modified by Ochoa 8
for the preparation of pig heart malate dehydrogenase. The precautions
outlined by Pfleiderer and Hohnholz 6 for the steps requiring calcium
chloride and ethanol have been incorporated. All operations are per-
formed at 0-5 ° unless otherwise specified.
Step I. Preparation of Acetone-Dried Powder. Fresh beef hearts kept
on ice are dissected free of gross fat and connective tissue, diced, and
passed through a mechanical meat grinder. One kilogram of mince is
suspended in 5 liters of ice cold 0.25 M sucrose buffered at pH 7.6 with
10 mM triethanolamine, and the mixture is stirred mechanically for 15
! O. H. Lowry, N. J. Rosebrough, A. G. Farr, and R. J. Randall, J. Biol. Chem. 193,

265 (1951).
2F. B. Straub, Z. Physiol. Chem. 275, 63 (1942).
'See Vol. 1 [123].
' R. G. Wolfe and J. B. Neilands, J. Biol. Chem. 221, 61 (1956).
s D. D. Davies and E. Kun, Biochem. J. 66, 307 (1957).
* G. Pfleiderer and E. Hohnholz, Biochem. Z. 331, 245 (1959).
VB. K. Joyce and S. Grisolia, J. Biol. Chem. 236, 725 (1961).
'F. C. Grimm and D. G. Doherty, J. Biol. Chem. 236, 1980 (1961).
* C. J. R. Thorne, Biochim. Biophys. Acta $9, 624 (1962).
"L. Siegel, Ph.D. Thesis, Albert Einstein College of Medicine, Yeshiva University,
New York, 1962.
,1L. Siegel and S. Englard, Biochim. Biophys. Aeta 54, 67 (1961).
[18] MITOCHONDRIALL-MALATE DEHYDROGENASE OF BEEF HEART 101
minutes. The suspension is passed through several layers of cheesecloth,

and the filtrate may be saved for preparation of extramitochondrial
malate dehydrogenase. The residue is washed two more times with 7
liters of ice cold distilled water, decanted through cheesecloth, and
squeezed with a Pexton hand press? 2 The washed tissue mince is next
stirred for 15 minutes with three volumes of acetone (precooled to
--10 °) and then filtered through cheesecloth with a Biichner funnel. The
residue is treated with a second portion of chilled acetone, collected on
a Biichner funnel with suction, and finally powdered and dried by
manual rubbing at room temperature. The yield of acetone-dried powder
from 1 kg of beef heart mince ranges from 160 to 200 g.
Step 2. Extraction of Acetone-Dried Powder. One hundred grams of
acetone-dried powder are suspended in 1000 ml of 0.1 M potassium
phosphate buffer, pH 7.4, and stirred vigorously at room temperature
(24-27 ° ) for 30 minutes. The suspension is centrifuged in the cold at
18,000 g for 20 minutes and the residue is reextracted as above with
600 ml of the same buffer. The first and second extracts are combined.
Step 3. Acidification with Calcium Chloride and Neutralization with
Trisodium Phosphate. For each 100 ml of combined extract, 10 ml of
0.5M sodium L-malate, pH 7.0-7.1, is added. The pH of the resulting
solution (7.3) is then lowered to 5.6 by addition, with mechanical stirring,
of a 15% solution of calcium chloride ((11.6 ml/100 ml of solution).
Hyflo Supercel (Johns-Manville) is added (1 g/100 ml of solution), and
the suspension is filtered rapidly with suction on a Biichner funnel (the
use of Reeve-Angel filter paper No. 202 is recommended). The precipitate
is discarded. The filtrate is neutralized to pH 7.4--7.5 by addition of 10%
trisodium phosphate. 12 H20 (21.5 ml/100 ml of solution). Hyflo Supercel
is added, the suspension is filtered by gravity through Eaton-Dikeman
flutted filter paper No. 192, and the precipitate is discarded.
Step 4. First Ammonium Sulfate Fractionation. To the clear orange-
red filtrate from step 3 is added solid ammonium sulfate to 49% satura-
tion (34.6 g/100 ml of solution). The salt is added over a period of 45
minutes with constant stirring. Equilibration is continued for an addi-
tional 30 minutes after the last addition of salt, the suspension is centri-
fuged at 18,000 g for 30 minutes, and the precipitate is discarded. The
clear supernatant solution is then brought to 70% saturation by the
further slow addition of solid ammonium sulfate (14.8 g/100 ml of
solution) over a period of 60 minutes. Mechanical stirring is continued
for an additional 15 minutes, the suspension is centrifuged as before,
and the supernatant solution is discarded. The residue is dissolved in
~Supplied by the Lee Engineering Company, Mihwmkee, Wisconsin.

0.1 M potassium phosphate buffer, pH 7.4, and yields a deep red solution
which is dialyzed against a 20% ammonium sulfate solution adjusted
previously to pH 7.4 with dilute ammonium hydroxide. Dialysis is
carried out for 18-24 hours with several changes of the ammonium
sulfate solution.
Step 5. Ethanol Fractionation. An equal volume of ethanol is added
dropwise over a period of 60 minutes to the dialyzed solution obtained
from step 4. Mechanical stirring is continued for an additional 30
minutes, and the bulky precipitate is then removed by centrifugation
at 18,000 g for 60 minutes at --5 °. The orange-yellow supernatant is
treated as before with an additional half volume of ethanol, and then
mechanical stirring is continued for 30 minutes. The precipitate is
collected by centrifugation at 18,000 g for 60 minutes. The fraction
precipitating between 50 and 67% ethanol is suspended in 20 mM potas-
sium phosphate buffer, pH 7.4, and dialyzed against this buffer for 18
hours with several changes of the dialyzing medium. After dialysis, the
insoluble material is removed by centrifugation at 32,000 g for 45
minutes. Occasionally, an appreciable loss of enzymatic activity occurs
during the first ethanol fractionation. Lowering of the temperature for
the ethanol fractionation from 0 ° to --10 ° does not prevent the loss
of activity.
Step 6. Second Ammonium Sul]ate Precipitation. The pale yellow
solution obtained from step 5 after dialysis and centrifugation is brought
to 70% saturation with ammonium sulfate (49.4 g/100 ml of solution).
The solid salt is added over a period of 1 hour. The suspension is allowed
to equilibrate with continual mechanical stirring for at least 45 minutes,
and the precipitate is then collected by centrifugation at 18,000 g for 30
minutes. The supernatant contains very little enzymatic activity and
is discarded. The residue is dissolved in 0.1 M potassium phosphate
buffer, pH 7.4, and dialyzed for 18-24 hours against 25% ammonium
sulfate dissolved in 0.2M potassium phosphate buffer, pH 7.4. The
dialyzing medium is changed twice during the period of dialysis.
Step 7. Heat Treatment. The dialyzed solution from step 6 is heated
rapidly to 60 ° in a boiling water bath while stirring is maintained
efficiently. The enzyme solution is kept at this temperature for 2 minutes
and then cooled rapidly to 0 ° in an ice bath. The heat-treated solution
is centrifuged at 32,000 g for 30 minutes, and the clear supernatant is
dialyzed exhaustively against 5 mM potassium phosphate buffer, pH 7.0.
Step 8a. Starch Block Electrophoresis. The supernatant from step 7
is ultrafiltered, and the pale yellow concentrate is dialyzed for 16 hours
against a solution of 20 mM sodium citrate and 1 mM EDTA, pH 6.1,
which is replaced several times. Electrophoresis is conducted in the same
[18] MITOCItONDRIAL L-MALATE DEHYDROGENASE OF BEEF HEART 103
buffer (citrate-EDTA) as follows: 500 g of Mallinkrodt potato starch

is washed successively with 750 ml, 500 ml, and 500 ml portions of buffer.
Each washing is carried out for 15 minutes with mechanical stirring.
After each washing, the suspension is filtered with suction on a Biiehner
funnel using Reeve-Angel paper No. 202. Tile starch block is prepared
by suspending the washed starch in about 200 ml of buffer and stirring
vigorously until a uniformly thick paste is obtained. This is poured into
a mold formed by folded linen towels oll a Lucite platform. After the
starch block is freed of excess buffer, the towels are removed and the
block is trimmed to 25 X 16 era. (1.5 cm in height). A ell'tnnel is formed
in the center of the block by cutting out a 0.5 cm strip parallel to
the direction of eleetrophoretie migration. A paste, formed by adding
4.0 ml of dialyzed enzyme to dry starch, is poured into the channel.
Electrophoresis is conducted for 22 hours at 0 ° using 300 V and 28 ma.
At conclusion of eleetrophorcsis, excess moisture from the starch block
is removed by means of a dry linen towel. Strips of 0.5 em are cut from
the block and eluted with 0.1 M potassium phosphate buffer, pH 7.4. Th(.
fraction containing the highest specific activity of malate dehydrogenase
is generally found in the region 2.5-3.5 cm from tile origin toward the
cathode. This fraction, which accounts for 80-85% of the total activity
recovered from the starch, is concentrated by ultrafiltration and stored as
a suspension in 80% saturated ammonium sulfate. Preparations with
specific activities of approximately 343 units per milligram of protein
and representing overall recoveries of 25% are routinely obtained.
Step 8b. Ion-Exchange Chromatography. This method, a less tedious
procedure, represents a convenient alternative to the starch block
eleetrophoretie separation described in step 8a. The dialyzed enzyme
solution from step 7 is placed on a AG-50W-X8 (BioRad, 200.400 mesh)
column, 4 X 45 cm. Before use, the ion-exchange resin is washed succes-
sively with 0.5 M hydrochloric acid, water, 0.5 21I potassium hydroxide,
water, 0.511I potassium phosphate buffer, pH 7.0, water and finally is
equilibrated with 5 mM potassium phosphate buffer, pH 7.0. The column
is washed with the 5 mM potassium phosphate buffer until protein can-
not be detected in the efttuent. Tile column is then connected to a mixing
flask containing 160 ml of 5 mM potassium phosphate buffer, pH 7.0; this
in turn is connected to a reservoir containing 0.3 M potassium phosphate
buffer, pH 7.0. A rapidly spinning magnetic bar ill the mixing flask
ensures thorough mixing of the dilute buffer with the more concentrated
buffer entering from the reservoir. A uniform and gradual increase in
potassium phosphate concentration of solution entering the ion-exchange
resin bed is thus achieved. Eluate is collected at a rate of 50-60 ml per
hour, and after the gradient is started, protein with malate dehydrogenase
104 REACTIONS ON THE CYCI~E [18]
activity begins to emerge at an effluent volume of approximately 200 m]

at a concentration of about 20 m M potassium phosphate. The active
fractions are pooled, and the solution is then brought to 80% saturation
with a m m o n i u m sulfate and stored in the cold.
A s u m m a r y of the purification is given in the table.
PURIFICATION OF MITOCHONDRIAL BEEF HEART

MUSCLE MALATE DEHYDROGENASE
Total Total Specific
Volume activity protein activity
Fraction (ml) (units) a (rag) (units/mg)
I. Acetone powder extract 1350 78,391 6380 12.2

II. Neutralized supernatant after 1550 38,228 2680 14.3
calcium chloride treatment
III. Dialyzed 49-70% ammonium 70 34,840 1290 27.0
sulfate fraction
IV. Dialyzed and centrifuged 40 27,098 264 103
50-67% ethanol fraction
V. Supernatant after heat 20 21,050 106 198
treatment
VI. AG-50W-X8 chromatography -- 15,969 49 325
a One unit is defined as amount of enzyme required to convert 1 micromole of NAD +
to NADH per minute under conditions specified.
Properties ~o,~
Kinetic Properties and Catalytic Specificity. At p H 6.7 in potassium
phosphate buffer, the rate of oxaloacetate reduction reaches a m a x i m u m
at a substrate concentration of approximately 0.13 m M ; inhibition by
oxaloacetate is already significant at a level of 0.26 mM. The K,, as
determined in the noninhibitory range of oxaloacetate concentration is 34
(at a 0.12 m M N A D H concentration). The K,, for N A D H , deter-
mined at a constant concentration of oxaloacetate of 0.13 m M at p H 6.7,
is 52 pit/. An anomalous accelerating effect of L-malate on the rate of
N A D ÷ reduction occurs at p H 10.0 in g l y c i n e - N a O H buffer. Under these
conditions of assay, at a constant concentration of N A D ÷ of 2.9 raM,
the Km for L-malate over a range of 0.15 to 7.5 m M is 0.37 mM. Over a
concentration range of 7.5 m M to 0.15 M, L-malate, a K,n of 1.8 m M is
obtained. In Tris buffer at p H 8.4, and at a constant N A D + concentra-
tion of 0.54 mM, a normal saturation curve is obtained for L-malate
within the range of 75 ~ M to 15 m M ; the K,, for L-malate under these
conditions is 0.25 mM. The K,, for N A D ÷ as determined with 16 m M
L-malate at p H 8.4 is 99 gM.
The oxidation of N A D H by mesoxalate at p H 6.7 is 8.5% of the rate
[18] MITOCHONDRIA.L L-MALATE DEHYDROGENASE OF BEEF HEART 105
of oxidation by optimum concentrations of oxaloacetate. Activity of the

enzyme toward a-kctoglutarate is detectable but slight, whereas a-keto-
butyrate and p y r u v a t e are not reduced at all. Reduction of N A D + at
p H 10.0 by either D-(--)-tartrate or mesotartrate is significant, although
the rate is less than 1% of t h a t observed with L-malate. Oxidation of
tartronate by N A D ~ is barely detectable; t . - ( ~ ) - t a r t r a t e is not oxidized
at all. The rate of oxidation of N A D P H by oxaloacetate at p H 7.5 in
glycylglycine buffer is approximately 1/125 that observed with N A D H .
However, the presence of 50 m M potassium phosphate buffer stimulates
the oxidation of N A D H by 2.6-fold 13 but inhibits the oxidation of
N A D P H by 44%. Finally, although reduction of N A D ÷ by L-malate at
p H 10.0 is measured readily, reduction of N A D P ÷ under similar condi-
tions is detectable only at very high concentrations of nucleotide co-
enzyme.
Molecular Properties. The enzyme appears to be homogeneous as
determined by ultracentrifugation studies and electrophoretic analysis
over a wide p H range. 11 The maximum electrophoretic mobility for the
enzyme (--2.20 X 10 -~ cm ~ sec -1 V -1) is observed at p H 7.4. Interpolation
of the curve of electrophoretic mobility as a function of p H yields an
approximate isoelectric point of 5.5-5.6. The enzyme has a sedimentation
velocity constant of 4.3 X 10 -13 sec at 20 ° and a diffusion constant of
6.45 X 10 -7 cm 2 scc -1, corresponding to a calculated molecular weight of
62,000 assuming a partial specific volume of 0.74 ml g-1. The molecular
constants agree closely with those obtained by G r i m m and D o h e r t y 8
for the same enzyme isolated from beef heart by another method of puri-
fication. The amino acid composition of the enzyme has been deter-
mined. ~ The close correspondence between the values of sulfhydryl
contents (12 residues per mole) calculated from p-chloromercuribenzoate
~The activity of the beef heart extramitochondrial malate dehydrogenase is un-

affected by addition of phosphate.'~ The oxaloacetate-sensitive malate dehydro-
genase from acetone-dried powders of whole pig heart is also stimulated by
phosphate as well as by sulfate, arsenate, maleate, or EDTA; u the effect appears
to be due to increased ionic strength of the assay medium. The latter observation
contradicts an earlier report s but agrees with the results of Joyce and Grisolia. '
Malate dehydrogenase obtained by direct extraction of acetone-dried powders of
beef heart mitochondria, and purified by the procedure of Davies and Kun, 5
also shows increased activity when assayed in glycylglycine buffer in the presence
of potassium phosphate.~ It thus appears that this stimulation of activity by
added salts is also characteristic of those enzymes which behave kinetically like
the malate dehydrogenases of mitochondrial origin as indicated by their inhibition
at elevated concentrations of oxaloacetate.
~ L. Siegel and S. Englard, Biochim. Biophys. Acta 64, 101 (1962).
'5S. Englard, L. Siegel, and H. H. Breiger, Biochem. Biophys. Res. Commun. 3,
323 (1960).
and N-ethylmaleimide titrations on the one hand with values for cysteic
acid determined after performic acid oxidation indicates the absence
of disulfide linkages in the protein. Although the sulfhydryl groups of
the native enzyme react very sluggishly with p-chloromercuribenzoate,
on prolonged incubation with this reagent one can titrate the full comple-
ment of these groups. Losses in enzymatic activity become evident after
the enzyme has reacted with 3 equivalents of p-chlormercuribenzoate.
Although the mitochondrial malate dehydrogenases from several species
have been reported to contain no tryptophan, TM the corresponding
enzyme from beef heart muscle contains one residue of tryptophan per
mole. 14 Based on the tryptophan content, a minimum molecular weight
of 65,000 may be calculated; this compares favorably with the value of
62,000 determined by physical methods.
'~G. B. Kitto and N. O. Kaplan, Biochemistry 5, 3966 (1966).
[19] I n t r a - a n d E x t r a m i t o c h o n d r i a l M a l a t e D e h y d r o g e n a s e s
from Chicken and Tuna Heart
[EC 1.1.1.37 L-Malate:NADoxidoreductase]
By G. BARRIEKITTO
L-Malate + NAD ~- oxaloacetate + NADH + H +
A s s a y Method
Principle
Malate dehydrogenase activity can conveniently be determined, in
either the forward or reverse reaction, by measuring the initial rates of
reduction or oxidation of NAD and NADH, respectively. The measure-
ment is carried out in a spectrophotometer equipped with a constant-
temperature cell holder maintained at 25 °, at wavelength 340 m/~, using
Pyrex or silica cuvettes of 10 mm light path.
Malate Oxidation 1
Reagents
Sodium glycinate buffer, 90 mM, pH 10.0
Sodium L-malateT- 1.0 M
NAD, 2 12.3 mM
~C. J. R. Thorne, L. I. Grossman, and N. O. Kaplan, Biochim. Biophys. Acta 73,
193 (1963).
Malate and NAD solutions are stored frozen.
and N-ethylmaleimide titrations on the one hand with values for cysteic
acid determined after performic acid oxidation indicates the absence
of disulfide linkages in the protein. Although the sulfhydryl groups of
the native enzyme react very sluggishly with p-chloromercuribenzoate,
on prolonged incubation with this reagent one can titrate the full comple-
ment of these groups. Losses in enzymatic activity become evident after
the enzyme has reacted with 3 equivalents of p-chlormercuribenzoate.
Although the mitochondrial malate dehydrogenases from several species
have been reported to contain no tryptophan, TM the corresponding
enzyme from beef heart muscle contains one residue of tryptophan per
mole. 14 Based on the tryptophan content, a minimum molecular weight
of 65,000 may be calculated; this compares favorably with the value of
62,000 determined by physical methods.
'~G. B. Kitto and N. O. Kaplan, Biochemistry 5, 3966 (1966).
[19] I n t r a - a n d E x t r a m i t o c h o n d r i a l M a l a t e D e h y d r o g e n a s e s
from Chicken and Tuna Heart
[EC 1.1.1.37 L-Malate:NADoxidoreductase]
By G. BARRIEKITTO
L-Malate + NAD ~- oxaloacetate + NADH + H +
A s s a y Method
Principle
Malate dehydrogenase activity can conveniently be determined, in
either the forward or reverse reaction, by measuring the initial rates of
reduction or oxidation of NAD and NADH, respectively. The measure-
ment is carried out in a spectrophotometer equipped with a constant-
temperature cell holder maintained at 25 °, at wavelength 340 m/~, using
Pyrex or silica cuvettes of 10 mm light path.
Malate Oxidation 1
Reagents
Sodium glycinate buffer, 90 mM, pH 10.0
Sodium L-malateT- 1.0 M
NAD, 2 12.3 mM
~C. J. R. Thorne, L. I. Grossman, and N. O. Kaplan, Biochim. Biophys. Acta 73,
193 (1963).
Malate and NAD solutions are stored frozen.
[19] MALATE DEHYDROGENASE FROM CHICKEN AND TUNA HEART 107
Procedure. The reaction mixture contains 0.1 ml of L-malate, 0.1 ml of

NAD, enzyme and buffer to a final volume of 3.0 ml. The reaction is
started by addition of either malate or enzyme. Readings of the optical
density at 340 mt~ are made, against a blank containing all the assay com-
ponents except NAD, at intervals of 15 seconds for 3 minutes. The initial
rate is used to calculate enzymatic "lctivity. Tile amount of enzyme used
in ttle test is adjusted so that tile initial itlcrease in optical density is not
greater than 0.04 per miaute.
Oxaloacetate Reduction
Reagents
Oxaloacetate,3 20 mM
NADH, ~ 14.3 mM
Procedure. The reaction mixture contains 0.03 ml of NADH, 0.05 ml
of oxaloacetate, enzyme and buffer to a final volume of 3.0 m!. The
reaction is started by addition of either oxaloacetate or enzyme. Readings
of optical density at 340 m~ are made, against a blank containing all
components of the assay mixture except NADH, every 15 seconds for 3
minutes. Enzyme activity is calculated from the initial rate of oxidation
of NADH. The amount of enzyme used is adjusted to give a decrease in
the optical density of approximately 0.04 per minute.
Units. A unit of malate dehydrogenase activity is defined as the
amount of enzyme required to oxidize or reduce 1 micromole of coenzyme
per minute under the conditions described above.
Purification Procedures
Intra- and extramitoehondrial malate dehydrogenases have been
prepared in this laboratory both by prior isolation of mitochondrial and
supernatant fractions and from total tissue extracts, with later separation
of the two types of enzyme. The differentmeans of preparation did not
lead to any detectable differences in the properties of the enzymes.
Because frozen tissues are more generally available than fresh material,
the procedure reported here is for the initial extraction of both extra- and
intramitochondrial enzyme from frozen hearts.
A . CHICKEN HEART MALATE DEHYDROGENASES 4'5
The following procedure is that used for the purification of chicken
malate dehydrogenases. The same method has been used for the purifica-
• Oxaloacetate and N A D H solutions are prepared fresh daily and kept at 0%
• G. B. K i t t o and N. 0 . Kaplan, Biochemistry 5~ 3966 (1966).
• G. B. Kitto, Biochim. Biophys. Acta 139, 16 (1967)
tion of malate dehydrogenases from a single ostrich (Struthio carnelus)

healS, 5 except that all procedures were scaled down in proportion to the
smaller amount of starting material available. This should not be taken
to imply that all avian malate dehydrogenases may be similarly prepared.
The separation procedures depend, in large measure, upon charge dif-
ferences between the intra- and extramitochondrial malate dehydro-
genases, and the chicken and ostrich enzymes differ little in this respect
as shown by starch gel electrophoresis. 6 An examination of the starch gel
electrophoretic patterns of malate dehydrogenases from a number of
galliform birds ~,7 suggests that the enzyme from most species of this
order can be purified by the procedure given below. Species from other
avian orders can show considerable difference in the charges on the intra-
and extramitochondrial malate dehydrogenases compared with the
chicken enzymes,~,7 and corresponding modifications of the purification
procedure will be required.
Step 1. Crude Extract. Frozen chicken hearts (20 pounds) stripped of
fat are ground three times in a mechanical meat grinder. The minced
tissue is then suspended in 10 liters of 5 mM potassium phosphate-1 mM
EDTA-1 mM fl-mercaptoethanol, pH 7.5, for 1 hour at 4 ° with occa-
sional stirring. The suspension is then homogenized in batches in a large
Waring blendor (30 seconds per batch) and allowed to stand in the cold
with continuous stirring for an additional 2 hours. The homogenate is
filtered through several layers of cheesecloth, and the solid residue is
placed in 5 liters of the above buffer, and stirred in the cold for 1 hour.
After the extracted homogenate is again passed through cheesecloth, the
solid residue is discarded and the two filtrates are combined. The com-
bined filtrates are clarified by centrifugation for 30 minutes at 1300 g at
4°; the residues are discarded. Starch gel electrophoresis at pH 7.0 is used
to determine that both intra- and extramitochondrial enzymes are
extracted by this procedure.
Step 2. First Ammonium Sulfate Precipitation. Solid ammonium sul-
fate is added to the combined filtrates to give 40% saturation. 8 In tbis
and other ammonium sulfate precipitations the pH is maintained at 7.5
by addition of ammonium hydroxide. The suspension is left at 4 ° for
several hours and then filtered overnight on fluted filter papers. The
precipitate is discarded. Additional solid ammonium sulfate is added to
the filtrate to give 85% saturation. The suspension is left at 4 ° for 2
hours and then centrifuged at 1300 g for 30 minutes. The precipitate is
eG. B. Kitto, unpublished observations (1968).
G. B. Kitto and A. C. Wilson, Science 153, 1408 (1966).
aPercentage saturation is based on Table I in A. A. Green and W. L. Hughes, Vol.
I, p. 67, even though the enzyme solutions are kept at 4°.
[19] MALATE DEHYDROGENASE FROM CHICKEN AND TUNA. HEAI~.T 109
dissolved in 5 mM potassium phosphate-1 nlM EDTA-1 mM fl-mer-

captoethanol, pH 7.5. Most of the malate dehydrogenase activity is
present in the precipitate. The supernatant is discarded.
Step 3. Secol,d Ammonium Sul]ate Precipitation. The dissolved pre-
cipitate from step 2 is dialyzed against three changes of 5 mM potassium
phosphate-1 mM EDTA-1 mM fl-mercaptoethanol, pH 7.5, 20 liter~,
about 6 hours for e~ch change. After dialysis, the enzyme solution is
clarified by centrifugation at 20,000 g for 15 minutes. To the clear super-
natant, solid ammonium sulfate is added to give 50% saturation. After
2 hours at 4 °, the suspension is centrifuged at 20,000 g for 15 minutes.
The precipitate contains only a small fraction of the total malate dehy-
drogenase activity and is discarded. Further ammonium sulfate is added
to give 80% saturation. The suspension is left at 4 ° for 2 hours and
centrifuged as before. The precipitate is dissolved in 50 mM potassium
phosphate-1 mM EDTA-10 mM fl-mercaptoethanol, pH 7.5, and dia-
lyzed at 4 ° against three changes of the same buffer (6 liters), 6 hours
per change. Starch gel electrophoresis at pH 7.0 is used to confirm the
presence of both intra- and extramitochondrial malate dehydrogenases.
Step 4. Negative Adsorption on DEAE-Cellulose. Neither intra- nor
extramitochondrial chicken malate dehydrogenase is adsorbed to DEAE-
cellulose at pH 7.5 in 50 mM potassium phosphate-1 mM EDTA-1 mM
fl-mercaptoethanol, although a considerable amount of other proteins is
retained by the resin under these conditions. DEAE-cellulose is prepared
as described by Pesce et al./ except that equilibration steps are carried
out in 0.5M potassium phosphate-1 mM EDTA-1 mM fl-mercapto-
ethanol, pH 7.5, and finally in 50 mM potassium phosphate-1 mM
EDTA-1 mM fl-mercaptoethanol, pH 7.5. The dialyzed enzyme from step
3 is placed on a DEAE-cellulose column (4.5 X 50 cm) and eluted with
the buffer used for the last change of dialysis. The malate dehydro-
genases are not retained on the column. The fractions containing malate
dehydrogenase activity are combined and dialyzed overnight against
saturated ammonium sulfate-10 mM fl-mercaptoethanol, pH 7.5, to con-
centrate the enzymes. The precipitated enzymes are collected by centrif-
ugation at 20,000 g for 15 minutes, and the precipitate is dissolved in
5 mM potassium phosphate-1 mM EDTA-1 mM fl-mercaptoethanol,
pH 6.5, and dialyzed overnight at 4 ° with three changes (6 liters) of the
same buffer.
St'ep 5. Carboxymethyl-CeUulose Chromatography. Carboxymethyl-
cellulose is prepared as described by Pesce et al., 9 except that the equili-
bration steps are carried out in 0.5M potassium phosphate-1 mM
o A. Pesce, R. H. McKay, F. Stolzenbach, R. D. C~hn, and N. O. Kaplan, J. Biol.
Chem. 239, 1753 (1964).
EDTA-1 mM fl-mercaptoethanol, pH 6.5, and finally in 5 mM potassium

phosphate-1 mM EDTA-1 mM fl-mercaptoethanol, pH 6.5. The dialyzed
enzyme solution from step 4 is placed on a 4.5 X 50 cm carboxymethyl-
cellulose column.
The extramitochondrial enzyme is not adsorbed to the resin and is
eluted from the column with 5 mM potassium phosphate-1 mM E D T A -
1 mM fl-mercaptoethanol, pH 6.5. The fractions containing malate
dehydrogenase activity arc combined and concentrated by dialysis
against concentrated ammonium sulfate as described abovc. Starch gel
electrophoresis at pH 7.0 is used to confirm that this fraction (CMC-1)
contains only the extramitochondrial form of malate dehydrogenase,
which is then further purified as described below.
The intramitochondrial malate dehydrogenase is adsorbed to the
carboxymethyl cellulose and is eluted with a linear gradient established
between 2 liters of 5 mM potassium phosphate-1 mM EDTA-1 mM
fl-mercaptoethanol, pH 6.5, and 2 liters of 0.2 M potassium phosphate-
1 mM EDTA-1 mM fl-mercaptoethanol, pH 6.5. The fractions containing
malate dehydrogenase activity are combined and concentrated by
dialysis against saturated ammonium sulfate, pH 7.5, as described above.
Starch gel eleetrophoresis at pH 7.0 is used to determine that this fraction
(CMC-2) contains only the intramitochondrial malate dehydrogenase.
This enzyme is further purified as described below.
Step 6. Partial Crystallization of Chicken Heart Extramitochondrial
Malate Dehydrogenase. The extramitochondrial enzyme (fraction CMC-
1 from step 5), aftel' ammonium sulfate concentration, is dialyzed over-
night at 4 ° against three changes (6 liters) of 5 mM potassium phos-
phate-1 mM EDTA-1 mM fl-mercaptoethanol, pH 7.8. The enzyme
solution is then placed on a 4.5 X 40 cm column of DEAE-cellulose
previously equilibrated in the same buffer and eluted with a linear
gradient established between 2 liters of 5 mM potassium phosphate-1
mM EDTA-1 mM fl-mercaptoethanol, pH 7.8, and 2 liters of 0.2M
potassium phosphate-1 mM EDTA-1 mM /~-mercaptoethanol, pH 7.8.
The enzyme is eluted at a salt concentration of approximately 25 mM;
fractions containing malate dehydrogenase activity are combined and
concentrated by dialysis against saturated ammonium sulfate as de-
scribed above. At this stage it is possible, by ammonium sulfate fraction-
ation, to obtain partly crystalline enzyme. Minor contaminants are most
easily removed by gel filtration as described below.
Step 7. Gel Filtration o] Chicken Extramitocho~drial Malate Dehy-
drogenase. The concentrated enzyme solution in ammonium sulfate
obtained in step 6 is dialyzed for 6 hours at 4 ° against three changes (3
liters) or 50 mM Tris-HC1 buffer, pH 7.0, containing 0.1 M KCI and
1 mM fl-mercaptoethanol. The enzyme solution (approximately 15 ml) is

then placed on a 2.5 X 80 cm column of Sephadex G-100, previously
equilibrated with the same buffer. The column is eluted with the same
buffer, and the fractions having the highest malate dehydrogenase
activity are combined and concentrated by dialysis against saturated
ammonium sulfate as described above. The leading and trailing edges
of the malate dehydrogenase peak are discarded.
Step 8. Crystallization o] Chicken Extramitochondrial Malate Dehy-
drogenase. The concentrated enzyme solution from step 7 is dialyzed for
6 hours against three changes (2 liters) of 0.1 M potassium phosphate-1
mM EDTA-5 mM fi-mercaptoethanol, pH 7.5. Solid ammonium sulfate
is added to give 50% saturation and any amorphous inactive protein
precipitate is removed by centrifugation.
Additional solid ammonium sulfate is added slowly to the solution
over a period of 4-5 hours until a slight turbidity is observed (at ap-
proximately 65% ammonium sulfate saturation). The enzyme solution is
stored at 4 °. Crystallization is apparent after about 8 hours. Crystalliza-
tion is allowed to continue for 3 or 4 days; the crystals are then harvested
by centrifugation and recrystallized four times in the same manner. In
our hands the yield is approximately 200 mg of crystalline enzyme,
representing some 15% of the total malate dehydrogenase activity present
in the crude tissue extract.
Step 9. Further Purification of Chicken Intramitochondrial Malate
Dehydrogenase. The intramitochondrial malate dehydrogenase fraction
obtained from carboxymethyl-cellulose chromatography (fraction CMC-
2 from step 5) is dialyzed against 0.1 M potassium phosphate-1 mM
EDTA-1 mM fl-mercaptoethanol, pH 7.5 (three changes of 3 liters each).
Solid ammonium sulfate is added to 45% saturation and the solution is
clarified by centrifugation. Additional ammonium sulfate is added slowly
over a period of about 4 hours until a slight turbidity is seen (at ap-
proximately 55% saturation). Crystallization is allowed to proceed at 4 °
for 3 days, and the crystals are harvested by centrifugation. At this stage
it is advisable to check for contamination by determining the fluorescence
spectrum of a solution of the enzyme (excitation at 280 m~). Pure
chicken intramitochondrial malate dehydrogenase contains no tryptophan
and has a fluorescence emission maximum at 307 m~. The presence of
tryptophan-containing contaminants, even in small amounts, is revealed
by a fluorescence emission maximum at approximately 335 mt~. Such
contaminants are effectively removed by gel filtration on Sephadex G-100
as described in step 7 for the extramitochondriat enzyme. Following gel
filtration, the enzyme is recrystallized four times as described above. The
yield is approximately 160 mg of crystalline enzyme, which represents
11% of the total malate dehydrogenase activity of the crude tissue

extract.
Properties 4
Physicochemical Characteristics. Both intra- and extramitochondrial
malate dehydrogenases have a molecular weight of approximately 67,000
and Seo,~, values of 4.3. The ~~.~% 1 am, 280 rn/~ for the intramitochondrial enzyme
is 2.9, this low value reflecting the lack of tryptophan in this enzyme.
The •~1%
t-~l cm, 280 mtt for the extramitoclmndrial enzyme is 13.1. From peptide
maps ~ and from reversible dissociation studies, 1°, 1~ both enzymes appear
to be composed of two identical, or nearly identical, subunits. From the
peptide maps, it is apparent that there are few common basic or acidic
peptides in the chicken intra- and extramitochondrial malate dehydro-
genases.
Catalytic Properties. The intramitochondrial enzyme has K~ values
of 0.9 mM for malate and 38 #M for oxaloacetate. The corresponding
values for the extramitochondrial enzyme are 0.8 mM and 50 ~M. Both
enzymes have pH optima of approximately 10 for malate oxidation. For
oxaloacetate reduction the intra- and extramitochondrial enzymes have
pH optima of 7.8 and 7.6, respectively. Neither enzyme shows any de-
tectable activity with D-malate or with NADP. The extramitochondrial
enzyme is markedly inhibited by high concentrations of L-malate. This
is not the case with the intramitochondrial enzyme. The intramitochon-
drial enzyme is more strongly inhibited by high concentrations of oxalo-
acetate than is the extramitochondrial enzyme. The intramitochondrial
enzyme is slightly more heat labile at 55 ° than is the extramitochondrial
form.
Immunological Properties. Rabbit antisera prepared against the
chicken intramitochondrial malate dehydrogenase inhibit the enzymatic
activity of the enzyme and show reaction by precipitin and complement
fixation procedures. No cross-reaction was found with the extramito-
chondrial enzyme. Exactly the reverse was true of antisera to the extra-
mitoehondrial enzyme which react well with this enzyme but not at all
with the intramitochondrial form. Antisera to the chicken intra- and
extramitochondrial enzyme do, however, show cross-reactions with their
respective forms of enzymes from species other than chickens.
Electrophoretic Properties. Although ultracentrifugal analysis of the
crystalline chicken intra- and extramitochondrial malate dehydrogenases
loO. P. Chilson, G. B. Kitto, and N. O. Kaplan, Proc. Natl. Acad. Sci. U.S. 53, 1006
(1965).
,1 O. P. Chilson, G. B. Kitto, J. Pudles, and N. O. Kaplan, J. Biol. Chem. 241, 2431
(1966).
[19] MALATEDEIIYDROGENASE FROM CIIICKEN AND TUNA HEART 113
indicate homogeneity, both enzymes exhibit multiple enzymatically

active bands when subjected to starch gel electrophoresis at pH 7.0.
Recent evidence 12,13 suggests that the multiple electrophorctie forms of
the intramitochondrial enzyme result from conformational differences
rather than differences in primary structure.
B. TUNA HEART MALATE DEHYDROGENASES
The following procedure was developed to allow the purification of
both lactate and malate dchydrogenases from the same batch of tissue.
The techniques employed were designed to give an efficient extraction of
the two enzymatic activities and to effect their early separation.
Purification
Step I. Extraction o/ Tissue. Frozen tuna hearts (18 pounds) are
thawed and then ground three times in a mechanical meat grinder. The
minced tissues are then suspended in 20 liters of 0.1 M Tris-{-1 mM
EDTA -t- 1 mM fl-mercaptoethanol, pH 7.6, at 4 ° and stirred slowly for
3 hours. The mince is filtered through cheesecloth and the filtrate is
clarified by centrifugation. The freeze-thawing and grinding process is
normally sufficient to disrupt most of the mitochondria and extract both
the intra- and extramitochondrial malate dehydrogenases. This can be
checked by sonicating a sample of the solid residue to determine whether
further malate dehydrogenase activity can be released. If so, the extrac-
tion procedure should be repeated. All subsquent purification is carried
out at a temperature of 4 °.
Step 2. Ammonium Sul]ate Fractionation. Solid ammonium sulfate is
slowly added to the tissue extract from step 1 to obtain 50% saturation.
In this, and in all subsequent steps involving ammonium sulfate frac-
tionation, the pH is kept at 7.5 by gradual addition of ammonium
hydroxide.
After 3 hours, the suspension is filtered on fluted filter papers, and the
precipitate discarded. More solid ammonium sulfate is added to the
filtrate to give 75% saturation and the suspension is left for 3 hours and
then filtered on fluted filter papers. Care should be taken that the precipi-
tate does not dry out on the filter paper. The moist precipitate is scraped
off and dissolved in 750 ml of 5 mM Tris-}-1 mM EDTA ~ 1 mM
fl-mercaptoethanol, pH 7.6. Little malate dehydrogcnase activity remains
in the filtrate, which is discarded.
,2 G. B. Kitto, P. M. Wassarman, J. Michejda, and N. O. Kaplan, Biochem. Biopby.~
Res. Commun. 22, (1966).
,3 G. B. Kitto, P. M. Wassarman, and N. O. Kaplan, Proc. Natl. Acad. Sci. U.S. 56,
578 (1966).
Step 3. First DEAE-Cellulose Chromatography. The dissolved pre-

cipitate from step 2 is dialyzed overnight against three changes (20 liters
each) of 5 mM Tris + 1 n ~ I EDTA + 1 mM/~-mercaptoethanol, pH 7.6.
The dialyzed enzyme is then placed on a 15 X 75 cm DEAE-cellulose
column, prepared as described by Pesce et al., 9 and previously equili-
brated with the above buffer. The column is eluted with this buffer until
the eluent shows negligible absorbance at 280 m~. Malate dehydrogenase
activity is not eluted. The column is then eluted with an exponential
NaC1 gradient (in 5 mM Tris + 1 mM EDTA + 1 mM fl-mercapto-
ethanol, pH 7.6), the final salt concentration being 0.2M. Lactate de-
hydrogenase is eluted early in the gradient, whereas the malate dehydro-
genases are more strongly adsorbed to the resin and are eluted at higher
salt concentrations, the two types of enzymatic activity being completely
separated. All fractions showing appreciable malate dehydrogenase ac-
tivity are combined, and solid ammonium sulfate is added to give 70%
saturation. After 3 hours the suspension is centrifuged at 20,000 g for 30
minutes and the precipitate is dissolved in a minimal volume of 5 mM
Tris + 1 mM EDTA + 1 mM fl-mereaptoethanol, pH 7.6. Little malate
dehydrogenase activity should be present in the supernatant fraction,
which is discarded. Starch gel electrophoresis, at pH 8.5, can be used to
demonstrate the presence of both intra- and extramitochondrial malate
dehydrogenases in the precipitate.
Step ~. Second DEAE-Cellulose Chromatography. The precipitate
from step 3 is dialyzed overnight against three changes (6 liters) of
5 mM Tris + 1 mM EDTA + 1 mM fl-mercaptoethanol, pH 7.6, and
applied to an 8 X 60 cm DEAE-cellulose column, previously equilibrated
with the same buffer. Elution is as in step 3, except that in this case
the gradient is linear. The linear gradient serves to separate the intra-
and extramitochondrial malate dehydrogenases. Two peaks of malate
dehydrogenase activity are eluted from the column. On the basis of
electrophoretic mobility on starch gels, at pH 8.5, the first peak to be
eluted can be identified as the extramitochondrial enzyme and the second
as the intramitochondrial enzyme. Fractions containing the extra- and
intramitochondrial malate dehydrogenase activities are separately pooled,
and these enzymes are precipitated by raising the ammonium sulfate
concentration to 70% saturation. The precipitates were dissoh'ed in 5
mM Tris + 1 mM EDTA + 1 mM fl-mercaptoethanol. At this point,
further attempts to purify aliquots of the tuna extramitochondrial malate
dehydrogenase in our hands only led to drastic losses of enzymatic
activity. Recent experiments suggest that some protective against inacti-
vation can be afforded by inclusion of NAD in the buffer and by raising
the concentration of fl-mercaptoethanol. The extramitochondrial enzyme
can be retained at this level of purification (approximately 30-fold) by

storing it at 4 ° in 50% saturated ammonium sulfate containing 10 mM
Tris + 1 mM EDTA + 1 mM fl-mercaptoethanol, pH 7.5, under which
conditions it is quite stable over a period of several weeks.
Step 5. Further Purification o] Tuna Intramitochondrial Malate
Dehydrogenase. The precipitated intramitochondrial malate dehydro-
genase fraction is normally colored dark brown. This coloration is re-
moved by gel filtration on Sephadex G-100. The precipitate is dialyzed
against three changes (6 liters) of 50 mM Tris + 0.1 M KC1 + 1 mM
fl-mercaptoethanol, pH 7.0, and the enzyme solution is applied to a
2.5 X 100 cm column of Sephadex G-100 previously equilibrated with the
same buffer. The column is eluted with the above buffer and fractions
containing malate dehydrogenase with the highest specific activity are
combined. The leading and trailing edges of the malate dehydrogenase
peak are discarded. The pooled malate dehydrogenase fractions are con-
centrated and the enzyme is precipitated by dialysis against saturated
ammonium sulfate containing 1 mM EDTA + 1 mM fl-mercaptoethanol
and adjusted to pH 7.5. The precipitate is collected by centrifugation at
20,000 g for 15 minutes and is dissolved in a minimal volume of 50 mM
Tris + 1 mM EDTA + 1 mM fl-mercaptoethanol, pH 7.5. Solid am-
monium sulfate is added to 50% saturation and any amorphous inactive
protein is removed by centrifugation. Further solid ammonium is added
slowly over a period of several hours until the enzyme starts to crystal-
lize (as evidenced by a slight sheen) at approximately 60% saturation.
Crystallization is allowed to proceed for 2 days; the crystals are then
harvested by centrifugation. The tuna intramitochondrial malate dehy-
drogenase is recrystallized several times by the same procedure to attain
constant specific activity and may be stored at 4 ° as a crystalline sus-
pension in ammonium sulfate containing 1 mM EDTA + 1 mM fl-mer-
captoethanol, adjusted to pH 7.5. In our hands, the yield of crystalline
tuna intramitochondrial malate dehydrogenase was approximately 150
mg. The gain in specific activity, over that of the crude tissue extract,
was 290-fold.
Properties ~
Physicochemical Characteristics. The intra- and extramitoehondrial
malate dehydrogenases of yellow-fin tuna heart closely resemble the prop-
erties of the respective chicken enzymes. Both the tuna malate dehydro-
genases have a molecular weight of 67,000 and the intramitochondrial
enzyme was found to have an $2o,~ value of 4.0. The intramitochondrial
,4G. B. Kitto and R. G. Lewis, Biochim. Biophys. Acta 139, 1 (1967).

enzymes from tuna, pig, 1~ and chicken heart 4 appear to be devoid of

tryptophan. The ~1%
a~ 1 om, 280 mt~ for the intramitochondrial tuna enzyme is 3.1.
Catalytic Properties. The tuna intramitochondrial malate dehydro-
genase is more susceptible to inhibition by high concentrations of oxalo-
acetate than is the extramitochondrial enzyme. However, this inhibition
is observed at markedly higher levels of oxaloacetate than those required
to inhibit the corresponding chicken enzymes. Similarly very high malate
concentrations are required to cause inhibition of the tuna malate
dehydrogenases. The intramitochondrial enzyme is more heat labile than
is the extramitochondrial form.
Immunological Properties. A rabbit antiserum, prepared against
crystalline tuna intramitochondrial malate dehydrogenase, reacts
strongly with this enzyme as judged by double diffusion and enzyme inhi-
bition tests. No cross-reaction of this antibody with the extramitochon-
drial enzyme could be observed.
Electrophoretic Properties. When subjected to starch gel electro-
phoresis at pH 8.5, the intramitochondrial tuna malate dehydrogenase
migrates faster toward the anode than does the extramitochondrial en-
zyme. This is the reverse of the situation found in avian and mammalian
species where the extramitochondrial enzyme moves faster toward the
anode. The native tuna intra- and extramitochondrial malate dehydro-
genases show no multiple electrophoretic forms comparable to those
found with the chicken and pig enzymes.1,12,13
,5C. J. R. Thorne and N. O. Kaplan, J. Biol. Chem. 238, 1861 (1963).
[20] C y t o p l a s m i c a n d M i t o c h o n d r i a l M a l a t e
Dehydrogenases from Beef Kidney
[EC 1.1.1~7 I,-Malate: NAD oxidoreductase]
By DANIEL DUPOURQUE and ERNEST gUN 1
DPNH -I- H + + oxaloacetate ~ malate q- DPN +
Isoenzymes of both cytoplasmic and mitochondrial malate deby-

drogenases were isolated from subcellular fractions of beef kidney.
A. Assay Methods for Both Isoenzymes

Activity. This was measured by following the rate of oxidation of
DPNH in the presence of oxaloacetate. The decrease in absorbance at
340 mtz was determined with a Gilford recording spectrophotometer. Silica
Recipient of Research Career Award of the U.S. Public Health Service.
enzymes from tuna, pig, 1~ and chicken heart 4 appear to be devoid of

tryptophan. The ~1%
a~ 1 om, 280 mt~ for the intramitochondrial tuna enzyme is 3.1.
Catalytic Properties. The tuna intramitochondrial malate dehydro-
genase is more susceptible to inhibition by high concentrations of oxalo-
acetate than is the extramitochondrial enzyme. However, this inhibition
is observed at markedly higher levels of oxaloacetate than those required
to inhibit the corresponding chicken enzymes. Similarly very high malate
concentrations are required to cause inhibition of the tuna malate
dehydrogenases. The intramitochondrial enzyme is more heat labile than
is the extramitochondrial form.
Immunological Properties. A rabbit antiserum, prepared against
crystalline tuna intramitochondrial malate dehydrogenase, reacts
strongly with this enzyme as judged by double diffusion and enzyme inhi-
bition tests. No cross-reaction of this antibody with the extramitochon-
drial enzyme could be observed.
Electrophoretic Properties. When subjected to starch gel electro-
phoresis at pH 8.5, the intramitochondrial tuna malate dehydrogenase
migrates faster toward the anode than does the extramitochondrial en-
zyme. This is the reverse of the situation found in avian and mammalian
species where the extramitochondrial enzyme moves faster toward the
anode. The native tuna intra- and extramitochondrial malate dehydro-
genases show no multiple electrophoretic forms comparable to those
found with the chicken and pig enzymes.1,12,13
,5C. J. R. Thorne and N. O. Kaplan, J. Biol. Chem. 238, 1861 (1963).
[20] C y t o p l a s m i c a n d M i t o c h o n d r i a l M a l a t e
Dehydrogenases from Beef Kidney
[EC 1.1.1~7 I,-Malate: NAD oxidoreductase]
By DANIEL DUPOURQUE and ERNEST gUN 1
DPNH -I- H + + oxaloacetate ~ malate q- DPN +
Isoenzymes of both cytoplasmic and mitochondrial malate deby-

drogenases were isolated from subcellular fractions of beef kidney.
A. Assay Methods for Both Isoenzymes

Activity. This was measured by following the rate of oxidation of
DPNH in the presence of oxaloacetate. The decrease in absorbance at
340 mtz was determined with a Gilford recording spectrophotometer. Silica
Recipient of Research Career Award of the U.S. Public Health Service.
[20] MALATE DEHYDROGENASES FROM BEEF KIDNEY 117
cells of 1 cm light path and 1 ml capacity were used. The enzymatic

reaction is described by the above equation.
A s s a y Procedure. (a) Test system for the mitochondrial enzyme was
composed of 100 micromoles of potassium phosphate (pH 7.2), 0.2 micro-
moles of oxaloacetate, and 0.1 micromole of DPNH, in a final volume
of 1 ml. (b) Test system for the cytoplasmic enzyme was the same as for
the mitochondrial one except that 2 micromoles of oxaloacetate was
present (instead of 0.2 micromoles).
The reaction was started by the addition of 5-20 ~l of enzyme solu-
tion. Measurement of reaction rates was carried out at room temperature
(22-25°). Activity was determined from initial velocity, read directly
from the slope obtained in the first 30 seconds of the reaction. The
amount of D P N H oxidized was calculated from the extinction coefficient
of 6.22 X 106 cm 2 mole-1 (footnote la).
Units. One unit of enzyme was defined as the quantity which oxidizes
1 micromole of DPNH per minute at 22 °. Specific activity was calculated
in terms of micromoles of D P N H oxidized per minute per milligram of
protein.
B. Other Methods
Protein was determined by the method of Lowry et alY Sucrose
gradients were carried out as described by Martin and Ames2 Progress
of the purification was followed by determination of specific activity
and by starch gel electrophoresis as described by Barrett, Friesen, and
Astwood.4 The gel was cut horizontally and the top layer stained with
Nigrosin dye for detection of protein. Enzyme activity was localized by
the tetrazolium method in the bottom layer of the gel, as described by
Fine and Costello. ~
C. Preparation of Mitochondrial Acetone Powder and

of Cytoplasmic Extract
Fifty pounds of beef kidney (not older than 2 days) was purchased
from Swift and Co. After removal of all fat, connective tissues and
most of the medulla, the cortical tissue was ground in a meat grinder.
All operations were carried out in the cold room (4-5°). Four hundred
gram batches were suspended in 800 ml of a solution of KC1 (0.9~)
z.B.L. Horecker and A. Kornberg, J. Biol. Chem. 175~ 385 (1948).

J O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193,
265 (1951).
• R. G. Martin and B. N. Ames, J. Biol. Chem. 236, 1372 (1961).
~R. J. Barrett, H. Friesen, and E. B. Astwood, J. Biol. Chem. 237, 432 (1962).
I. H. Fine and L. A. Costello,Vol. VI, p. 958.
containing 5 mM phosphate buffer (pH 7.4). This suspension was blended

for 60 seconds in an overhead-type blendor, and the pH was readjusted
to 7.4 with 6 N solution of KOH. After centrifugation at 1300 g, the
suspension was filtered through four layers of cheesecloth. The filtered
solution was passed through a refrigerated continuous-flow Sharples
centrifuge at 60,000 g in batches of 10 liters. The turbid supernatant
can be stored for more than 6 months at --20 ° without loss of enzymatic
activity.
Approximately 50 pounds of kidney could be processed in 1 day by
this technique. An average of 800 g of mitochondrial paste was obtained,
and 25 liters of supernatant, from this amount of starting material.
The mitochondrial paste was poured into 1 liter of reagent grade
acetone (--10 °) and mixed vigorously in a Waring blendor operated
in the cold room for 15-20 seconds. This suspension of mitochondria was
poured immediately into a second batch of 5 liters of acetone (--10°),
mixed, and centrifuged at 1000 g at --10 ° for 10 minutes. It is of
importance to maintain subzero temperatures during these operations.
The precipitate was resuspended and stirred in 4 liters of acetone,
filtered by suction through Whatman paper No. 1 (in the cold room)
and washed quickly with approximately 200 ml of ether (--10°). The
mitochondrial cake was spread on a sheet of heavy paper and dried at
room temperature. Drying is promoted by repeated redistribution of the
acetone powder on fresh sheets of paper by means of a large spatula.
The average yield of dry acetone powder was 180 g. This preparation can
ke.~p for months at --20 ° and is stable at room temperature for more
than 6 hours.
D. Purification of Cytoplasmic Enzyme

The cytoplasmic enzyme was purified from centrifugal supernatant
in batches of 2 liters, stored frozen, and thawed at 5 ° overnight. This
turbid supernatant contained 90 units/ml with a specific activity of 1.5.
Step 1. Ammonium Precipitation. Solid ammonium sulfate was added
to achieve 50% saturation. After 1 hour the pH was adjusted to 6.8 and
the solution was centrifuged. The precipitate was discarded and the
supernatant was brought to 85% of saturation with (NH4)~SO~ and left
standing for 2 hours at 4 °. The precipitate contained the enzyme and
was dissolved in 400 ml of phosphate buffer, 5 mM, pH 7.4. The specific
activity of this solution was 8 and the yield 50%.
Step 2. First DEAE Column. The protein fraction obtained in step 1
was dialyzed for 2 days against four successively changed volumes of 10
liters each, of 5 mM potassium phosphate, pH 7.4. DEAE (100 g, BioRad
Cellex-D) was washed with large amounts of distilled water; the pH
was adjusted to 7.4 with HC1 and again washed 6 times (4 liters each)
with phosphate buffer, pH 7.4, 5 raM, then left to equilibrate overnight
against this buffer. The protein solution was applied to a column (8 X 20
cm) and washed with 5 mM phosphate buffer, pH 7.4. The cytoplasmic
enzyme is adsorbed by the column, but not the mitochondrial one. The
cytoplasmic extract contains about one-third of the total mitochondrial
enzyme as a contaminant. In order to remove the mitochondrial enzyme
and other proteins, the column is washed with 12-16 liters of 5 mM
phosphate, pH 7.4, until elution of all red-colored proteins is complete.
At this point the cytoplasmic enzyme is eluted by 50 mM phosphate
buffer (pH 7.4). The brownish-yellow protein fraction containing the
cytoplasmic malic dehydrogenasc was pooled and precipitated at 80%
saturation of ammonium sulfate. The precipitate was collected by centrif-
ugation and dissolved in 40 ml of 10 mM phosphate buffer (pH 7.4) and
dialyzed free from (NHD2SO~ against 10 mM phosphate. In this step
approximately 50% of the cytoplasmic enzyme was recovered.
Step 3. Second DEAE Column. The dialyzed solution obtained in step
2 (50 ml) was further purified on a second DEAE column (3.5 X 25 cm).
After the adsorption of the protein, the column was washed with 200 ml
of 5 mM phosphate buffer, pH 7.4, and a linear gradient of increasing
P04 concentration (potassium phosphate buffer, pH 7.4, 800 ml, 5 mM
plus 800 ml, 50 mM) was applied; fractions were collected in 25 ml
portions. The enzyme was eluted around the 55th tube. All fractions
containing M D H (400 ml, 25,000 units, specific activity 180) were pooled,
and the enzyme was reprecipitated by ammonium sulfate (80% satura-
tion). After centrifugation the precipitate was dissolved in 20 ml of
phosphate buffer and dialyzed against 20 volumes of 5 mM phosphate
buffer overnight (with three changes).
Step 4. Hydroxylapatite Column. Hydroxylapatite was prepared by
the method of Siegelman et al. 6 at pH 6.8. The dialyzed solution obtained
in step 3 was applied on a 3.5 )< 10 cm column. After adsorption of
protein, the column was washed with 150 ml of 5 mM phosphate buffer.
Thereafter, a gradient (800 ml, 5 mM plus 500 ml, 50 mM) was applied
and the eluent was collected in 20 ml fractions. A yellow peak was first
obtained, followed immediately by a slightly brown fraction which con-
tained the bulk of malate dehydrogenase activity. All the active fractions
(300 ml, 15,000 units with specific activity of 320) were pooled and
reprecipitated by ammonium sulfate (80% saturation).
Step 5. Crystallization. The precipitate was washed carefully with
phosphate buffer 10 mM, pH 6.8, and dissolved in 2 ml of the same
*H. W. Siegelman, C. A. Wieczoreck, and B. C. Turner, Anal. Biochem. 13, 402
(1965).
buffer. Solid (NH,)2S04 was added until 4 5 ~ saturation, and this solu-
tion was stirred for 30 minutes. This solution was recentrifuged and
dialyzed against a progressively increasing concentration of ammonium
sulfate, as described by Englard and Breiger 7 and Thorne2 During this
procedure an impurity which appears as a turbid brown precipitate was
removed by centrifugation. Crystallization started at 60% saturation of
(NH4) 2S0~. After a period of 12 hours, needlelike crystals were harvested
by centrifugation and resuspended in 60% saturated ammonium sulfate.
Approximately 5800 units of enzyme with a specific activity between 350
and 380 was obtained. The protein was homogeneous and migrated as a
single band on starch gel electrophoresis, and as a symmetrical peak
(S~o.w = 5.2) in the analytical centrifuge. A summary of the purification
is given in Table I.
TABLE I
PURIFICATION OF CYTOPLASMIC ]~NZYME
Specific
protein activity (units/rag Yield
Step (rag) (units) ~ protein) (%)
Crude extract 125,000 180,000 1.5

1. Ammonium sulfate 12,400 100,000 8.3 56
2. First DEAE 800 32,000 40 20
3. Second DEAE 130 25,000 180 14
4. Hydroxylapatite 46 15,000 320 8
5. First crystallization 15 5,800 380 3
6. Second crystallization 8 3,000 300 1.7
o A unit is the quantity which oxidizes 1 micromole of DPNH per minute under the
assay conditions.
E. Mitochondrial E n z y m e
The mitochondrial acetone powder was worked up in batches of 50 g.
Step 1. Extraction. F i f t y grams of acetone powder was suspended in
1 liter of potassium phosphate buffer, 0.1 M, p H 7.4, and stirred for 2
hours. After centrifugation a clear yellow supernatant fluid was obtained,
which contained 280,000 units of M D H with a specific activity of 15.
Step ~. Ammonium Sul]ate Precipitation. Ammonium sulfate was
added to 40% saturation. After 1 hour, the p H was adjusted to 7.4 and
the solution was centrifuged. The supernatant was brought to 80% of
saturation and allowed to stand for 2 hours. After centrifugation, the
' S. Englard and H. H. Breiger, Biochim. Biophys. Acta 56, 571 (1962).
*C. J. R. Thorne and P. M. Cooper, Biochim. Biophys. Acta 81, 397 (1964).
precipitate containing the enzyme was dissolved in 200 ml of potassium

phosphate buffer. The specific activity was 55, and the yield 88%.
Step 8. Mixed CM and DEAE Column. The enzyme solution (step 2}
was dialyzed for 2 days against three changes (10 liters each) of 5 mM
potassium phosphate. CM and DEAE were treated as described for the
cytoplasmic enzyme. A column was prepared from a mixture of CM and
DEAE, containing both in equal amounts. At pH between 7 and 7.4, the
mitochondrial enzyme is not adsorbed by either CM or DEAE. The
dialyzed solution passed through the column with a 5 mM potassium
phosphate buffer, pH 7.4. A slight brownish-yellow enzyme solution was
obtained which was pooled (yield 60~'o, specific activity 220), reprecipi-
tared with ammonium sulfate (80% saturation), and centrifuged; the
precipitate was dissolved in 15 ml of phosphate buffer. This procedure
removes a large portion of mitochondrial proteins and yields about 5-fold
purification of MDH.
Step ~. Hydroxylapatite Column. A 3.5 X 15 cm column was prepared
as described for the cytoplasmic enzyme. The preparation obtained in
step 3: was dialyzed overnight against 10 liters of 10 mM phosphate
buffer. The protein solution was then applied on the column. After
adsorption, an elution gradient of potassium phosphate buffer, pH ?.6
(800 ml, 10 mM plus 800 ml, 0.5 M) was carried out. Twenty-milliliter
fractions were collected. Highest enzymatic activity was present in the
last fractions, which were pooled (yield 40%, specific activity 680) and
reprecipitated by ammonium sulfate (80% saturation); the precipitate
was dissolved in 10 ml of phosphate buffer.
Step 5. Alcohol Fractionation in Presence o] ~0% Ammonium Sulfate
(c]. ]ootnote 9). The protein solution obtained in step 4 was dialyzed
overnight against 10 mM phosphate buffer, then brought to 20%
saturation by adding a solution of saturated ammonium sulfate. Ethanol
concentration was brought to 40% (8 ml absolute ethanol for each 12 ml
of protein solution) by slow addition of chilled (--10 °) absolute ethanol,
admixed with continuous stirring. The precipitate formed during this
procedure was centrifuged at M10 ° and discarded. The remaining super-
natant solution was brought to 70% ethanol concentration (20 ml
absolute ethanol to the 20 ml supernatant) by the same cautious proce-
dure. The precipitate formed (containing MDH) was collected by centrif-
ugation, dissolved in 10 ml of 0.1 M phosphate buffer, dialyzed against 1
liter of the same buffer for 3 hours, and cleared up by centrifugation.
This clear supernatant solution contained 32% of MDH activity ob-
tained by the first extraction of acetone powder. Specific activity was
1400.
' D. Davies and E. Kun, Biochem. J. 66, 307 (1957).
TABLE II
PURIFICATION OF THE MITOCHONDRIAL ENZYME
Specific
protein activity (units/rag Yield
Step (mg) (units) ~ protein) (%)
1. Crude extract 19,000 280,000 15 100

2. Ammonium sulfate 4,500 250,000 55 84
3. CM -4- DEAE 800 170,000 220 60
4. Hydroxylapatite 178 115,000 650 40
5. Alcohol 65 90,000 1400 32
See footnote Table I.
Purification is summarized in Table II. The mitochondrial enzyme
appears as a symmetrical peak (S~o,w ~ 4.2) in the analytical ultra-
centrifuge. The mitochondrial enzyme migrates toward the cathode at
pH 6.5 (starch gel). Two to three enzymatically active bands were
observed.
F. Comparison of Properties
Both cytoplasmic and mitochondrial enzymes are stable for months
at --20 ° and for several hours at room temperature. The two enzymes
differ widely in their physical and kinetic properties. The mitochondrial
enzyme is a basic protein with an isoelectric point greater than 7. I t
exhibits substrate inhibition by oxaloaeetate and activation by phosphate.
These kinetic characteristics vary with changes in p H and ionic
strength. I° The p H maximum for the mitoehondrial enzyme is 7.8--8.
Sedimentation and electrophoresis studies indicate the presence of sub-
units in the mitochondrial enzyme.
The cytoplasmic enzyme has a lower specific activity. I t does not
exhibit abnormal kinetics with respect to oxaloacetate, nor is it influenced
by small changes in ionic strength. Its p H maximum is close to 7.4, and
its isoeleetrie point less than 6.0.
Acknowledgment
This work was supported by research grants from the National Science Founda-
tion (GB-5749 and GB-3488), the U.S. Public Health Service (RO1-HD-01239-11
and RO1-CA-07955-03), and the American Heart Association, Inc. (66-652).
1. E. Kun, R. Z. Eanes, and P. Volfin, Nature 214, 5095 (1967).

[21] CYTOPLASMICL-MALATE DEHYDROGENASE OF BEEF HEART 123
[ 2 1 ] E x t r a m i t o c h o n d r i a l L - M a l a t e D e h y d r o g e n a s e of
Beef Heart
[EC 1.1.1.37 ~.-Malate:NAD oxidoreductase]
By SASHA ENGLARD
L-Malate -t- NAD+ ~- oxaloacetate -b N A D H -b H +
Assay Method
Principle. Extramitoehondrial malate dehydrogenase activity is meas-
ured spectrophotometrically by the decrease in absorption at 340 m/~ due
to N A D H oxidation in presence of oxaloacetate.
Reagents
Triethanolamine-HC1, 60 mM, containing 6 mM EDTA, pH 7.6
Oxaloacetic acid, 1.25 mM, unneutralized solution, prepared freshly
each day and stored in ice
NADH, 2.25 mM, prepared freshly each day and stored in ice
Procedure. Assays are carried out in cuvettes of 10 mm light path

maintained at 30 ° and containing 2.5 ml of triethanolamine-EDTA
buffer, 0.3 ml of oxaloacetic acid, and 0.2 ml of NADH. Reactions are
initiated by addition of 10 ~I of the various enzyme fractions diluted
appropriately with 0.1 M potassium phosphate buffer, pH 7.4. At inter-
vals of 30 seconds, absorbancy at 340 m~ is measured with a model
PMQII Zeiss spectrophotometer or recorded continuously using a Gilford
model 2000 multiple sample absorbance recorder. A linear relationship
generally is maintained for the first 5-6 minutes provided the changes in
absorbance do not exceed 0.035 per minute.
Units. A unit of enzyme activity is the amount of enzyme required to
oxidize 1 micromole of N A D H per minute under the conditions of assay
specified above. Specific activity is the number of units of enzyme activity
per milligram of protein; protein is determined by the method of Lowry
et al., 1 using crystalline bovine serum albumin as a standard.
Purification Procedure. A procedure for the preparation of crystalline
pig heart supernatant malate dehydrogenase has been reported. 2 More
recently, crystals of supernatant malate dehydrogenase have been
10. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem
193, 265 (1951).
2C. J. R. Thorne and P. M. Cooper, Biochim. Biophys. Acta 81, 397 (1963).
prepared from several other mammalian species2 The enzymes were

isolated in a form suitable for growing reasonably large crystals by
using a combination of the procedures of Englard and Breiger4 and that
of Thorne and Cooper, ~ followed by chromatography on DEAE-Sephadex.
The method reported for the preparation of crystalline beef heart muscle
extramitochondrial malate dehydrogenase4 is described below.
Unless otherwise indicated all operations are carried out at 3-5 ° . All
additions of solid ammonium sulfate are carried out at 0 °, the pH of the
mixture being maintained between 7.1 and 7.3 by dropwise addition of
approximately 2 N ammonium hydroxide.
Step 1. Fresh beef hearts, kept on ice prior to use, are dissected from
fat and connective tissue, diced, and passed through a mechanical meat
grinder. One kilogram of mince is suspended in 5 liters of ice-cold 0.25 M
sucrose buffered at pH 7.6 with 10 mM triethanolamine, and the mixture
stirred mechanically for 15 minutes. The suspension is passed through
several layers of cheesecloth and the residue is squeezed with a Pexton
hand press2 The initial extract usually contains a total of 30-36 g of
protein with a specific activity of 8.6-11.4. From the residue, after addi-
tional washings with cold distilled water, an acetone-dried powder is
prepared from which the mitochondrial malate dehydrogenase subse-
quently can be extracted and purified2-s
Step ~. The pH of the crude extract is raised to 7.2 by addition of
dilute ammonium hydroxide. Solid ammonium sulfate is added slowly to
4 0 ~ saturation (28.3 g/100 ml), and the mixture is stirred for 45
minutes. Hyflo Supercel, 1 g, (Johns-Manville Co.) is then added for
each 100 ml of suspension, and the mixture is filtered through Eaton and
Dikeman grade No. 192 folded filter paper. Ammonium sulfate (30.4
g/100 ml of initial extract) is added gradually to the clear filtrate to 82%
saturation; the mixture is then stirred for 60 minutes. The suspension is
centrifuged for 40 minutes at 18,000 g or alternately, is passed through a
KSB-R Servall 8-tube continuous-flow system at a rate of approxi-
mately 40 ml per minute at a rotor speed of 12,500 rpm. The super-
natant is discarded; the precipitate is dissolved in a solution of 50 mM
potassium phosphate buffer, pH 7.4, in 1 mM EDTA to a total volume
equivalent to 40% of the initial weight of the mincc (400 ml). This
fraction has a specific activity of 21.1-31.6.
L. J. Banaszak, J. Mol. Biol. 22, 389 (1966).

' S . Englard and H. H. Breiger, Biochim. Biophys. Acla 56, 571 (1962).
s Supplied by the Lee Engineering Company, Milwaukee, Wisconsin.
• L. Siegel, Ph.D. Thesis, Albert Einstein College of Medicine, Yeshiva University,
New York, 1962.
L. Siegel and S. Englard, Biochim. Biophl/s. Acta 54, 67 (1961).
SSee this volume [18].
[21] CYTOPLASMIC L-MALATE DEHYDROGENASE OF BEEF HEART 125
Step 3. The solution obtained in step 2 is next brought to approxi-

mately 35% saturation of ammonium sulfate by addition of the solid
salt (24.7 g/100 ml). The mixture is stirred for 45 minutes and centri-
fuged for 1 hour at 18,000 g; the residue is discarded. The clear super-
natant is heated rapidly to 60 ° in a boiling water bath (2¼ to 2~/~
minutes), and maintained between 62 ° and 64 ° for 10 more minutes.
After rapid cooling in a salt-ice bath at --10 °, the heat-treated solution
is kept at 0 ° for 30-45 minutes and centrifuged as before. Solid am-
monium sulfate is added to the supernatant to 43% saturation (5.7 g/100
ml), and the solution is stirred for 45 minutes. The suspension is centri-
fuged and the precipitate is discarded. The ammonium sulfate concen-
tration of the supernatant is raised to 72% saturation by further addition
of salt (20.4 g/100 ml), and the solution is stirred for 1 hour. The
precipitate is collected by centrifugation at 18#00 g for 90 minutes, dis-
solved in a minimum volume of 10 mM potassium phosphate buffer, pH
6.9, and dialyzed against this buffer for 18-24 hours; several changes of
dialyzing medium are made. The dialyzed solution is clarified by centrif-
ugation at 18,000 g for 75-90 minutes. Specific activity, 40.7-65.3.
Step 4. DEAE-cellulose with an exchange capacity of 0.7 meq per
gram is washed successively with potassium phosphate buffers, pH 6.7-6.9,
of decreasing concentrations starting with 200 mM. The anion exchange
cellulose finally is equilibrated with 10 mM potassium phosphate buffer,
pH 6.9. A slurry containing 50 g of equilibrated DEAE-cellulose is
poured onto a column of 4 cm internal diameter, and the cellulose is
permitted to pack by gravity and occasional gentle tapping to liberate
trapped air bubbles. Under these conditions a column height of 21.5-22
cm generally is obtained. The dialyzed solution from step 3 is passed
through this column, and 40 ml of 10 mM potassium phosphate buffer,
pH 6.9, is added above the DEAE-cellulose bed. A dropping funnel
containing the same buffer is then attached to the column and approxi-
mately 660 ml of effluent is collected (including the effluent obtained by
the initial passage of the dialyzed enzyme solution through the column).
This solution, deep red in color, contains 31-40% of the protein and
2.4-6.2% of the total units of malate dehydrogenase activity placed ini-
tially on the column. The enzyme in this fraction is sensitive to elevated
concentrations of oxaloacetate, and therefore appears to be malate dehy-
drogenase of mitochondrial origin. Pig heart and rat liver mitochondrial
malate dehydrogenases have been reported -~'9 to behave similarly on
DEAE-cellulose, being unretarded at pH 6.9 in potassium phosphate
and pH 8.2 in Tris buffer, respectively. The column is next connected to
a mixing flask containing 400 ml of 10 mM potassium phosphate buffer,
pH 6.9, which in turn is connected to a reservoir containing 50 mM
PC. J. R. Thorne, Biochim. Biophys. Acta 42, 175 (1960).
potassium buffer, pH 6.9. The mixing flask contains a rapidly spinning

magnetic bar, and in this manner the concentration of potassium phos-
phate in solution entering the DEAE-cellulose is increased gradually
and uniformly. Eluate is collected at a rate of 30-35 ml per hour. A 500
ml fraction containing insignificant malate dehydrogenase activity is
first collected. Small fractions (15-20 ml) are then collected with an
automatic fraction collector. Malate dehydrogenase of increased specific
activity emerges from the column in effluent fluid which has a phosphate
concentration of 28 raM. Fractions with increased specific activity (5-fold
or greater) are pooled, and the combined solution is reduced in volume
to 3-5 ml by means of pressure dialysis. The concentrated enzyme solu-
tion, slightly red in color, is dialyzed against sodium barbital buffer at
0.038 ionic strength at pH 8.6 for 22-24 hours; at least two changes of
buffer are made during the dialysis. At this stage of purification, the
specific activity ranges from 297.9 to 329.0.
Step 5. Mallinckrodt potato starch (500 g) is washed and equil-
ibrated with sodium barbital buffer of 0.038 ionic strength, pH 8.6. After
equilibration, a thick paste is made by suspending the starch in 225-250
ml of the same buffer. The paste is poured onto a hollow Lucite platform
with water at 0 ° circulating beneath it. Excess buffer is removed and
the starch block is trimmed to a size of 25.5 X 15 cm (height 1.0-1.2
cm). A strip 0.3-0.5 cm in width is removed from the center of the block.
The dialyzed solution obtained in step 4 is mixed with dry starch and
the heavy paste is poured into the prepared channel. Electrophoresis is
performed for 18-24 hours at a constant potential gradient of 350 V
(current initially, 27-29 ma). At termination of electrophoresis, the block
is sliced into 0.5 cm strips starting at the center and moving toward the
anode. Each starch sample is eluted by washing it several times with 3
ml portions of 100 mM potassium phosphate buffer, pH 7.4, on a sintered-
glass filter of medium porosity. The fractions obtained in this way are
adjusted to a total volume of 25.0 ml and assayed for enzymatic activity
and for protein content. Under normal conditions, malate dehydrogenase
with the highest specific activity and in greatest amount is located in a
1.5 cm band approximately 4.5 cm from the origin in the direction of the
anode, separated clearly from a slower moving diffuse light red band.
Since no loss of enzymatic activity is experienced in this step, the frac-
tions of lower specific activity can be combined, concentrated, and after
dialysis again subjected to electrophoretic separation in order to increase
further the total yield of material of high specific activity. 1°
'° The more active fractions, after a second electrophoretic run often yield some cuts
with specific activities equaling t h a t of the crystalline enzyme obtained subse-
quently. The total units of activity in these fractions, however, indicate a poorer
recovery t h a n t h a t obtained by inclusion of step 6 in the purification procedure.
[21] CYTOPLASMIC L-MALATE DEIIYDROGENASE OF BEEF HEART 127
Step 6. The electrophoretically separated fractions with similar

highest specific activities are combined (specific activity ranging from
493 to 541) and reduced in volume by means of pressure dialysis so that
a solution containing 25-30 mg of protein per milliliter is obtained. The
concentrated enzyme solution is then equilibrated by dialysis with 55%
saturated ammonium sulfate in 50 m M potassium phosphate buffer, pH
6.2 (prepared by diluting a neutralized solution of ammonium sulfate
saturated at 3-5 ° appropriately with 0 . 5 M potassium phosphate, pH
6.2, and water). If a turbidity develops at this stage, the solution is
clarified by centrifugation. The dialyzing medium is then changed to 5 8 ~
PURIFICATION OF EXTRAMITOCtIONDRIAL BEEF HEART
MUSCLE MALATE DEHYI)ROGENASE

activity protein activity Yield
Steps (units) a (rag) (units/rag) (%)
1. Crude extract 371,893 32,510 11.4 --

2. Ammonium sulfate fractionation 279,120 10,816 25.8 75.1
3. Heat inactivation and ammonium 292,518 5,769 50.7 78.7
sulfate fractionations
4. DEAE fractionation 164,737 552.9 298 44.3
5. Starch electrophoresis 125,604 232.3 541 33.8
6. Crystallization
First crystalline crop 79,996 131.6 608 21.5
First mother liquor 28,875 66.5 434 --
First recrystallization 52,495 72.7 722 14.1
Mother liquor of recrystallization 30,724 45.9 669 --
A unit is the amount of enzyme required to oxidize 1 micromole of NADH per
minute under the conditions specified.
ammonium sulfate in 50 m M potassium phosphate buffer, p H 6.2, and
dialysis is continued for at least 24 hours. The solution is centrifuged for
1 hour at 41,000 g to remove a slight amorphous precipitate that forms.
The clear supernatant is then dialyzed against 60% ammonium sulfate
in 50 m M potassium phosphate buffer, p H 6.2, and slow crystallization
ensues as evidenced by appearance of a characteristic silky sheen when
the dialysis bag is swirled. (Crystallization usually begins within 24-36
hours.) The crystals settle rapidly and are precipitated fully under these
conditions within 48-72 hours fl'om the time of appearance of the first
turbidity. 11 The crystals are harvested by centrifugation, dissolved in
0.1 M potassium phosphate buffer, p H 7.4, and recrystallized between 58
u If crystallization is incomplete, as revealed by the presence of significant levels of
malate dehydrogenase activity in the mother ~iquor, additional quantities of
crystalline material can be obtained by slowly increasing the ammonium sulfate
concentration of the dialyzing medium to 62--63% saturation.
128 REACTIONS ON TIIE CYCLE [21]
and 60% saturated ammonium sulfate in 50 mM potassium phosphate

buffer, pH 6.2, by means of the procedure just described. The specific
activity of the enzyme in the first crystalline crop and after one re-
crystallization is increased significantly, and repeated recrystallizations
do not lead to significant increases in specific activity. The specific
activity of the first mother liquor is substantially lower than that of the
first crystalline material, but mother liquors obtained after repeated
recrystallizations finally approach the specific activity of the crystals.
A summary of the purification is given in the table.
Properties 4,~-0
Kinetic Properties and Catalytic Specificity. At pH 6.7, the rate of
NADH oxidation reaches a maximum when oxaloacctate concentration
is 0.13 mM, and no inhibitory effects are observed at substrate concen-
trations as high as 1.9 raM. In presence of a constant concentration of
NADH (0.136 raM), the K,~ value for oxaloacetate is 42 ~M. The K,,
value for NADH, determined at a constant concentration of oxaloacetate
of 0.25 mM at pH 6.7, is 27 ~d~.
The rate of NAD ÷ reduction at pH 8.4 in Tris buffer reaches a
maximum at L-malate concentration of 16 raM, and inhibitory effects
are already evident at substrate levels of 39 raM. The K~, value, deter-
mined in the noninhibitory range of L-malate concentrations, is 0.47
mM at a constant NAD ÷ concentration of 0.535 mM. Under similar
conditions of assay, at a constant optimum concentration for L-malate
of 15.5 mM, a Km of 99 ~M for NAD ÷ is obtained.
a-Ketobutyrate and pyruvate are inactive as substrates. Although
NADH oxidation and NAD + reduction are detectable at high enzyme
concentrations in the presence of a-ketoglutarate or tartronate, respec-
tively, rates are extremely slow. With L-malate (0,1 M) the rate of NAD +
reduction is 94 micromoles per minute per milligram of protein at pH
10.0 in glycine-NaOH, compared with corresponding specific activities
of 0.32 for mesotartrate (0.16 M) and 0.44 for D- (--)-tartrate (0.16 M).
The enzyme is completely inactive with L-(+)-tartrate. At pH 6.7,
relative specific activities of 487 and 13 are obtained with oxaloacetate
(0.25 raM), and mesoxalate (8.3 raM), respectively. The rate of oxida-
tion of NADPH by oxaloacetate in glycylglycine at pH 7.5 is 1.1% of
that observed with NADH.
Molecular Properties. Thc enzyme appears to be homogeneous as
determined by ultracentrifugation and electrophoretic criteria. The maxi-
mum electrophoretic mobility for the enzyme is --5.62 X 10-~ cm 2 see-'
'' L. Siegel and S. EngIard, Biochim. Biophys. Acta 64, 101 (1962).
[22] MALATEDEHYDROGENASE (FAD-LINKED) FROM A. xylinum 129
V -1 (at pH 7.1), a value 2.5 times greater than that obtained for the
corresponding mitochondrial enzyme. The isoelectric point determined
from a pH vs mobility curve (pH of zero mobility) is 4.6-4.7. The
enzyme has an S~o,~, of 5.1 X 10-la sec and a D2o,~ of 9.1 X 10-7 cm2/sec.
From these data, assuming a partial specific volume of 0.74 ml/g, a
molecular weight of 52,000 is calculated. The amino acid composition of
the enzyme has been determined. TM The extramitochondrial malate de-
hydrogenase contains significantly more lysine, arginine, tyrosine, methi-
onine, aspartic acid, and tryptophan than does the mitochondrial enzyme,
and less phenylalanine, glycine, proline, and threonine. The enzyme
contains 6 sulfhydryl groups per mole and no disulfide linkages. Only
half of the sulfhydryl groups of the native protein can be titrated even
in the presence of excess p-chloromercuribenzoate with essentially no
loss of enzymatic activity.
[22] Malate Dehydrogenase (FAD-Linked) from

Acetobacter xylinum
By MO~HE BENZlMAN
b-Malate ~ oxaloacetate W 2 H + W 2 e
Assay Method
Principle. The routine method utilizes ferricyanide as the electron
acceptor for malate oxidation to oxaloacetate. The velocity of oxidation
is determined by following spectrophotometrically at 400 m~ the rate
of ferrieyanide reduction to ferrocyanide. Though, as outlined below,
other electron acceptors may be used also, ferricyanide has proved to be
the most effective oxidant with both the particulate and soluble enzyme?
Reagents
L-Malate, 0.5 M, pH 7.4
Tris-H~S04 buffer, 1 M, pH 7.4
KC1, 1 M
KCN, 0.1 M, pH 7.4, freshly prepared
Potassium ferricyanide, 6 mM. This reagent was prepared every 2
days and stored in tightly stoppered brown bottles.
Enzyme. A solution containing 1-4 units/ml (see definition of unit
below) was prepared by dilutiu~ with 50 mM Tris-H~SO~ pit 7.4,
containing 0.1 M KCl.
I M. Benziman and Y. Galanter, J. Bacteriol. 88, 1010 (1964).
V -1 (at pH 7.1), a value 2.5 times greater than that obtained for the
corresponding mitochondrial enzyme. The isoelectric point determined
from a pH vs mobility curve (pH of zero mobility) is 4.6-4.7. The
enzyme has an S~o,~, of 5.1 X 10-la sec and a D2o,~ of 9.1 X 10-7 cm2/sec.
From these data, assuming a partial specific volume of 0.74 ml/g, a
molecular weight of 52,000 is calculated. The amino acid composition of
the enzyme has been determined. TM The extramitochondrial malate de-
hydrogenase contains significantly more lysine, arginine, tyrosine, methi-
onine, aspartic acid, and tryptophan than does the mitochondrial enzyme,
and less phenylalanine, glycine, proline, and threonine. The enzyme
contains 6 sulfhydryl groups per mole and no disulfide linkages. Only
half of the sulfhydryl groups of the native protein can be titrated even
in the presence of excess p-chloromercuribenzoate with essentially no
loss of enzymatic activity.
[22] Malate Dehydrogenase (FAD-Linked) from

Acetobacter xylinum
By MO~HE BENZlMAN
b-Malate ~ oxaloacetate W 2 H + W 2 e
Assay Method
Principle. The routine method utilizes ferricyanide as the electron
acceptor for malate oxidation to oxaloacetate. The velocity of oxidation
is determined by following spectrophotometrically at 400 m~ the rate
of ferrieyanide reduction to ferrocyanide. Though, as outlined below,
other electron acceptors may be used also, ferricyanide has proved to be
the most effective oxidant with both the particulate and soluble enzyme?
Reagents
L-Malate, 0.5 M, pH 7.4
Tris-H~S04 buffer, 1 M, pH 7.4
KC1, 1 M
KCN, 0.1 M, pH 7.4, freshly prepared
Potassium ferricyanide, 6 mM. This reagent was prepared every 2
days and stored in tightly stoppered brown bottles.
Enzyme. A solution containing 1-4 units/ml (see definition of unit
below) was prepared by dilutiu~ with 50 mM Tris-H~SO~ pit 7.4,
containing 0.1 M KCl.
I M. Benziman and Y. Galanter, J. Bacteriol. 88, 1010 (1964).
Procedure. The following components were added in the order listed

to a cuvette of 1 cm light path and 1.2 ml capacity: Tris-H2S04 buffer,
0.1 ml; KCI, 0.1 ml; KCN, 0.1 ml; ferricyanide, 0.1 ml; water 0.48 ml;
suitably diluted enzyme, 0.1 ml; and L-malate, 0.02 ml. Absorbancy read-
ings were taken at 400 n ~ at 15 second intervals, over a 4 minute period.
At the proper concentration of the enzyme the reaction proceeded at a
linear rate over this period of time.
Units. One unit of enzyme is defined as that amount which catalyzes
the oxidation of 0.1 micromole of L-malate, or the reduction of 0.2 micro-
mole of ferricyanide (/xOD4~o,~ ~ 0.2) in 1 minute under the assay
conditions described. Specific activity is expressed as units per milligram
of protein. Protein is determined by the colorimetric method of Lowry
et al., 2 with crystalline bovine serum albumin as standard. The assay
method is equally applicable to particulate and soluble preparations and
is linear with enzyme concentration.
Other Assay Procedures. In particulate preparations oxidation of
malate by the respiratory chain could be determined with oxygen, with
2,6-dichlorophenol-indophenol, or with phenazine methosulfate as elec-
tron acceptors. 1 The rates of malate oxidation with these acceptors was
75, 40, and 65%, respectively, of the rate observed with ferricyanide.
However, after solubilization of the enzyme, reactivity with oxygen was
completely lost and that with dichlorophenol-indophenol was greatly
reduced. Reactivity with phenazine methosulfate remained unchanged.
Malate oxidation by beef heart cytochrome c was very low (4% that of
ferricyanide) even in crude preparations.
Growth of the Organism. Cellulose-synthesizing cells of A. xylinum
previously grown on succinate 8 were cultivated in the following medium
prepared with glass-distilled water: suceinic acid, 2%; yeast extract
(Difeo), 0.5%; Bacto peptone (Difco), 0.5%; and monopotassium phos-
phate, 0.3%. The final pH was adjusted to 4.0 with NaOH. The medium
was transferred into Roux flasks (100 ml per flask) and autoclaved.
Then 1.5 ml of culture stock in a test tube was transferred to 100 ml of
medium and incubated statically in a Roux flask (layer-thickness of
medium 1.0 cm) for 42 hours at 30 °. The contents of the flask was then
shaken vigorously to enhance the release into the medium of the cells
embedded in the surface pellicle. Ten-milliliter portions of the mixed
medium were used to inolculate other flasks, which were then incubated
for 42 hours, as described previously.
O. YI. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193,
265 (1951).
s M. Benziman and IT. Burger-Rachamimov, J. Bacteriol. 84, 625 (1962).
[22] M&LATEDEHYDROGENASE (FAD-LINKED) FROM A. xylinum 131
Harvest o] Cells. All operations were carried out at temperatures

below 6 °. The pellicles formed on the medium surface in the flasks were
cooled on ice (prepared from distilled water), drained, and then pulped
rapidly in a Waring blendor (1 minute, high speed) in 5 volumes of 50
mM Tris-H2S04 buffer, pH 7.4. The foam was skimmed off, and the
suspension of bacteria and cellulose fibrils was passed through 8 layers
of gauze cloth. Residues on the cloth and the separated foam were stirred
into fresh buffer and the procedure was repeated. The filtrates were
centrifuged at 10,000 g for 15 minutes, and the precipitate was washed
twice with the buffer and centrifuged. The packed cells were transferred
into glass tubes, frozen rapidly by dipping the tubes into a mixture of dry
ice and acetone, and stored at --20 °. The yield was 250 mg (dry weight)
of bacteria per liter of medium.
Preparation o] Crude Extract. Frozen cells were thawed and sus-
pended in 50 mM Tris-H2SO, buffer, pH 7.4, to a concentration of 30-60
mg (dry weight) of cells per milliliter. Portions of 20 ml of this suspen-
sion were treated in a cooled French pressure cell (American Instrument
Co., Silver Spring, Maryland) under 15,000 psi pressure. Cellular debris
and unbroken cells were removed by centrifugation at 9000 g for 10
minutes. The supernatant fluid, carefully removed with a pasteur pipette,
contained 15--30 mg of protein per milliliter (crude extract).
High-Speed Centri]ugation. The extract was centrifuged in a Spinco
model L preparative ultracentrifuge at 105,000 g for 60 minutes at 0 °.
The supernatant contained less than 10% of total activity and was
discarded. The precipitate containing most of the original activity was
suspended with a hand homogenizer in 50 mM Tris-H~S04 buffer, pH
7.4, to a protein concentration of 10-20 mg/ml.
Solubilization o] the Dehydrogenase. To the suspended particles was
added a solution of sodium desoxycholate to a final concentration of
0.5%, and the mixture was kept in ice for 30 minutes.
Ammonium Sul]ate Treatment. To the mixture of the previous step
was added solid ammonium sulfate to 30% of saturation (176 g per
liter). The salt was added slowly while the mixture was being stirred, and
precipitation was allowed to proceed for 15 minutes prior to centrifuga-
tion at 15,000 g for 15 minutes. The viscous precipitate which contained
no activity was discarded. The supernatant contained 50-55% of the
activity of the particulate fraction.
Sephadex Column. The ammonium sulfate supernatant was then
passed through a Sephadex column (Sephadex G-25, Pharmacia, Uppsala,
Sweden). The latter was prepared by suspending 20 g of Sephadex into
a chromatographic tube of 2 cm diameter. The column was washed with
5 mM Tris-H2S04 buffer, pH 7.4, then the protein was placed on the
Sephadex and eluted with the same buffer. The clear yellowish protein
132 REACTIONS ON TtIE CYCLE [22]
fraction contains the dehydrogenase, which remained in solution even

after 4 hours of centrifugation at 105,000 g.
A summary of the solubilization and purification procedures are
given in the table.
PURIFICATION PROCEDURE
Specific
Total activity
Total Units/ activity Protein (units/rag Recovery
Fraction volume ml (units)" (mg/ml) protein) (%)
Crude extract 37 52 1924 30.8 1.7 100

Precipitate of high-speed 33 57 1880 20.5 2.8 98
centrifugation
Suspension after 35 20 700 20.0 10 36
deoxycholate treatment
(NH~)2SO~ supernatant 35 22 750 1.8 12 39
Sephadex eluate 48 12.5 600 1.25 10 31
o See definition in section on Assay Method.
Properties
Prosthetic Group. The enzyme has a flavoprotein spectrum, exhibiting
in the oxidized form a major peak at 410 m s and a shoulder at 435-455
m~. Malate and more intensively hydrosulfite caused a bleaching with
a difference spectrum centering at 455 m~. The decrease in the intensity
of the 410 n ~ peak upon the addition of malate has been interpreted to
suggest the involvement of nonheme iron. 1 The activity of the soluble
enzyme was increased 20% by adding FAD (0.5 raM). Complete de-
pendency on added FAD was obtained by exposing the enzyme to p H 4.0
in strong ammonium sulfate solution. Such treatment resulted in com-
plete loss of malate oxidizing activity which was completely restored by
addition of FAD, but n o t F M N or riboflavin. The apparent Km for
FAD for the dissociated enzyme was 2 #M. 1
Malate-Quinone Reductase Activity. The enzyme in the particulate
form oxidized malate with the endogenous quinone (Qlo) or with mena-
dione at rates compatible with the overall oxidase activity. 4,s
Ef/ect o] pH on Activity. The p H optimum in the ferricyanide assay
in Tris buffer was in the range 7.2-8.0.
E)~ect o] Substrate Concentration. In the ferricyanide assay the Km
for L-malate was 0.5 raM. 1
' M. Benziman and L. Perez, Biochem. Biophys. Res. Commun. 19, 127 (1965).
5M. Benziman and H. Goldhamer, Bacteriol. Proc. p. 103 (1967).
Stability. The crude extracts could be stored in the frozen state for a
few months without appreciable loss of activity. The solubilized enzyme
stored in the presence of 0.1 M KC1 was stable for 5 days at 4 °.
Activators. Activity of the enzyme both in its particulate and soluble
forms was significantly stimulated by monovalent anions in the following
order: NO~- > Br- > C1- > CN- > CH~COO-2 At 0.1 M concentration
in the ferricyanide assay the stimulation was 7.6-, 5.7-, 4.3-, 3.5-, and
3.0-fold, respectively. The maximum effective concentration for any anion
or of a combination of anions was approximately 0.2 M. The nature of
the cation (K ÷, Na ÷, Tris ÷) did not affect the degree of stimulation.
Divalent anions as well as malate were without effect. Phosphate stimu-
lated to a degree compatible with its dissociation to a monovalent anion.
A similar pattern of monovalent anion stimulation was observed in crude
preparations assayed with oxygen as terminal acceptor. Additions of the
anions did not affect the K,~ for malate or the pH activity curve. Malate
oxidation by the particulate and soluble enzyme was stimulated up to
two times by 10 mM imidazole3 ,6 Imidazole stimulated even in the
presence of a high concentration of monovalent anions.
Inhibitors. Enzyme activity was not affected by oxaloacetate even
at 20 mM concentration. 7 Atabrine 0.3 mM inhibited activity by 60%.
At 0.9 mM it completely inhibited activity with ferricyanide or oxygen
as acceptors. The inhibition was reversed completely by FAD, but not
by F M N or riboflavin3 Chlorpromazine (1.2 raM) inhibited the par-
ticulate and soluble enzyme by 80%. Activity was completely restored
by FAD. Hematin (1 mM) inhibited 80%. Preincubation of the enzyme
with globin or imidazole prior to addition of hematin prevented the
inhibition. 1
Metal-Binding Reagents. 10 mM o-phenathroline, 3 mM 8-hydroxy-
quinoline, and 10 mM ad-dipyridyl when preincubated with the enzyme
caused, respectively, 60, 70, and 45% inhibition?
Thiol Reagents. p-Hydroxymercury benzoate (0.4 raM) caused a 75%
inhibition. With N-ethylmaleimide (3 mM) the inhibition was only
25%.~
Electron Tra~sport Inhibitors. The malatc dehydrogenase ill A.
x!]linum is linked to the cytochrome chain, probably through a quinone
(Q~o).5 The oxidation of malate via the respiratory chain was inhibited
completely by cyanide (1 mM) and azide (10 mM). Antimycin A was
not inhibitory even at a concentration of 10 ~g/ml. On the other hand
2-n-heptyl 4-hydroxyquinoline-N-oxide (HQNO), which is generally
believed to block electron transport in microorganisms between cyto-
Y. Karnieli and M. Benziman, Israel d. Cllem. 4, 72 (1966).
7M. Benziman and A. Abeliovitz, J. Bacteriol. 87, 270 (1964).
chrome b and c, 8 caused 85% inhibition of malate oxidase activity at

40 pit//. Malate oxidation with oxygen, endogenous or exogenous quinone,
phenazine methosulfate, or 2,6-dichlorophenol-indophenol, but not with
ferrieyanide, was 90% inhibited by 2 mM sodium amytal 1,5 in spite of
the nonparticipation of pyridine nucleotides in the reaction. ~ Irradiation
of crude extracts with light at 360 m/~ for 90 minutes decreased malate
oxidase activity by 80%. Full activity could be restored by addition of
0.10 mM menadione. The restored activity was inhibited completely by
1 mM cyanide. 4
Substrate Specificity. Oxidation of malate was 60% inhibited by
citrate or malonate at concentration equal to that of the substrate,
whereas m-tartrate, DL-tartrate, and isocitrate were inhibitory only when
present at a higher concentration. Of these acids only m-tartrate was
oxidized slightly (at 5% of the rate of malate) by the enzyme. Fumarate,
suceinate, and L-lactate were not oxidized. DL-Malate at a concentration
of 0.4 mM was oxidized at half the rate of L-malate. 1
Other Solubilizing Methods. Treatment of the particulate enzyme with
phospholipase A from snake venom (Crotalus atrox) or sonication of the
enzyme at alkaline or acid pH with different buffer systems were found
to be completely without effect. Incubation of the enzyme with sodium
lauryl sulfate or treatment with various organic solvents resulted in
complete loss of activity.
Other FAD-Malate Dehydrogenases. Other microorganisms have been
reported to contain nicotinamide nucleotide-independent enzyme systems
capable of oxidizing L-malate to oxaloacetate. In addition to A. xylinum
these systems have been demonstrated in Micrococcus lysodeikticus, 9
Mycobacterium avium, ~°,n Mycobacterium phlei, ~2 Pseudomonas ovalis
Chester, ~3 and Azotobacter vinelandii. ~4 In the first, second, and last cases
the organisms contain in addition a soluble NAD-linked malate dehy-
drogenase. The enzyme from Pseudomonas ovalis Chester was obtained
in soluble form by sonication and partially purified. In this form it
exhibited a requirement for FAD, quinone (Qg), and phospholipid.
Similar requirements were reported for the Mycobacterium phlei enzyme
with vitamin KgH as the natural acceptor, and for the M. avium enzyme
partially purified as the apoenzyme.
8j. W. Lightbown and F. L. Jackson, Biochem. J. 63, 130 (1956).

9 D. V. Cohn, J. Biol. Chem. 233, 299 (1958).
~"T. Kimura and J. Tobari, Biochim. Biophys. Acla 73, 399 (1963).
~'J. Tobari, Biochem. Biophys. Res. Commun. 15, 50 (1964).
12A. Asano, T. Kaneshiro, and A. F. Brodie, J. Biol. Chem. 240, 895 (1965).
'~P. J. R. Phizaekerley and J. O. Franc.is, Bioclwm. J. 101, 524 (1966).
" C . W. Jones and E. R. Redfearn, Biochim. Biopl, us. Acta 113, 467 (1966).
[23] MALATE DEHYDROGENASE FROM P. ovalis 135
[23] M a l a t e D e h y d r o g e n a s e ( F A D - L i n k e d ) f r o m
Pseudomonas ovalis C h e s t e r
B y P. J. R. PHIZACKERLEY
L-M~late --* oxaloacetate + 2 H + + 2 e
In Pseudomonas ovalis Chester, L-malate dehydrogenase is bound

firmly to the cell wall membrane, and the aerobic oxidation of L-malate
to oxaloacetate is dependent upon a chain of carriers which includes
fiavin, coenzyme Qg, and cytochromes.1 This organism does not contain
NAD-dependent L-malate dehydrogenase, EC 1.1.1.37; L-malate dehydro-
genase (decarboxylating), EC 1.1.1.40, is found in the soluble fraction
obtained by disrupting whole cells with ultrasound or by crushing in a
Hughes press. 2
Assay Method
Principle. The assay method is based upon the oxidation of L-ri~alatc
to oxaloacetate. The appropriate electron acceptor and the cofactor re-
quirements depend upon the degree of purification of the enzyme. The
particulate oxidase can utilize a variety of electron acceptors, including
2,6-dichlorophenol-indophenol, phenazine methosulfate, ferricyanide, or
02. Supplementation with cofactors is not required, though activity is
usually increased by including FAD and a quinone in the assay system.
The purified dehydrogenase, on the other hand, is inactive in the
absence of FAD, quinone, and phospholipid, and of the electron ac-
ceptors listed above can utilize only 2,6-dichlorophenol-indophenol and,
providing the quinone used in the assay system is autoxidizable, oxygen.
FAD cannot be replaced by FMN, but the enzyme is relatively un-
specific in its requirement for quinone and phospholipid. There is, how-
ever, a relationship between the nature of the quinone used in the assay
system and the nature of the phospholipid required for activation2 If
2-methyl-l,4-naphthaquinone (vitamin K3) or 2,3-dimethoxy-5-methyl-
1,4-benzoquinone (coenzyme Qo) is used, all phospholipids tested have
similar activating effects, but if a quinone with a long aliphatic side
chain, such as 2-methyl-3-phytyl-l,4-naphthaquinone (vitamin K~), or
(,oenzyme Q6 or coenzyme Q9 is used, only unsaturated phosphatidyl-
ethanolamine effectively activates the enzyme.
~M. J. O. Francis, D. E. Hughes, H. L. Kornberg, and P. J. R. Phizackerley,
Biochem. ]. 89, 430 (1963).
" D. E. Hughes, Brit. J. Exptl. Pathol. 32, 97 (1951).
~P. J. R. Phizaekerley and M. J. 0. Francis, Biochem. ]. 101, 524 (1966).
Spectrophotometric Assay o] Purified L-Malate Dehydrogenasc

This assay is carried out in cells of 1.8 ml capacity, 1 cm light path,
using a recording spectrophotometer. It is essential to clean the cells with
organic solvents after each assay to remove traces of phospholipid, and
the sequence water, ethanol, chloroform, ethanol, water is satisfactory.
Enzyme activity is affected by the sequence of addition of reagents, and
the order given has been found to give maximum activity. The technique
of mixing reagents is important; the most satisfactory method is to seal
the cell with thin aluminum foil (not Parafilm) and to mix by inversion
three times after each addition. If the cell contents are not mixed until
after the last reagent has been added, enzyme activity is reduced by
more than 50~.
Reagents
Tris-H3P04 buffer, 1 M, pH 7.2 (footnote 4)
Potassium L-malate, 0.1 M
Phospholipid-P in ethanol, ~ 1 mM
FAD, 0.1 mM
K:, in ethanol, 0.02 M
Freshly prepared 2,6-dichlorophenol-indophenol, 0.1%
Procedure. To the test and control cells are added water to bring the
final volume to 1.5 ml, Tris-phosphate buffer, 0.15 ml; phospholipid, 0.01
ml; enzyme; K3, 0.05 ml; FAD, 0.1 ml; L-malate, 0.15 ml (omitted from
control cell); and 2,6-dichlorophenol-indophenol, 0.05 ml. Enzyme ac-
tivity is calculated from the initial rate of decrease of extinction at 600
mt~ in the test cell read against the control cell. The initial reaction
velocity is constant until about 40% of the dye is reduced.
Units. One unit of enzyme catalyzes the oxidation of 1 micromole of
L-malate to oxaloacetate per minute. Since the millimolar extinction co-
efficient at 600 mtL for the reduction of 2,6-dichlorophenol-indophenol is
19.1,6 1 unit of enzyme in the present assay catalyzes a AEGooof 12.7 and
conversely AE6oo of 0.100 per minute is equivalent to 0.0079 unit of
4All pH measurements made at 20°.

~The apparent Michaelis constants of phospholipids found to be active in this
assay are: unsaturated phosphatidylethanolamine,0.1 #M; dipalmityl phospha-
tidylethanolamine, 2.4 t~M; unsaturated phosphatidylcholine from egg yolk, 0.1
~M; dipalmityl phosphatidylcholine, 0.6 t,M; sphingomyelin, 4.0 #M; phospha-
tidic acid, 0.03 t~M; cardiolipin from beef heart 0.02 pM. Phosphatidic acid and
cardiolipin inhibit at higher concentrations.
D. E. Green, S. Mii, and P. M. Kohout, J. Biol. Chem. 217, 551 (1955).
[23] MALATE DEHYDROGENASE FROM P. ovalis 137
enzyme. Specific activity is expressed as units of enzyme per milligram of

protein per milliliter.
Assay Using Other" Quinones. K~ can be replaced by coenzyme Qo
(0.05 ml of a 20 m M solution in water) without otherwise modifying the
assay. If however K1 (0.05 ml of a 1 m M solution in ethanol) or co-
enzyme Q~ or coenzyme Q9 (0.05 ml of a 20 ~M solution in ethanol) is
used, three changes are necessary. In the first place, unsaturated phos-
phatidylethanolamine must be used as the activating phospholipid. ~
Second, the sequence of addition of reagents should be altered so that
the enzyme is exposed to both phospholipid and quinone simultaneously
i.e., b y adding both phospholipid and quinone to the cells before adding
enzyme. Third, in order to obtain initial rates of reaction t h a t are
constan~ with time and proportional to e n z y m e c o n c e n t r a t i o n , it is nec-
essary to preincubate the enzyme with its cofactors. This is achieved
most simply by adding all the reactants except 2,6-dichlorophenol-
indophenol to the cells, which are then allowed to stand at room tem-
perature (about 20 °) for 5 minutes. D y e is then added and the assay is
completed.
Ultrasonic disruption is carried out in two stages, and with two
different objects in view. I n stage 2 of the purification procedure, the
object is to disrupt whole cells in such a way t h a t the m a x i m u m yield
of particulate L-malate oxidase is obtained, and in stag~ 3 the object is
to disrupt the particulate oxidase so as to give the m a x i m u m yield of
soluble L-malate dehydrogenase. The conditions required to achieve
these objects have been explored systematically, and it is important to
adhere to the conditions given below. The main purpose of the am-
7Unsaturated phosphatidylethanolamine constitutes about 65% of the phospholipid
in Pseudomonas ovalis Chester and can be conveniently prepared from the particles
obtained at step 2 of the purification procedure. The particles are extracted with
20 volumes of chloroform-methanol 2:1 (v/v) for 4 hours at room temperature with
shaking; after filtration the extract is washed as described by J. Folch, M. Lees,
and G. H. Sloane-Stanley [J. Biol. Chem. 226, 497 (1957)]. Silicic acid 20 g (100
mesh, Mallinckrodt Chemical Works) is washed as described by E. J. Barron and
D. J. Hanahan [J. Biol. Chem. 231, 493 (1958)], activated by heating for 14 hours
at ll0 °, well shaken in chloroform, and packed under gravity in a column 8.5
cm X 5 cm? The column is washed with 3 volumes of chloroform, and a volume of
the lipid extract containing 20 mg of phospholipid-P is applied to the column.
The column is eluted successively with 370 ral of chloroform, 180 ml of chloro-
form-methanol (20:1 v/v), and 270 ml of chloroform-methanol (9:1 v/v). The
flow rate is lff0 ml per hour, and 15 ml fractions are collected. The early fractions
eluted with chloroform-methanol 9:1 contain unsaturated phosphatidylethanol-
amine uncontaminated by other lipids.
monium sulfate fractionation is to remove much of the nucleic acid

from the enzyme preparation, and if this step is omitted, no purification
is achieved on DEAE-ccllulosc chromatography. The purification pro-
cedure has been repeated many times and has been found to be re-
producible.
Step 1. Maintenance and Growth of Organism. Pseudomonas ovalis
Chester is maintained on agar slopes consisting of: potassium phosphate
buffer, pH 7.2 (50 mM), ammonium chloride (50 mM), sodium succinate
(50 mM), essential salts (4 mg of CaC12.6 H20, 8 mg of MgS04.7 H.~O,
0.4 mg of MnS04.4 H20, and 0.4 mg of FeS04"7 H20 per 100 ml of
medium), solidified with 2% (w/v) of agar. Stock cultures of the or-
ganism are subcultured every 2 weeks, grown at 30 °, and stored at 2 °.
For growth in liquid medium, a loopful of organisms from a freshly
grown slope is transferred to a 2 liter conical flask containing 1 liter of
the above-mentioned medium with the agar omitted. Flasks are shaken
at 30 ° for 16--24 hours, and the cells are harvested by centrifugation at
2 °, resuspended in 10 volumes of 10 mM potassium phosphate buffer,
pH 7.2, centrifuged and stored at --15 ° until required. The yield is about
3 g .(wet weight) of hard-packed bacteria per liter of growth medium.
Step 2. Disruption of Cells with Ultrasound. The cells (100 mg dry
weight/ml) are suspended in 50 mM Tris adjusted to pH 6.0 with 1 M
HaPO~ and exposed to ultrasound in an M.S.E. ultrasonic disintegrator
operating at 1.5 A for 9 minutes at 0 °. The suspension is centrifuged at
12,000 g for 10 minutes at 2 ° to remove intact cells. The supernatant
fluid is then centrifuged at 100,000 g for 1 hour at 0 °. The supernatant
is discarded and the translucent red sediment, which consists of particles
derived from the cell wall membrane, is suspended in 3-5 volumes of 50
mM Tris-phosphate buffer pH 7.0, a loose-fitting Potter homogenizer be-
ing used. The centrifugation is repeated and the supernatant is again dis-
carded. The washed particles contain the L-malate oxidase activity of the
cells and can be stored at --15 ° for many months with little loss of
activity.
Step 3. Disruption o] Particles with Ultrasound. The particles (4.0 mg
of protein/ml 8) are suspended in 50 mM Tris-phosphate buffer pH 8.5 and
exposed to ultrasound as described above for 5 minutes at 0 °. The extract
is centrifuged at 100,000 g for 4 hours at 0 ° and the residue is discarded.
Step ~. Ammonium Sulfate Precipitation and Dialysis. The super-
natant fluid from step 3 is brought to 50% saturation with ammonium
sulfate by adding slowly with mechanical stirring a saturated solution
of metal-free ammonium sulfate at 0 °. After the preparation has stood
O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem.
193, 265 (1951); see Vol. III [73].
[23] MALATE DEHYDROGENASE FROM P , ovalis 139
for 30 minutes, the suspension is centrifuged and the superliatant is dis-

carded. The sediment is dissolved in the minimum volume of 50 mM
Tris-phosphate buffer pH 8.5 and dialyzed overnight in Cellophane tub-
ing against 20 mM Tris-phosphate buffer pH 7.0, at 2 °. The dialysis
residue is centrifuged on a bench centrifuge and the small precipitate is
discarded. It is convenient at this point to determine the content of
protein and nucleic acid in the supernatant fluid, by the method of
Warburg and Christian." The enzyme loses about 10% activity on stor-
age for 24 hours at 0 ° at this stage in purification.
Step 5. Column Chromatography on DEAE-cellulose. About 3.5 g of
diethylaminoethyl cellulose (DEAE-cellulose Whatman D E 50) re-
peatedly washed with 2 M NaC1 is used as a column 10.5 cm X 2.8 cm. ~
A layer of washed sand about 1 cm deep is placed on top of the DEAE-
cellulose, and the column is washed with 20 mM Tris-phosphate buffer,
pH 7.0, until the eluate runs constantly at pH 7.0. A volume of the
PURIFICATION PROCEDURE
Specific
volume Units/ activity Protein (units/mg Yield
Preparation (ml) ml (units) ~ (mg/ml) protein) (%)
Ultrasonic extract of cellsb . . . . 0. 017 --

Particles (step 2) ¢ 25 1.72 43 4.0 0.43 100
Supematant (step 3) 22.5 1.26 28,5 0.86 1.46 66
0-50% (NH4)~SO~ precipitate 17.5 1.28 22,4 1.0 1.28 52
after dialysis
NaCI eluate, 0.10 M ~ 30 0.28 8.4 0.03 9.3 19
One unit catalyzes oxidation of 1 micromole of L-malate per minute.

b L-Malate dehydrogenase activity was measured on a sample of cells disrupted by
ultrasound as described in step 2. The assay was carried out as described in the
text, but without added cofactors.
One gram dry weight of cells yields about 70 mg of particle protein.
The values refer to the most active fractions.
dialyzed fluid from step 4 is applied to the column such that the load of
protein is about 5 mg per gram of DEAE-cellulose and the load of
nucleic acid is less than 2 mg per gram of DEAE-cellulose. The column
is eluted stepwise at 4 ° with 20 mM Tris-phosphate buffer, pH 7.0, 50
mM Tris-phosphate buffer, pH 7.0, and then with 50 mM Tris-phosphate
buffer, pH 7.0, containing either 0.05 M NaC1 or 0.1 M :NaCh The col-
umn is run at a constant flow rate of 60 ml per hour maintained by a
micropump, and 10 ml fractions are collected. The extinctions at 260 m~
0. Warburg and W. Christian, Biochem. Z. 310, 384 (1941); see Vol. I I I [73].
and 280 m~ of each fraction is measured, and elution under a particular

condition is continued until no more UV-absorbing material is released.
About 100-150 ml of each eluting solvent is required. L-Malate dehydro-
genase is eluted in each of the eluates containing NaC1. The enzyme
eluted with 50 mM sodium chloride has the highest specific activity and
is virtually free of phospholipid. The enzyme eluted with 0.1 M NaC, I is
obtained in greater yield, but contains some phospholipid.
A summary of the purification procedure is given in the table.
Properties
Stability. The purified enzyme is unstable, and loses 85% of its
activity after 24 hours at 20 °, 50% at 0 °, and 70% at --15 °.
E n z y m a t i c Purity. The enzyme is free of all contaminating enzymes
for which tests have been made, and in particular does not contain
L-malate dehydrogenase (decarboxylating), DPNH dehydrogenase, suc-
cinate dehydrogenase, or cytochrome.
Kinetic Properties. The apparent Michaelis constant for L-malate is
0.45 m M ; for FAD, 0.40 #M; for phosphatidylethanolamine, 0.6 ~M; for
coenzyme Qg, 2.4 p2l/; and for vitamin K~, 3.0 mM.
Inhibition by Sodium Amytal. The enzyme is inhibited by 1.5 mM
sodium amytal. The inhibition markedly decreases as the concentration
of L-malate is reduced, and at concentrations of L-malate lower than
about 0.1 mM, amytal has a slight activating effect. The inhibition by
amytal is competitive with respect to K3, uncompetitive with respect to
phosphatidylethanolamine, and noncompetitive with respect to FAD.
Other Sources of Enzyme

Enzyme systems capable of oxidizing L-malate to oxaloacetate in the
absence of added nicotinamide nucleotides have been described in Pseudo-
monas fluorescens, 1° Serratia marcescens, n Azotobacter agilis/~ Micrococ-
cus lysodeikticus/3 Mycobacterium a v i u m / ' Mycobacterium phlei/5 and
Acetobacter xylinum. 16 The L-malate dehydrogenases of M. phlei ~7 and
M. avium TM have been purified and have been shown to have a triple
cofactor requirement for FAD, quinone, and phospholipid.
~*R. Y. Stanier, I. C. Gunsalus, and C. F. Gunsalus, J. Bacteriol. 66, 543 (1953).
*~A. W. Linnane and J. W. Still, Biochim. Biophys. Acla 16, 305 (1955).
t, M. Alexander and P. W. Wilson, J. Bacteriol. 71, 252 (1956).
~*D. V. Cohn, J. Biol. Chem. 233, 299 (1958).
~'T. Kimura and J. Tobari, Biochim. Biophys. Acta 73, 399 (1963).
15A. Asano and A. F. Brodie, Biochem. Biophys. Res. Commun. 13, 423 (1963).
~*M. Benziman and L. Perez, Biochem. Biophys. Res. Commun. 19, 127 (1965).
1:A. Asano, T. Kaneshiro, and A. F. Brodie, J. Biol. Chem. 240, 895 (1965).
~J. Tobari, Biochem. Biophys. Res. Commun. 15, 50 (1964).
[24] L-MALATIg. DEttYDI{.OGENASE FROM B. subtilis 141
[ 2 4 ] L - M a l a t e D e h y d r o g e n a s e f r o m Bacillus subtilis 1
[EC 1.1.1.37 I,-Malate:NAD oxidoreductase]
By AKIRA YOSHIDA
L-Malate + NAD+ ~ Oxaloacetate + NADtI + H +
Assay Method
Prinaip[e. The oxidation of L-malate is measured by the increase of
optical density at 340 n ~ caused by the reduction of NAD. Alternatively,
the reduction of oxaloacetate may be measured by the decrease in optical
density arising from the oxidation of NADH.
Reagents. For oxidation of L-malate:

L-Malic acid, monosodium salt, 0.1 M
NAD, 10 mM
Tris-HCl buffer, 0.1 M, pH 8.8
For reduction of oxalacetate:

Oxaloacetic acid, 0.1 M, neutralized with sodium hydroxide solution
NADH, 10 mM
Enzyme. The enzyme is dissolved in 10 mM Na2HPO~-KH2PO,, pH
7.2 to obtain a concentration of 0.05-5 units of enzyme per milliliter (see
definition below).
Procedure. For the oxidation of L-malate: 2.5 ml of 0.1 M Tris-HC1
buffer, pH 8.8; 0.1 ml of 0.1M sodium malate; 0.1 ml of 10 mM NAD;
0.01-0.1 ml of enzyme solution; and water to a final volume of 3.0 ml
are placed in a silica cell having a 1 cm light path. The increase of
absorbancy at 340 m~ is recorded on a logarithmic recorder connected to
an output spectrophotometer.
For reduction of oxaloacetate: 2.5 ml of 0.1 M Tris-HC1 buffer, pH
8.8; 0.05 ml of 0.1M oxaloacetate; 0.05 ml of 10 mM NADH, 0.014}.1
ml of enzyme solution, and water to a final volume 3.0 ml are placed in
a silica cell. The decrease of absorbancy at 340 m~ is recorded as
described previously.
Units. One unit of activity causes NAD to be reduced (oxidation of
malate) or N A D H to be oxidized (reduction of oxaloacetate) at an
initial rate of one micromole per minute at 25 °. The units of enzyme in
' S. Ochoa, Vol. I, p. 735.
the reaction mixture are obtained from the initial rate of increment or
diminution of absorbancy at 340 m~ using the following equation:
AOD in 1 minute
Units of enzyme in assay mixture = 6.22 X 3
Specific activity is expressed as the number of enzyme units per milli-

gram of protein.
Purification Procedure TM
Bacillus subtilis strain 60-180 (a prototroph, a derivative of the
Marburg strain) was grown in minimal glucose medium 2 consisting of
K~HP04, 14 g; KH2P0,, 6 g; sodium citrate, 1 g; ammonium sulfate,
2 g; MgS04-7 H20, 0.25 g, in 1 liter (adjust pH 6.8-6.9) supplemented
with 50 ml of 10% solution of glucose in 20 liter batches at 44-45 ° with
good aeration. An inoculum of 2 X 105 cells per milliliter gave a titer of
approximately 109 cells per milliliter after 16-18 hours of cultivation.
The cells were harvested in a Sharples centrifuge. The bacterial cells
were dried by acetone cooled at --20 °. Fifteen to twenty g of acetone-
dried cells were obtained. The acetone-dried cells can be stored in a
freezer for years without loss of the enzyme activity.
Step 1. Extraction. About 30 g of freshly harvested cells (or 10 g of
acetone-dried cells) were suspended in 200 ml of 50 mM Tris-HC1 buffer,
pH 8.0, containing 1 mM mereaptoethanol and digested with lysozyme
(0.5 mg/ml) at 37 ° for 30 minutes. Subsequently the cell suspension
was digested by 30 minutes of further incubation in the presence
of pancreatic ribonuclease (25 #g/ml), pancreatic deoxyribonuclease
(0.1 ~g/ml), and 2 mM MgC12. The liquefied extract was centri-
fuged at 30,000 g for 30 minutes. After addition of MnS04 to the super-
natant at a concentration of 50 raM, a precipitate formed which was
removed by centrifugation. The yield was about 104 units (reduction of
oxaloacetate) in 1.5 g of protein. All the subsequent procedures, including
chromatography and dialysis, were carried out in the cold.
Step 2. Fractionation with Ammonium Sul]ate. Ammonium sulfate
was added to the supernatant (60 g/100 ml) ; after the preparation had
stood for 1 hour, the precipitate was collected by centrifugation. After
removal of material which was soluble in 65% saturated ammonium
sulfate (saturated ammonium sulfate: water = 65:35, v/v), the insolu-
ble fraction was extracted with 45% saturated ammonium sulfate. The
extraction was repeated once more and the combined extract (about 120
ml) was reprecipitated by adding solid ammonium sulfate (30 g/100 ml
1~A. Yoshida, J. Biol. Chem. 240, 1113 (1965).
2C. Anagnostopoulos and J. Spizizen, J. Baeteriol. 81, 741 (1961).
[24] L-MALATE DEHYDROGENASEFROM B. subtilis 143
of the extract). The precipitate was suspended in about 50 ml of 10 mM

Na2HPO~-KH2P04 buffer, pH 7.7, and dialyzed against the same buffer.
Step 8. Calcium Phosphate Gel Column Chromatography. The di-
alyzed material was placed on a calcium phosphate gel (hydroxylapatite
prepared by the method of Tiselius, Hjert~n, and Levin 3) column
(2.5 X 30 cm) buffered with 10 mM Na_~HPO4-KH2PO4 (pH 6.8),
and was eluted with phosphate buffer, pH 6.8, at concentrations which
were gradually increasing from 10 mM to 0.2M at a flow rate of
25 ml per hour. The gradient was produced by adding 0.2 M phosphate
buffer to a mixing chamber which contained 500 ml of 10 mM buffer
(fixed initial buffer volume). Malate dehydrogenase was eluted at phos-
phate buffer concentrations ranging from 50 mM to 0.1 M. Enzyme
activity of the effluents was measured and the bulk of the enzyme frac-
tion was precipitated with ammonium sulfate (60 g/100 ml). The pre-
cipitate was resuspended in about 10 ml of 10 mM phosphate buffer, pH
7.7, containing 1 mM EDTA and dialyzed against the same buffer.
The yield of the enzyme was 9 X 103 units (reduction of oxaloacetate)
in about 200 mg of protein.
Step 4. DEAE-Sephadex Column Chromatography. The dialyzed
solution was placed on a DEAE-Sephadex column (1 X 30 cm) buffered
with 20 mM phosphate buffer, pH 7.7, containing 1 mM EDTA, and it
was eluted with increasing concentration of NaC1, from 0 to 0.4 M. The
gradient was produced by adding phosphate buffer containing 0.5M
NaC1 to a mixing chamber which contained 250 ml of phosphate buffer
(fixed initial buffer volume). The enzyme was eluted at NaCl concentra-
tions ranging from 0.3 M to 0.4 M. Enzyme activity of the effluents was
measured and the bulk of the enzyme fraction was precipitated with
ammonium sulfate (60 g/100 ml), redissolved in several milliliters of l0
mM phosphate buffer, pH 7.7, and dialyzed against the same buffer con-
taining 1 mM EDTA. The yield of the enzyme was about 7.2 X 10~ units
(reduction of oxaloacetate) in about 40 mg of protein.
Step 5. ECTEOLA Cellulose Column Chromatography. The dialyzed
solution was placed on an ECTEOLA cellulose column (1 X 30 cm)
buffered with 20 mM phosphate buffer, pH 7.7, containing 1 mM
EDTA. The protein was eluted by gradually increasing NaCl from 0 to
0.3 M. The gradient was produced by adding phosphate buffer containing
0.4 M NaC1 to a mixing chamber which contained 250 ml of phosphate
buffer (fixed initial buffer volume). The dehydrogenase was eluted at
N'aCl concentrations ranging from 0.15 M to 0.2 M. Enzyme activity of
the effluents was measured and the bulk of the enzyme fraction was
collected by precipitation with ammonium sulfate (60 g/100 ml).
3A. Tiselius, S. Hjert~n, and D. Levin, Arch. Biochem. Biophys. 65, 132 (1956).
Step 6. Crystallization. The precipitate was redissolved in the smallest

feasible amount of 50 mM phosphate buffer, pH 7.7, containing 1
mM EDTA. After a small amount of insoluble material had been
discarded by centrifugation, saturated ammonium sulfate solution was
added until the solution became slightly turbid. The crystallization was
accomplished after the solution had been kept overnight in the cold.
Recrystallization could be performed without a significant loss in
activity. Needlelike crystals were obtained. The yield of the enzyme was
about 3 X 103 units (reduction of oxaloacetate) in about 9 mg of protein
after reerystallization three times.
Properties
Enzymatic Properties 4
Specificity. None of the following analogs of L-malate was oxidized
by the enzyme: L-alanine, D-lactate, L-lactate, citrate, isocitrate, tar-
tronate, L-(+)-tartrate, D-(--)-tartarate, DL-fl-hydroxybutyrate, DL-t~-
hydroxyisovalerate, and fumarate. Mesotartrate was oxidized slowly
(0.5~ of the rate of L-malate at a concentration of 7.7 raM). None of
the following ketoacids were used as substrate: a-ketobutyrate, a-keto-
caproate, a-ketoisocaproate, oxalosuccinate, and pyruvate. Mesoxalate
was a weak substrate (maximum rate was 1.1~ of that of oxaloacetrate).
Since the enzyme has no transaminase activity ( < 0 . 0 0 1 ~ ) , it can be
used for the assay of transaminase activity. NADP and N A D P H were
inactive as substrates. 3-Acetyl-pyridineadenine dinucleotide (K,, = 50
tdl//, V ~ , relative to NAD = 1.4) and 3-acetyl pyridinedeaminoadenine
dinucleotide (K,, ----0.27 raM, V~,.~ relative to NAD ---- 1.9) were better
substrates than NAD (K~ ---=0.14 raM) itself.
Inhibitor. D-Malate (K~ -----7.7 mM), D-tartrate (Ks = 11 mM), L-
tartrate (Ks = 54 mM), and tartronate (Ks -----19 mM) are competitive
inhibitors. Various metal ions (Mg *÷, Ca**, Fe 3÷, Cu *+, Zn ÷*, Ag ÷, Hg ÷÷)
showed no, or very weak, inhibition, p-Chloromercuribenzoate had no
significant inhibitory effect after incubation for 30 minutes at 25 ° .
Effect o] pH. The optimal pH for the oxidation of L-malate was 9.0
(sodium carbonate-bicarbonate buffer) and that for the reduction of
oxaloaeetate was 9.6 (sodium carbonate-bicarbonate buffer).
Ef]ect o] Substrate Concentration. The Michaelis constants (K~) for
the primary substrates were oxaloacetate 61 ~M, N A D H 27 #M, L-malate
0.9 mM, and NAD 0.14 mM at pH 8.8. When the concentration of oxalo-
acetate exceeded 1 mM, partial suppression of the reaction was observed.
Turnover Number. Reduction of oxaloacetate: The approximate maxi-
mum specific activity that could be obtained at optimal pH (pH 9.6)
and under conditions of saturation of the enzyme with substrates is
'A. Yoshid~, J. Biol. Chem. ~,40~ 1118 (1~5).
[25] MALATI~. DEHYDROGENASE FROM E. coli 145
estimated as 810 at 25 ° . Since the molecular weight of the enzyme is

about 150,000, the maximal turnover number is about 120,000 moles of
substrate per minute per mole of enzyme.
Oxidation of L-malate: The approximatc maximum specific activity
of 210 at 25 ° at pH 9.0, and the maximal turnover number is 31,000 moles
of substrate per minute per mole of enzyme.
Stability. The enzyme can be stored in crystalline form in partially
saturated ammonium sulfate without loss of the activity for more than
a year. The enzyme is stable at neutral pH (pH 6.8-7.2) at 4 ° for at
least a month, but it is rapidly inactivated at 58 °, especially in the
absence of I~ADH. The enzyme is inactivated reversibly by dilution in
alkaline pH (above pH 8) as a result of dissociation of the active tet-
ramer into smaller subunits. N A D H and, although less effectively, adeno-
sine di- and triphosphates and Zn+÷ protected against the inactivation.
Physical and Chemical Properties. The molecular weight of the
enzyme is 148,000 ± 2000 (by sedimentation equilibrium method). The
sedimentation constant (S~o,~) is 6.15 S at a concentration of 0.3% of
the protein. The enzyme is composed of four identical or nearly identical
subunits of molecular weight 37,0002, ~ The subunits have the following
amino acid composition3: Asp3~, Thr27, Ser~3, Glu3s, Pro15, Gly3G, Ala29,
CysH1, Val3~, Mets, I1e22, Leu31, Tyr15, Phes, Lys21, His2 Arg15. The
enzyme has no tryptophan.
A. Yoshida, Biochim. Biophys. Acta 105, 70 (1965).
[ 2 5 ] M a l a t e D e h y d r o g e n a s e f r o m Escherichia coli
[EC 1.1.1.37 L-Malate:NAD oxidoreductase]
By WILLIAM H. MURPHEY and G. BAaRm KITTO
L-Malate + NAD ~ oxaloacetate + NADH + H +
Assay Method
The reagents and procedures for assaying malate dehydrogenase
activity are the same as those described for the preparation of malate
dehydrogenases from chicken and tuna hearts (see this volume [19]).
Twenty pounds of Escherichia coli strain B cells2 are thawed, washed,
and broken by sonication in 10 mM potassium phosphate buffer, pH 7.0,
I W. H. Murphey, C. Barnaby, F. J. Lin, and N. O. Kaplan, J. Biol. Chem. 242, 1548
(1967).
Obtained from Grain Processing Corp., Muscatine, Iowa.
[25] MALATI~. DEHYDROGENASE FROM E. coli 145
estimated as 810 at 25 ° . Since the molecular weight of the enzyme is

about 150,000, the maximal turnover number is about 120,000 moles of
substrate per minute per mole of enzyme.
Oxidation of L-malate: The approximatc maximum specific activity
of 210 at 25 ° at pH 9.0, and the maximal turnover number is 31,000 moles
of substrate per minute per mole of enzyme.
Stability. The enzyme can be stored in crystalline form in partially
saturated ammonium sulfate without loss of the activity for more than
a year. The enzyme is stable at neutral pH (pH 6.8-7.2) at 4 ° for at
least a month, but it is rapidly inactivated at 58 °, especially in the
absence of I~ADH. The enzyme is inactivated reversibly by dilution in
alkaline pH (above pH 8) as a result of dissociation of the active tet-
ramer into smaller subunits. N A D H and, although less effectively, adeno-
sine di- and triphosphates and Zn+÷ protected against the inactivation.
Physical and Chemical Properties. The molecular weight of the
enzyme is 148,000 ± 2000 (by sedimentation equilibrium method). The
sedimentation constant (S~o,~) is 6.15 S at a concentration of 0.3% of
the protein. The enzyme is composed of four identical or nearly identical
subunits of molecular weight 37,0002, ~ The subunits have the following
amino acid composition3: Asp3~, Thr27, Ser~3, Glu3s, Pro15, Gly3G, Ala29,
CysH1, Val3~, Mets, I1e22, Leu31, Tyr15, Phes, Lys21, His2 Arg15. The
enzyme has no tryptophan.
A. Yoshida, Biochim. Biophys. Acta 105, 70 (1965).
[ 2 5 ] M a l a t e D e h y d r o g e n a s e f r o m Escherichia coli
[EC 1.1.1.37 L-Malate:NAD oxidoreductase]
By WILLIAM H. MURPHEY and G. BAaRm KITTO
L-Malate + NAD ~ oxaloacetate + NADH + H +
Assay Method
The reagents and procedures for assaying malate dehydrogenase
activity are the same as those described for the preparation of malate
dehydrogenases from chicken and tuna hearts (see this volume [19]).
Twenty pounds of Escherichia coli strain B cells2 are thawed, washed,
and broken by sonication in 10 mM potassium phosphate buffer, pH 7.0,
I W. H. Murphey, C. Barnaby, F. J. Lin, and N. O. Kaplan, J. Biol. Chem. 242, 1548
(1967).
Obtained from Grain Processing Corp., Muscatine, Iowa.
containing 0.1 M fl-mercaptocthanol. The cellular debris is removed by

centrifugation, and the supernatant crude extract is treated in three steps
with protamine sulfate (1 ml of 1% solution per milligram of protein).
The resultant precipitated proteins are removed by centrifugation. The
protamine sulfate precipitates contain lactate dehydrogenase activity and
this procedure has been employed in the purification of this enzyme from
E. colt2 The supernatant fraction contains most of the malate dehydro-
genase activity of the initial cell extract.
The volume of the supernatant fraction is reduced, and the enzyme
is purified by dialysis in the cold against appropriate amounts of satu-
rated ammonium sulfate to yield at equilibrium a 50% saturated solu-
tion. 4 The precipitate is removed by centrifugation and the supernatant
fluid is brought to 70% saturation by dialysis against an appropriate
amount of saturated ammonium sulfate. The enzymatic activity during
this step is partially inhibited by the high concentrations of ammonium
sulfate, but full activity is restored upon dialysis against ammonium
sulfate-free buffers. The malate dehydrogenase activity is found in the
50-70% precipitate. The precipitate is dissolved in a minimal quantity
of 50 mM potassium phosphate buffer, pH 7.0, and dialyzed against
twenty volumes of the same buffer.
Sufficient DEAE-cellulose, equilibrated in 50 mM potassium phos-
phate buffer, pH 7.0, is added in a batch to the supernatant fluid to
absorb 95-99% of the malate dehydrogenase activity. The ion-exchange
resin is washed on a Biichner funnel with the above buffer until no further
protein is eluted. The enzyme is then eluted from the resin in a single
wash with 50 mM potassium phosphate buffer containing 50 mM potas-
sium chloride. The malate dehyrogenase is precipitated by dialysis in the
cold against saturated ammonium sulfate. The precipitate is dissolved in,
and dialyzed against, 50 mM Tris, 0.1 M KC1, pH 7.5, and then passed
through a Sephadex G-100 column (8 X 60 cm) equilibrated and eluted
with the same buffer. The fractions containing malate dehydrogenase
activity are combined, the enzyme is precipitated with ammonium sulfate,
as described above, and the precipitate is dissolved in, and dialyzed
against, 0.1 M potassium phosphate, pH 7.0, for assay. The preparation
is then dialyzed exhaustively against l0 mM Tris-HC1 buffer at pH 7.6
and chromatographed on a DEAE-cellulose column equilibrated with the
same buffer. After the column has been washed with 10 mM Tris-HCl, pH
7.6, the malate dehydrogenase activity is eluted by a linear gradient of
0-0.15 M NaCl ill the same buffer.
E. Tarmy, Ph.D. dissertation, Brandeis University, Waltham, Massachusetts, 1967.

Percentage saturation is based on Table I in A. A. Green and W. L. Hughes, Vol. I,
p. 67, even though the enzymesolutions are kept at 4°.
[25] M&LATE DEHYDROGENASE FROM E. coli 147
The malate dehydrogenase activity is precipitated by dialysis against

ammonium sulfate, as described above, and the precipitate is dissolved in
0.1 potassium phosphate buffer, pH 7.0. In our hands, the preparation
at this stage contained two minor contaminants as demonstrated by
electrophoresis. By slow additions of solid ammonium sulfate, these
contaminants were removed as amorphous inactive precipitates. Addition
of further ammonium sulfate led to the formation of crystalline E. coli
malate dehydrogenase. To ensure maximum purity, the enzyme is then
recrystallized twice more by the same procedure.
Properties ~
Physicochemical Characteristics. The malate dehydrogenase of E. coli
has a molecular weight of approximately 61,000 and an S 20,w ° value of
4.4. The size of this enzyme is therefore similar to that reported for
malate dehydrogenases of higher organisms and a number of other micro-
organisms. The E. coli enzyme has a molecular weight approximately half
that reported for the enzyme from Bacillus subtilis and other Bacillus
species. The E. coli enzyme has a E 1% lcm.28o,,~ = 3.39. This enzyme
contains 6 residues of cysteine per mole, in contrast to the B. swbtilis
enzyme which lacks this amino acid. Although the B. subtilis malate
dehydrogenase and mitochondrial malate dehydrogenases from verte-
brates lack tryptophan, the enzyme from E. coli contains approximately
4 residues per mole. The difference in tryptophan content is reflected in
the fluorescence spectrum of these enzymes. The proteins lacking trypto-
phan exhibit an emission maximum at about 307 m~ (excitation at 280
mt~), whereas the malate dehydrogenase from E. coli has a maximum at
330 n~. Reversible dissociation studies indicate that the E. coli enzyme
consists of two subunits.
Catalytic Properties. Under the routine assay conditions at 22 ° , the
crystalline E. coli malate dehydrogenase has a maximum specific activity
of 542 micromoles of D P N H oxidized per minute per milligram of pro-
tein. The apparent Km of oxaloacetate measured in 0.1 M potassium
phosphate, pH 7.3, in the presence of 0.14 mM D P N H was 50 td~r. The
pH optimum for oxaloacetate reduction is 9.0.
Immunological Properties. Rabbit antisera prepared against the E.
coli malate dehydrogenase show a single band of precipitation in
Ouchterlony double diffusion plates when tested against either the puri-
fied protein or against a crude, cell-free extract. The antisera to the E.
coli enzyme cross-react with malate dehydrogenases in crude extracts of
other bacterial species known to have malate dehydrogenases of 60,000-
70,000 molecular weight. No cross-reaction has been observed with the
malate dehydrogenases of Bacillus species which contain a malate
dehydrogenase of approximately 120,000 molecular weight.
[26] M a l a t ¢ D e h y d r o g e n a s e f r o m P e a Epicotyls
[EC 1.1.1.37 L-Malate: NAD oxidoreductase]
By DAVID D. DAVIES
Malate + NAD ~ Oxaloacetate ~ NADH..
Assay Method
Principle. Since the equilibrium favors the reduction of oxaloacetate
to malate, the reaction is measured by the fall in optical density at
340 rn~ due to the oxidation of NADH.
Reagents
NADH, 6 mM
Oxaloacetic acid, 3 raM, in 0.6?~ disodium ethylenediaminetetra-
acetic acid (EDTA)
Procedure. Activity is measured at 30 ° by following the oxidation

of NADH in a 1 cm light path cuvette containing buffer, 1 ml; NADH,
0.5 ml (0.4 micromole); oxaloaeetic acid and EDTA solution, 0.5 ml;
water and enzyme to give a final volume of 3 ml.
Provided the rate of change of optical density does not exceed 0.07
min -1 the velocity is proportional to the enzyme concentration.
Units. A unit of activity is the amount of enzyme producing an
extinction change of 1.0 min -1, and specific activity is the number of units
per milligram of protein.
Application o] Assay Method to Crude Tissue Extracts. Malate dehy-
drogenase activity is relatively high in plants and the assay can often
be applied directly to dilute homogenates provided NADH is not oxi-
dized or destroyed. Endogenous substrates are removed by passing the
crude extract through a column of BioGel P-10.
Peas are grown in darkness at 25 ° for 7 days. The epieotyls arc
removed and blended with ice-cold potassium phosphate buffer (pH 7.4,
0.2 M) in a Waring blendor, to give a 1:1 (w/v) homogenate.
Step 1. Ammonium Sul]ate Precipitation. The homogenate is
strained through a linen towel and cooled to 3-5 °, then ammonium
sulfate is added to give 50% saturation. After 10 minutes the precipitated
[25] MALATE DEItYDROGENASE FROM PEA EPICOTYLS 149
protein is collectcd by eentrifugation at 25,000 g for 5 minutes, dissolved

in potassium phosphate buffer (pH 7.4, 0.2 M), and dialyzed for 1 hour
against a flow of phosphate buffer (pH 7.4, 50 raM).
Step ~. Ammonium Sul]ate Fractionation. The dialyzed solution is
centrifuged at 25,000 g for 15 minutes then treated serially with am-
monium sulfate to give fractions precipitating at 0-25, 25-35, 35-45, and
45-55% saturation. The precipitate from each fraction is collected by
centrifugation at 25,000 g for 5 minutes, dissolved in phosphate buffer
(pH 7.4, 50 mM) and assayed for activity. The fraction with the main
activity (35-45~) is stored overnight at --15 °.
Step 3. Treatment with Calcium Phosphate. The active fraction is
thawed and dialyzed for 2 hours against a flow of 15 liters of dipotassium
hydrogen phosphate (1 raM). Calcium phosphate gel (8.3 mg/ml, dry
weight) is added in successive small portions (0.1 volume); after each
addition the suspension is kept for 10 minutes before the gel is removed
by centrifugation at 10,000 g for 3 minutes and the supernatant is as-
sayed for enzyme activity. Further additions of gel are made until the
specific activity of malate dehydrogenase reaches a maximum.
Step ~. Ion-Exchange Chromatography. The clear solution is poured
on a column of DEAE-cellulose, previously equilibrated with a solution
of dipotassium hydrogen phosphate (2 raM). The column is subjected to
gradient elution with a mixing volume of 1 liter of dipotassium hydrogen
phosphate (2 raM) and reservoir of phosphate buffer (pH 8.0, 0.2 M).
The column eluate is collected in 10-ml fractions and the fractions are
assayed for activity. Two peaks of activity are obtained. Tubes contain-
ing the peaks of activity are stored at --15 °. The result of the purifica-
tion is shown in Table I.
TABLE I
PURIFICATION OF MALATE DEHYDROGENASEFROM PEA ~,PICOTYL8

Volume activity protein activity Recovery Purifi-
Step (ml) (units) (mg) (units/rag) (%) cation
Extract 1500 75,400 16,200 4.6 -- --

First (NH~)~SO4 220 22,500 2,100 10.7 30 2.3
precipitate
Second (NH4)~SO4 50 14,200 350 40.6 19 9
fractionation
Gel supematant 72 12,300 180 67 16 15
Combined fractions 60 4,100 17 240 6 52
from first peak on
DEAE-cellulose
Purification ]rom Other Plant Materials. Malate dehydrogenase may
be purified from the florets of cauliflower by the method described for pea
epicotyls2 A purification procedure for barley seedling, which omits
the calcium phosphate step, has been published; 2 it gives a 46-fold
purification. The enzyme has also been purified 100-fold from acetone
powders of spinach2
Properties
Isoenzymes. At least three isocnzymes of malic dehydrogenase can be
isolated from higher plants. The three isoenzymes of maize roots can be
separated by starch gel electrophoresis.~ All three isoenzymes are
found in the supernatant after the mitochondria have been removed, and
two of the three isoenzymes are associated with the mitoehondria. The
first peak of malate dehydrogenase activity eluted from the DEAE
column probably corresponds to the mitochondrial forms of the enzyme
and the second peak to the supernatant forms.
Kinetic Constants. Differences between malic dehydrogenase prepared
from the mitochondria and from the supernatant have been noted
(Table II).
TABLE II
KINETIC CONSTANTS OF MALATE DEHYDROGENASE
K 0AA K NADH K malate K NAD

Source of enzyme pH (~M) (~M) (raM) (mM)
Pea mitochondria 7.0 125 120 -- --

Pea supernatant 7.0 74 120 -- --
Barley mitochondria 8.2 -- -- 50 1.7
Barley supernatant 8.2 -- -- 8.2 0.3
Substrate Specificity. Malate dehydrogenase is an a-hydroxydicarbox-

ylic acid dehydrogenase. A preparation from pea epicotyls (specific
activity 240) gave the following K~ values: K~ oxaloacetate = 90 #M,
K~ dioxosuccinate ---- 7 mM, K~ oxomalonate ---- 25 mM.
Stability. The purified enzyme retains its activity for at least 9
months when stored at --15 ° .
' D. D. Davies, Biochem. J. 80, 93 (1961).

2S. B. Yue, Phytochemistry 5, 1147 (1966).
SA. J. Hiatt and It. J. Evans, Plant Physiol. 35, 662 (1960).
~I. P. Ting, I. W. Sherman, and W. M. Dugger, Plant Physiol. 41, 1083 (1966).
PREVIOUSLY PUBLISHED ARTICLES FttOM METIIODS IN ENZYMOLOGY
RELATED TO SECTION II
Vol. I [124]. "Malic" Enzyme. Severo Ochoa.

Vol. I [125]. Oxalacetic Carboxylase of Micrococcus lysodeiklicws. Denis Herbert.
Vol. I [126]. Oxalacetate Synthesizing Enzyme. Merton F. Utter and Kiyoshi
Kurashashi.
Vol. II [53]. Aspartase. Artturi I. Virtanen and Nils Ellfolk.
Vol. V [81]. Itaconic Acid Enzymes. Ronald Bentley.
Vol. V [84]. Conversion of P-Pyruvate to Oxalacetate (Plant). Birgit Vennesland.
Vol. V [85]. Citratase and Isocitratase. H. YI. Daron and I. C. Gunsalus.
Vol. V [86]. Malate Synthetase from Baker's Yeast. G. H. Dixon and 1:t".L. Komberg.
Vol. V [87]. Oxalate Decarboxylation. William B. Jakoby.
Vol. V [88]. Citrate-Cleavage Enzyme. Paul A. Stere.
VoL IX [63]. Glyoxylate Dehydrogenase. J. R. Quayle.
[27] ATP CITRATE LYASE 153
[27] A T P C i t r a t e L y a s e ( C i t r a t e - C l e a v a g e E n z y m e )
[EC 4.1.3.8 ATP: citrate oxaloacetate-lyase (CoA-acetylating and
ATP-dephosphorylating)]
By YOSHIRO TAKEDA, FUJIO SUZUKI, a n d HIDEO INOUE
Mg++
Citrate -t- ATP -t- CoA ~ acetyl-CoA -~ oxaloacetate -t~ ADP + P,
Assay Methods
Principle. ATP citrate lyase is assayed by determining the amount
of acetyl-CoA or oxaloacetate formed. Three" methods have been em-
ployed. (1) The hydroxamate method: The acetyl-CoA formed is trapped
as acetylhydroxamate and the latter is determined by the color produced
with FeCls. 1 (2) The spectrophotometric method: The oxaloacetate
formed is measured by its reaction with NADH in the presence of malate
dehydrogenase.2 (3) The isotopic method: Citrate-l,5-1'C is incubated
with ATP, CoA, Mg ÷÷, and enzyme, and the oxaloacetate-14C formed is
degraded according to the method of Krebs and Eggleston2 The ~4C02
evolved is trapped and counted.', 5 The hydroxamate method is applicable
to only a narrow range of enzyme concentrations, but it is useful in
purification steps 1 through 3 because cruder preparations contain a
significant activity of endogenous NADH oxidation which sometimes
interferes with the use of the spectrophotometric method. The spectro-
photometric method is used in the subsequent steps of purification {steps
4 through 7). Because of its high sensitivity, the isotopic method is
employed when the ATP citrate lyase activity in some tissue is so low
that the methods (1) and (2) cannot be applied. It can also be used when
one of the reaction products, acetyl-CoA or oxaloacetate, is present in the
reaction system.
The Hydroxamate Method
Reagents
Tris buffer, 0.5 M, pH 8.4
MgCI2, 0.2 M
l p. A. Srere and F. Lipmann, J. Am. Chem. Soc. 75, 4874 (1953). See also Vol. V
[ss].
s p. A. Srere, J. Biol. Chem. ~ 2544 (1959).
' H. A. Krebs and L. V. Eggleston, Biochem. J. 39, 408 (1945).
H. Inoue, F. Suzuki, K. Fukunishi, K. Adachi, and Y. Takeda, J. Biochem. 60, 543
(1966).
F. Suzuki, K. Fukunishi, Y. Daikuhara, and Y. Takeda, J. Biochem. 62, 170 (1967).
154 REACTIONS LEADING TO AND FROM THE CYCLE [27]
2-Mercaptoethanol, 0.2 M
Potassium citrate, 0.2 M
CoA, 2 mM
Hydroxylaminc, 2 M, adjusted to pH 8.4 with KOH
ATP, 0.1 M
Trichloroacetic acid, 20%
FeC13, 2 M
Procedure. The following components are added: Tris buffer, 0.4 m];
MgCl:, 0.05 ml; 2-mercaptoethanol, 0.05 ml; potassium citrate, 0.1 ml;
CoA, 0.05 ml; hydroxylamine, 0.1 ml; enzyme to be assayed; and water
to make a total volume of 0.95 ml. A blank tube is prepared with all
components except CoA. The reaction is started by adding 0.05 ml of
ATP. After 30 minutes at 37 °, 1.2 ml of trichloroacetic acid and then
0.3 ml of FeC13 are added; the acetylhydroxamate formed is measured at
520 mt~.
The Spectrophotometric Method

Reagents
MgCl~, 0.2 M
Potassium citrate, 0.2 M
CoA, 2 mM
ATP, 0.1 M
NADH, l0 mM
Malate dehydrogenase, 20 units/ml
Procedure. The following components are added to a 1.5 ml silica cell
(length = 1 cm): Tris buffer, 0.4 ml; MgCl~, 0.05 ml; 2-mercaptoethanol,
0.05 ml; potassium citrate, 0.1 ml; ATP, 0.1 ml; NADH, 0.02 ml;
malate dehydrogenase, 0.01 ml; enzyme to be assayed; and water to
make a total volume of 0.90 ml. A blank cell is prepared with all
components except ATP and CoA. The reaction is started by adding
0.1 ml of CoA. The decrease in absorption at 340 n ~ at 37 ° is measured.
The Isotopic Method

Reagents
MgCI2, 0.2 M
[27] ATP CITR&TE LYASE 155
Potassium citrate-l,5-~4C (0.5 ~C per micromole), 20 mM

CoA, 2 mM
ATP, 0.1 M
Oxaloacetic acid, 0.1 M, in 2 N HC1
Phthalate buffer, 0.75 M: 15.3 g of potassium hydrogen phthalate
and 1.8 g of N a 0 H in 100 ml of water
A12(SO4)~" 18 H20, 33.3~'o
Hyamine, 1 M, in methanol
Procedure. Incubation is carried out in a double-armed Warburg flask

equipped with a serum bottle stopper. The flask contains Tris buffer, 0.2
ml; MgC12, 0.05 ml; 2-mereaptoethanol, 0.05 ml; CoA, 0.05 ml; potas-
sium citrate-l,5-14C, 0.1 ml; enzyme to be assayed; and water to make
a total volume of 0.90 ml in the main compartment, and 0.1 ml of ATP
in a side arm. A blank flask is prepared with all components except
CoA. The reaction is started by adding ATP. After incubation for an
appropriate time at 37 °, the reaction is stopped by addition of 0.2 ml of
oxaloacetic acid in 2 N HC1. For the degradation of oxaloacetic acid,
0.5 ml of phthalate buffer is placed in one side arm, 0.5 ml of aluminum
sulfate solution in the other, and 0.2 ml of methanotic hyamine solution
in the center well. After equilibration for 10 minutes at 25 °, the tap is
closed and the phthalate buffer is introduced from the side arm followed
by the aluminum sulfate solution. The flask is shaken for another 75
minutes at 25 °. The 1~C02 evolved is trapped by absorption on methan-
olic hyamine solution in the center well. Then the hyamine solution is
transferred to scintillator-toluene solution with 0.5 ml of methanol.
Radioactivities are measured in a liquid scintillation spectrometer.
Protein Determination. Protein concentrations are determined by the
biuret method 6 in the crude extract and by the procedure of Lowry
et al. 7 in the subsequent steps.
Units. One unit of enzyme is defined as the amount of enzyme that
forms 1 micromole of acetylhydroxamate, oxidizes 1 micromole of
NADH, or evolves 1 micromolc of 1~C02 per minute at 37 °.
Treatment of Animals. Adult albino rats are pretreated with a high-
sucrose diet in order to induce ATP citrate lyase in the liver. The
animals are starved for 2 days and then fed on a high-sucrose diet for
8A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949).
See also Vol. III, p. 450.
265 (1951). See also Vol. III, p. 448.
3 days before being killed.'. 8 The high-sucrose diet contains 63% sucrose,
30% casein, 4% salt mixture, 2% cellulose powder, l~b vitamin mixture,
and 0.1% choline chloride. This diet results in about 10-fold the normal
level of the enzyme in the liver.
Step 1. Preparation o] the Crude Extract. All operations are carried
out at 0-4 °. Fresh livers (550 g), from rats which have been fed on a
high-sucrose diet, are homogenized in 4 volumes of 0.25 M sucrose con-
taining 20 mM Tris buffer (pH 8.0) with the aid of a Potter-
Elvehjem glass homogenizer. The homogenate is centrifuged at 15,000 g
for 30 minutes and then 105,000 g for 30 minutes. To the supernatant
fluid thus obtained (crude extract), 1 M Tris buffer (pH 8.0) is added
to adjust the pH to about 7.6 and then 2-mereaptoethanol and MgCI2 are
added to give final concentrations of 10 mM and 1 raM, respectively, in
order to stabilize the enzyme. All subsequent steps are performed in the
presence of these two agents.
Step ~. Ammonium Sulfate Fractionation. To the crude extract, am-
monium sulfate is added to 25% saturation. The precipitate formed is
removed by centrifugation and discarded. Further ammonium sulfate
is then added to the supernatant to 45~ saturation. The resulting
precipitate is collected by eentrifugation and dissolved in 0.01 M Tris
buffer (pH 7.8). The enzyme solution is dialyzed overnight against
30 volumes of the same buffer.
Step 3. DEAE-Cellulose Column Chromatography. The dialyzed en-
zyme solution containing 25.3 g of protein is diluted with 0.01 M Tris
buffer (pH 7.8), to give 20 mg of protein per milliliter, and then applied
to a DEAE-cellulose column (Sigma, medium mesh; 8 cm in diameter
and 35 cm in height), equilibrated with 0.005 M Tris buffer (pH 7.8).
Elution is effected by using a continuous gradient (convex shape) of KC1
in 0.005 M Tris buffer (pH 7.4) with an initial concentration of 0.02 M
in the mixing chamber (3 liters) and 0.4 M in the reservoir (2 liters). The
eluate is collected in 20-ml fractions in tubes containing 1 ml of 1 M Tris
buffer (pH 7.4) at a flow rate of 4 ml per minute. The enzyme activity is
usually eluted between 2000 and 2500 ml. Fractions having a specific
activity of over 0.4 are pooled and concentrated with ammonium sulfate
at 40% saturation. After centrifugation, the precipitate is dissolved in a
small volume of 0.005 M Tris buffer (pH 7.4) and dialyzed overnight
against 200 volumes of the same buffer.
Step ~. Alumina C~ Gel Treatment. Step 3 enzyme is adjusted to pH
6.8 with 1 M acetate buffer (pH 5.0) and then mixed with an amount
of alumina Cv gel equivalent to that of the protein. The mixture is
a M. S. Kornacker and J. M. Lowenstein, Biochem. J. 94, 209 (1965); ibid. 95, 832
(1965).
stirred for 15 minutes, then centrifuged; supernatant fluid is discarded.

The precipitate is washed twice with 200 ml portions of 10 mM potas-
sium citrate (pH 7.4), and then the enzyme is eluted successively with
three 200 ml portions of 0.15M and finally with 200 ml of 0.3M
potassium citrate (pH 7.4). The eluates obtained with 0.15 M and 0.3 M
potassium citrate are combined and concentrated with ammonium sulfate
at 40% saturation. After centrifugation, the precipitate is taken up in a
small volume of 5 mM Tris buffer (pH 7.4) and dialyzed overnight
against 200 volumes of the same buffer.
Step 5. Brushite Column Chromatography. The dialyzed enzyme of
step 4 is applied to a column of brushite (1 cm 3 of brushite per milligram
of protein) equilibrated with 5 mM potassium phosphate buffer (pH 7.4).
After washing the column with a bed volume of the same buffer, elution
is carried out with 70 mM potassium phosphate buffer (pH 7.4) at a
flow rate of about 1 ml per minute. Fractions having a specific activity
of over 3.8 are collected.
Step 6. First Gel Filtration on Sephadex G-200. Step 5 enzyme is
precipitated by addition of ammonium sulfate to 40% saturation and
dissolved in a small volume of 10 mM Tris buffer (pH 7.4) containing
50 mM potassium citrate (pH 7.4). Then the enzyme is applied to a
column of heat-treated Sephadex G-2009 (equivalent to 50 volumes of the
enzyme solution), which has been equilibrated with the same buffer.
The column is eluted with the same buffer in 3 ml fractions at a flow
rate of about 15 ml per hour. The elution pattern of protein usually
gives two peaks. The first is the major peak and the second is a minor
peak. The enzyme activity is associated with the first major peak, and
colored impurities, if any, with the second minor peak. Active fractions,
having a specific activity of over 5.5, are combined.
Step 7. Second Gel Filtration on Sephadex G-200. The combined
active fractions of step 6 are concentrated by treatment with 40%
saturation of ammonium sulfate and then subjected to a second gel
filtration treatment on Sephadex G-200 in the same way as described
in step 6. Active fractions with the same specific activity (6.25) are
collected and combined. The purification of the enzyme is summarized in
the table.
Comments. During the course of purification, some variations have
been encountered in the brushite step. The separation of the enzyme and
colored impurities (brownish yellow) in this step is satisfactory in most
' S e p h a d e x G-200 (Pharmaeia) is suspended in distilled water and boiled for 30

minutes. Then, the particles are w:(shed 20 times by decantation with warm dis-
tilled water at 60 ° to remove fine material and stored in 10 m M Tris buffer (pH 7.4)
containing 50 m M potassium citrate ( p H 7.4) and 1 m M MgCl~.
SUMMARY OF PURIFICATION PROCEDURE
Total
Protein activity Specific Recovery
Step (rag) (units) ° activity (%)
1. Crude extract 56,100 6,452 0.12 100

2. Ammonium sulfate 25,300 5,060 0.20 78.4
fractionation
3. DEAE-cellulose column 3,210 4,350 1.36 67.4
chromatography
4. Alumina C~ gel 2,040 4,202 2.06 65.1
treatment
5. Brushite column 541 2,299 4.25 35.6
chromatography
6. First gel filtration on 288 1,713 5.95 26.5
Sephadex G-200
7. Second gel filtration 220 1,375 6.25 b 21.3
on Sephadex G-200
o See section on units under assay methods.
b This value is slightly higher than that previously reported (see text footnote 4).
However, the dilution factor of the enzyme at the last step is so great that the
highest specific activities vary from 5.9 to 6.3 under the assay conditions.
cases, although on some occasions the enzyme is retained more tightly on

the column and is eluted not with 70 mM, but with 0.3 M, potassium
phosphate buffer (pH 7.4). In this case, there is more trailing of the
enzyme peak and a higher percentage of its activity, up to 3 0 - 4 0 ~ , is
eluted with the colored impurities. The reason for this is not clear, but
these impurities can be removed in the subsequent step, step 6. The
DEAE-cellulose and the Sephadex G-200 column steps are highly re-
producible.
Crystallization. Crystallization of the enzyme is carried out as fol-
lows. Step 7 enzyme is precipitated by addition of ammonium sulfate to
5 0 ~ saturation. After centrifugation, the precipitate is extracted suc-
cessively with 40, 35, 30, and 25% ammonium sulfate solutions (3 ml
each) containing 10 m M Tris buffer (pH 7.4), 50 m M potassium citrate
(pH 7.4), 10 m M 2-mercaptoethanol and 1 m M MgCl~. Each extract is
stored at 4 ° . After several days, small colorless plates appear in the
extract with 30% ammonium sulfate. The specific activity of the enzyme
remains the same after crystallization.
Properties
Purity. The purified enzyme moves as a single homogeneous protein
on sedimentation and on moving boundary electrophoresis. The homo-
geneity of the enzyme is also demonstrated by immunological criteria.

The purified enzyme contains no chromophore and is also free of nucleo-
tides and nucleic acids. Assays for citrate synthase, isocitrate, dehydro-
genase (NADP), malate dehydrogenase, aconitate hydratase, reduced
NADP dehydrogenase, adenylate kinase, acetyl-CoA carboxylase, and
fatty acid synthase are all negative.
Molecular Weight. The sedimentation coefficient calculated for water
at 20 ° and zero protein concentration is 13.5 S and the diffusion constant
(D2o, w) is found to be 2.62 X 10-7 cm2/sec. From these values, the
molecular weight of the enzyme is calculated to be approximately 500,000,
assuming a partial specific volume of 0.75.
Specificity. The enzyme is highly specific for citrate. Other tri-
carboxylic acids tested, such as tricarballylate, c/s- and trans-aconitates
and DL-isocitrate, are not attacked at any detectable rate. The enzyme
is specific also for ATP, and other triphosphonucleotides tested, such as
CTP, GTP, and UTP, have little or no effect. The activity of this
enzyme depends on the presence of Mg +÷. Mn ++ can partially substitute
for Mg +÷, but Ni ÷÷, Fe ÷÷, Fe ~÷, Cu *+ and Zn +÷ are all inactive. The
enzyme requires sulfhydryl compounds for maximal activity. 2-Mercap-
toethanol, cysteine, glutathione, and dithiothreitol are all nearly equally
effective.
Stability. The purified enzyme preparation is labile on storage and
retains only about a quarter of its activity after 3 days and one-tenth
after 5 days when stored under air at 3 ° in 10 mM Tris buffer (pH 7.8).
The inactivation of the enzyme can be effectively prevented by the
addition of sulfhydryl compounds, such as 2-mercaptoethanol, gluta-
thione, or cysteine, together with MgC12. The most satisfactory method
for storage is the use of dithiothreitol. In the presence of this reagent
at a concentration of 10 mM together with 1 mM MgCl~, the enzyme is
quite stable at 3 ° under an atmosphere of nitrogen for at least 2 weeks.
Freezing of the enzyme in the presence of sulfhydryl compounds is not
recommended for storage.
pH Optimum. With Tris buffer, maximal activity is obtained at about
pH 8.4. At pH 7.0 and 9.0, the activity is about 60% of that seen at pH
8.4.
K,~ Values. The Km values for citrate and ATP at pH 8.4 are found
to be 0.56 mM and 0.172 raM, respectively.
Inhibitors. ADP, one of the reaction products, inhibits the reaction
competitively with respect to ATP. The Ki value for ADP at pH 8.4 is
0.172 mM. Inorganic orthophosphate at a concentration of 20 mM lowers
the enzyme activity to about 30% of the control value.
Distribution. ATP citrate lyase is widely distributed in the soluble
fraction of a variety of animal tissues, e.g., liver, brain, heart, kidney,

and adipose tissue; it is found also in certain bacteria.
Organ and Species ~pecificities. The enzymes from rat tissues, e.g.,
kidney, heart, brain, and adipose tissue, cannot be distinguished from
rat liver enzyme on the basis of their reactivity with rat liver anti-
enzyme, whereas the enzymes from the livers of chick, dog, guinea pig,
and rabbit react only partially with the same antienzyme.
[28] Citrate Lyase

[EC 4.1.3.6 Citrate oxaloacetate-lyase]
By S. "[')AGLEY
Citrate ~ oxaloacetate -{- acetate
The preparation of citrate lyase from Klebsiella aerogenes (Aero-
bacter aerogenes) described previously 1 has been improved to provide an
enzyme that sediments with a single symmetrical peak having an S2o.,
value of 16 S in the analytical ultracentrifuge? The purified enzyme, like
cruder preparations, 3 is activated by a range of divalent metal ions
(Mg ÷÷, Mn ++, Fe ++, and Zn÷+), shows optimal activity at pH 8.0-9.0, and
is powerfully inhibited by oxaloacetate. 4 The keto form of this compound
is a substrate for the enzyme, but the enol form is not and may be
responsible for the inhibition observed2 On account of this inhibition,
true equilibrium can be reached in the direction of citrate cleavage only
when the initial concentration of citrate does not exceed 2 mM and when
enzyme, Mg ++, and acetate are provided in excess2 Apparent equilibrium
constants expressed in terms of total citrate, oxaloacetate, and acetate
concentrations are markedly affected by the initial concentrations of the
reactants2 This is due to the ability of Mg *÷ ions, added as cofactor, to
complex with citrate and the isomers of oxaloacetate. For this reason,
and also because Mg ~ and oxaloacetate complex, respectively, with
phosphate and Tris which were used as buffers, earlier values of equi-
librium constants 1,~ appear to be in error, e When the reaction is per-
formed in triethanolamine-hydrochloric acid buffer, pH 8.4, which does
1H. H. Daron and I. C. Gunsalus, Vol. V [85].

' C. Siva Raman, Bioehim. Biophys. Aeta 52, 212 (1961).
sS. Dagley and E. A. Dawes, Biochlm. Biophys. Aeta 17, 177 (1955).
T. J. Bowen and L. J. Rogers, Biochim. Biophys. Aeta 77, 685 (1963).
'S. S. Tate and S. P. Datta, Bioehem. J, 911 18c (1964).
'S. S. Tate and S. P. Datta, Biochem. J. 94, 470 (1965).
'R. J. Harvey and E. B. Collins, J. Biol. Chem. 238, 2648 (1963).
fraction of a variety of animal tissues, e.g., liver, brain, heart, kidney,

and adipose tissue; it is found also in certain bacteria.
Organ and Species ~pecificities. The enzymes from rat tissues, e.g.,
kidney, heart, brain, and adipose tissue, cannot be distinguished from
rat liver enzyme on the basis of their reactivity with rat liver anti-
enzyme, whereas the enzymes from the livers of chick, dog, guinea pig,
and rabbit react only partially with the same antienzyme.
[28] Citrate Lyase

[EC 4.1.3.6 Citrate oxaloacetate-lyase]
By S. "[')AGLEY
Citrate ~ oxaloacetate -{- acetate
The preparation of citrate lyase from Klebsiella aerogenes (Aero-
bacter aerogenes) described previously 1 has been improved to provide an
enzyme that sediments with a single symmetrical peak having an S2o.,
value of 16 S in the analytical ultracentrifuge? The purified enzyme, like
cruder preparations, 3 is activated by a range of divalent metal ions
(Mg ÷÷, Mn ++, Fe ++, and Zn÷+), shows optimal activity at pH 8.0-9.0, and
is powerfully inhibited by oxaloacetate. 4 The keto form of this compound
is a substrate for the enzyme, but the enol form is not and may be
responsible for the inhibition observed2 On account of this inhibition,
true equilibrium can be reached in the direction of citrate cleavage only
when the initial concentration of citrate does not exceed 2 mM and when
enzyme, Mg ++, and acetate are provided in excess2 Apparent equilibrium
constants expressed in terms of total citrate, oxaloacetate, and acetate
concentrations are markedly affected by the initial concentrations of the
reactants2 This is due to the ability of Mg *÷ ions, added as cofactor, to
complex with citrate and the isomers of oxaloacetate. For this reason,
and also because Mg ~ and oxaloacetate complex, respectively, with
phosphate and Tris which were used as buffers, earlier values of equi-
librium constants 1,~ appear to be in error, e When the reaction is per-
formed in triethanolamine-hydrochloric acid buffer, pH 8.4, which does
1H. H. Daron and I. C. Gunsalus, Vol. V [85].

' C. Siva Raman, Bioehim. Biophys. Aeta 52, 212 (1961).
sS. Dagley and E. A. Dawes, Biochlm. Biophys. Aeta 17, 177 (1955).
T. J. Bowen and L. J. Rogers, Biochim. Biophys. Aeta 77, 685 (1963).
'S. S. Tate and S. P. Datta, Bioehem. J, 911 18c (1964).
'S. S. Tate and S. P. Datta, Biochem. J. 94, 470 (1965).
'R. J. Harvey and E. B. Collins, J. Biol. Chem. 238, 2648 (1963).
[28] CITRATE LYASE 161
not complex with M g ÷+, and when allowance is made for the equilibria
existing between M g *+ and the substrates, a value of K = 3.08 ± 0.72 is
obtained which is constant over a range of concentrations of M g *+ ions
and substrates.6
Assay Method
Principle. The spectrophotometric assay of citrate lyase is based on
measurement of oxaloacetate accumulation. I The method has been modi-
fied by using triethanolamine-hydrochloric acid buffer which does not
form complexes with magnesium. 6
Reagents
Triethanolamine-HCl buffer, 0.1 M, pH 7.4
MgC12, 30 mM
Trisodium citrate, 0.15 M
Enzyme source
Procedure. Into a 3-ml (1 cm light path) quartz cuvette were pipetted
2.4 ml of buffer, pH 7.4; 0.2 ml of MgCl~; 0.2 ml of enzyme. After
measurement of the optical density at 280 m~ the reaction was started by
addition of 0.2 ml of the trisodium citrate solution. The increase in
optical density was measured during 1 minute.
Units. One unit of citrate lyase activity is defined as that amount of
enzyme catalyzing the formation of 1 micromole of oxaloacetate per
minute under the conditions specified. The molar extinction coefficient of
enol oxaloacetate at 280 m/~ is 3600,8 and the enol isomers are stabilized
by M g ÷÷.
Enzyme Purification
Growth o] Cells and Extract Preparation. 3'~ Klebsiella aerogenes,
NCIB 418 (British), was grown without aeration at 37 ° in 10 liter flasks
filled to the neck with medium of the following composition (all in
grams per liter): 9 trisodium citrate.2 H~O; 2KH2PO,; 1 (NI~)2SO,;
0.4 MgSO,. 7 H20; and adjusted to pH 7.0 with sodium hydroxide. After
harvesting, cells, 50 g wet weight from about 40 liters of culture, were
crushed without abrasive in a Hughes bacterial press precooled to --15 °
and were extracted with 200 ml of 30 mM potassium phosphate buffer,
pH 7.0. The cells may alternatively be disrupted by sonic vibration.
Enzyme Fractionation ~,6
Removal o] Nucleic Acids. The crude extract was diluted with cold
30 mM phosphate buffer to 400 ml {protein content, 1%). Streptomycin
'8. 8. 'rate, A. K. Grzybowski,and S. P. Datta, J. Chem. 8oc. p. 1372 (1964).
sulfate (1.4 g/100 ml) was added, and the precipitated nucleic acids
were removed by centrifugation. The clear solution (380 ml) contained
approximately 0.8% of protein.
Treatment with Alumina C~ Gel. To the streptomycin-treated solu-
tion was added 15.2 ml of alumina Cr suspension (1 mg dry weight of
alumina per milliliter of extract). The gel was centrifuged and discarded.
To the supernatant solution was added a further 45.6 ml of gel suspension
and the mixture was kept at 2 ° for 30 minutes with stirring. At this stage
the enzyme was absorbed. The gel was removed by centrifugation,
washed with 80 ml of 10 mM phosphate buffer, pH 7.4, and the enzyme
was eluted from the gel by 80 ml of 75 mM phosphate buffer, pH 7.4.
After centrifugation, the supernatant solution contained 0.3~ of
protein.
Fractionation with Ammonium Sul]ate. The solution was brought to
0.1 saturation by addition of ammonium sulfate, and the slight precipi-
tate was discarded. More ammonium sulfate was added to bring to 0.6
saturation; the resulting precipitate was centrifuged, dissolved in 14 ml
of cold 10 mM phosphate buffer, pH 7.4, and dialyzed in the cold for 4
hours against 3 liters of the same buffer. The volume of the resulting
solution was 17 ml and contained 0.95% of protein.
Treatment with DEAE-Cellulose. The solution (14 ml) was applied to
a column (30 cm X 2 cm) of DEAE-cellulose that had been equilibrated
with 10 mM phosphate buffer, pH 7.4. The protein was washed on to
the column with 100 ml of the same buffer. Gradient elution was then
carried out from a closed mixing chamber (capacity 300 ml), filled with
10 mM phosphate buffer, pH 7.4, to which was connected a reservoir
CITRATE LYASE: FRACTIONATION OF K. aerogenes EXTRACTSa'b
Specific activity
Volume Protein (~moles/min/mg
Fraction (ml) (mg/ml) of protein)
Extract 215 19.86 1.16

Streptomycin-treated extract 380 8.32 1.63
Supernatant from first 395 6.85 1.84
alumina treatment
Extract from second 80 2.95 11.23
alumina treatment
AmSO~, 0.1-0.6 sat. 17 9.45 14.0
DEAE-cellulose and 15 2.75 35.16
AmSO~ treatments
° Based on 50 g wet weight of cells.

b Values given in this table are those of S. S. Tate and S. P. Datta, Biochem. J. 94, 470
(1965).
[29] ISOCITRATE LYASE 163
containing a solution of KH~PO, (10 raM) and NaCI(0.5 M) adjusted to

pH 7.4 with sodium hydroxide. The eluate.was collected in 4 ml fractions.
Enzymatic activity was eluted as a single peak between 0.15M and
0.25 M Na ÷. Fractions were pooled, dialyzed for 4 hours against 10 mM
phosphate buffer, pH 7.4, and precipitated by bringing to 0.9 saturation
with ammonium sulfate. The precipitate was collected by centrifuging,
dissolved in 13 ml of the same buffer, and stored as a suspension ma(le by
bringing to 0.9 saturation with ammonium sulfate. The results of a
purification procedure" are shown in the table.
[29] I s o c i t r a t e L y a s e 1
[EC 4.1.3.1 threo-n.-Isocitrate glyoxylate-lyase]
By BRUCE A. MCFADDEN
Mg++
threo-D,-Isocitrate 3- ~ glyoxylate- -t- succinate2-
The anaplerotie function of isocitrate lyase and malate synthase
during microbial growth on acetate is well documented,la Catalysis by
these enzymes is also vital in the conversion of lipid reserves to carbo-
hydrates, as found, for example, early in the germination of fatty plant
seedlings? Catalysis by isocitrate lyase also may provide glyoxylate for
condensation with higher fatty acids during microbial growth on these
compounds,s for glycine synthesis in nematodes,4 and for oxalate ac-
cumulation in Oxalis2
In general, microbial growth on acetate enhances formation of iso-
citrate lyase in contrast to tricarboxylic acid cycle intermediates, glucose,
or complex media which markedly suppress the formation2 A much more
relaxed repressive control of isocitrate lyase exists in P s e u d o m o n a s
indigofera. 7
1See also Vol. V, pp. 628-633.

~aH. L. Kornberg, in "Essays in Biochemistry" (P. N. Campbell and G. D. Greville,
eds.), pp. 1-32. Academic Press, New York, 1966.
H. Beevers, Nature 191, 433 (1961).
SR. Rabin, H. C. Reeves, W. S. Wegener, R. E. Mcgraw, and S. J. Ajl, Science 150,
1548 (1965).
M. Rothstein and H. Mayoh, Arch. Biochem. Biophys. 108, 134 (1964).
A. Millerd, R. K. Morton, and J. R. E. Wells, Biochem. J. 88, 281 (1963).
'H. L. Kornberg and S. R. Elsden, Advan. Enzymol. 23, 401 (1961).
TB. A. McFadden and W. V. Howes, J. Bacteriol. 84, 72 (1962).
[29] ISOCITRATE LYASE 163
containing a solution of KH~PO, (10 raM) and NaCI(0.5 M) adjusted to

pH 7.4 with sodium hydroxide. The eluate.was collected in 4 ml fractions.
Enzymatic activity was eluted as a single peak between 0.15M and
0.25 M Na ÷. Fractions were pooled, dialyzed for 4 hours against 10 mM
phosphate buffer, pH 7.4, and precipitated by bringing to 0.9 saturation
with ammonium sulfate. The precipitate was collected by centrifuging,
dissolved in 13 ml of the same buffer, and stored as a suspension ma(le by
bringing to 0.9 saturation with ammonium sulfate. The results of a
purification procedure" are shown in the table.
[29] I s o c i t r a t e L y a s e 1
[EC 4.1.3.1 threo-n.-Isocitrate glyoxylate-lyase]
By BRUCE A. MCFADDEN
Mg++
threo-D,-Isocitrate 3- ~ glyoxylate- -t- succinate2-
The anaplerotie function of isocitrate lyase and malate synthase
during microbial growth on acetate is well documented,la Catalysis by
these enzymes is also vital in the conversion of lipid reserves to carbo-
hydrates, as found, for example, early in the germination of fatty plant
seedlings? Catalysis by isocitrate lyase also may provide glyoxylate for
condensation with higher fatty acids during microbial growth on these
compounds,s for glycine synthesis in nematodes,4 and for oxalate ac-
cumulation in Oxalis2
In general, microbial growth on acetate enhances formation of iso-
citrate lyase in contrast to tricarboxylic acid cycle intermediates, glucose,
or complex media which markedly suppress the formation2 A much more
relaxed repressive control of isocitrate lyase exists in P s e u d o m o n a s
indigofera. 7
1See also Vol. V, pp. 628-633.

~aH. L. Kornberg, in "Essays in Biochemistry" (P. N. Campbell and G. D. Greville,
eds.), pp. 1-32. Academic Press, New York, 1966.
H. Beevers, Nature 191, 433 (1961).
SR. Rabin, H. C. Reeves, W. S. Wegener, R. E. Mcgraw, and S. J. Ajl, Science 150,
1548 (1965).
M. Rothstein and H. Mayoh, Arch. Biochem. Biophys. 108, 134 (1964).
A. Millerd, R. K. Morton, and J. R. E. Wells, Biochem. J. 88, 281 (1963).
'H. L. Kornberg and S. R. Elsden, Advan. Enzymol. 23, 401 (1961).
TB. A. McFadden and W. V. Howes, J. Bacteriol. 84, 72 (1962).
164 REACTIONS LEADII#G TO AND FROM THE CYCLE [29]
Assay Method
Principle. Enzymatic activity is measured by determining the amount
of glyoxylate formed from threo-vs-isocitrate s in 10 minutes at 30 °. A
slight modification of a highly sensitive and relatively specific colori-
metric assay for glyoxylate9 is employed.
Reagents
Tris-HC1 buffer, 0.1 M, pH 7.7 (25°), containing 3 mM MgCl2
Glutathione (GSH), 0.125M, freshly prepared in the Tris-Mg**
buffer above
Trisodium vL-isocitrate, 40 raM, prepared in the Tris-Mg ÷÷ buffer
above (store at 2 ° )
Pseudomonas indigo]era extract containing 0.0075-0.075 unit of
isocitrate lyase. For dilution of the enzyme, use cold Tris-Mg ÷÷
buffer as before
Trichloroaeetic acid, 10~ (w/v)
Mixture of 5 parts 10 mM oxalic acid and 1 part freshly prepared
1 ~ phenylhydrazine hydroehloride
Potassium ferricyanide, 5%, freshly prepared
Procedure. A mixture containing the following is preineubated in
10 cm or 15 cm test tubes at 30 ° for 10 minutes: Tris-Mg ~ buffer, 1.5 ml;
GSH, 0.2 ml; extract, 0.1 ml. The reaction is initiated with the addition
of 0.2 ml of the isocitrate solution followed by thorough mixing. After
incubation for 10.0 minutes at 30 °, the reaction is stopped with the addi-
tion of 1.0 ml of trichloroacetie acid. At this stage, the reaction mixture
may be stored overnight at 2 °. From the reaction mixture, 1.0 ml is
transferred to a 30- or 50-ml beaker. To this, 6.0 ml of the oxalic acid-
phenylhydrazine hydrochloride mixture is added and the mixture is
heated until just boiling on a preheated hot plate. The solution is removed
immediately, cooled at room temperature for 5 minutes, and then chilled
in an ice bath for 2 minutes. The beaker is removed from the ice bath,
4.0 ml of concentrated HCI is added, then 1.0 ml of the potassium ferri-
cyanide; the preparation is mixed thoroughly. If a series of samples is be-
ing run, the ferricyanide solution should be added in a known, timed
sequence. Seven minutes after the addition of ferricyanide, the optical
density (OD) is read at 520 m~ against a water blank in a suitable col-
orimeter (e.g., Bausch & Lomb Speetronie 2{)). If the path length is 1.0
era, the yield of glyoxylate in mieromoles per reaction vessel (i.e., 2.0 m!
of original incubation mixture) is given by: (0D52o-0.05)/1.15.
• H. B. Vickery, J. Biol. Chem. 237, 1739 (1962).
°B. A. M c F a d d e n a n d W. V. Howes, Anal. Biochem.'l, 240 (1960).
[29] 1SOCITRATE LYASE 165
Units. One unit of enzyme is that amount which catalyzes the dis-
appearance of 1 micromole of threo-Ds-isocitrate per minute at 30 ° under
conditions of the assay. The amount of isocitrate that disappears is
equivalent to the glyoxylate produced. 1° Specific activity is defined as
units per milligram of protein. The estimation of protein is based upon
the ultraviolet absorption of pure isocitrate lyase 11 and is given by the
equation:
Protein concentration (mg/ml) : 0.77 OD~8o- 0.38 OD~o
where 0D28o and OD2eo are optical densities at 280 and 260 intL.
Culture and Preparation of Extract
Culture. Cells from a fresh agar slant of Pseudomonas indigofera M1
(American Type Culture Collection 19706) maintained n on 1% Difco
yeast extract, 0.5% glucose, and 0.05% sodium acetate-3 H20 are grown
successively under the following conditions at 30°: first culture, in 36 ml
of 0.3% Difco yeast extract on a shaker for 24 hours; second culture, in
1.2 liters of a medium, pH 7.0, containing 0.54% K2HP04, 0.3% KN0.~,
0.1~ MgS0~.7 H20, 0.3% sodium butyrate (Matheson, Coleman and
Bell), and 0.1% Difco yeast extract with forced aeration for 24 hours;
third and main culture, in 40 liters of an identical medium but with
0.05% Difco yeast extract and for 20-24 hours with forced aeration. In
preparing the second two media, it is necessary to adjust the pH to 7.0
(with HC1) of an appropriate phosphate concentrate and to autoclave it
separately from the remaining components. After cooling, the solutions
are mixed aseptically. If growth response is inadequate, the main medium
can be fortified with 0.01 volume of a trace minerals solution. 1~
The cells (wet weight about 100 g) are harvested by continuous
ccntrifugation, washed once by centrifugation (10,000 g for 20 minutes)
with 50 mM Tris, pH 7.7 (25°), and after removal of the washings, stored
at --20 °. Isocitrate lyase is stable indefinitely in the frozen cell mass.
Preparation of the Extract. The cell mass is weighed and a 40% cell
suspension (40 g of wet packed cells per 60 ml) in 50 mM Tris, pH 7.7
(25°), containing 10 mM MgCI2 is prepared. After blending in a Waring
blendor, the suspension is divided into 50-ml portions and each is treated
for 6 minutes with a Raytheon 10-kc sonic oscillator operating at full
power with tap water as a coolant. Alternatively, 50-100-ml portions can
be treated with a Bronwill Biosonik oscillator for 15 minutes in a beaker
I°B. A. McFadden and W. V. Howes, Biochim. Biophys. Acta 50, 179 (1961).
,11. Shiio, T. Shiio, and B. A. McFadden, Biocbim. Biopllys. Acta 96, 114 (1965).
'~B. A. McFadden and W. V. Howes, J. Baclcriol. 81, 858 (1961).
'3E. A. Wolin, M. J. Wolin, :md R. S. Wolfe, J. Biol. Chem. $38, 2882 (1963).
immersed in an ice bath; the temperature of the suspension should not

exceed 20 °. The resulting suspension is centrifuged at 105,000 g for 1
hour. The high-speed supernatant fluid (fraction E) may be stored in
a freezer.
Enzyme Purification
Step 1. Heat Treatment. The protein concentration in fraction E
should be in the range of 8-10 mg/ml. If it exceeds l0 mg/ml, the con-
centration should be diluted to 10 mg/ml with the same buffer used for
preparation of the extract. Fraction E is then placed in a water bath at
50 °, swirled continually for 10 minutes, and then centrifuged at 8000 g
for 15 minutes at 2 ° to remove extraneous protein.
All subsequent operations are conducted at 0-4 ° . Where solutions
containing Tris are used, the pH is adjusted with HC1 and measured at
25 ° .
Step 2. First Ammonium Sulfate Fractionation. To the supernatant
solution resulting from the heat treatment step (SH), solid (NH4)~S04
(277 g per liter of SH) is slowly added with stirring to achieve 0.45
saturation. The precipitate is removed by centrifugation at 12,000 g for
15 minutes. The volume of the supernatant is measured and 65 g of
(NH4) 2S04 is added per liter to achieve 0.55 saturation. After centrifuga-
tion, the precipitate is dissolved in 0.05 M Tris, pH 7.7 (final volume 50
ml) and dialyzed overnight against 3 liters of a solution (hereafter
referred to as TEM), pH 7.7, containing 0.01 M Tris-HC1, 1 mM EDTA
and 1 mM mercaptoethanol to yield fraction P.
Step 3. Protamine Sulfate Step. The protein concentration of this
fraction (P) is then brought to 5-7 mg/ml by the addition of TEM, and
the pH to 6.2-6.4 by the dropwise addition of 1 M acetic acid with stir-
ring. The slight precipitate that may be present is not removed. To that
solution (or suspension) is slowly added 0.1 volume of another solution
prepared 1-2 hours in advance by slowly dissolving unfrozen protamine
sulfate in H.~O (15 mg/ml) and subsequently adjusting the pH to 4.5-5.0
with 0.2 M Tris. The precipitate is removed by centrifugation at 8000 g
for 15 minutes. The pH of the supernatant solution is adjusted to 7.7-8.0
by the addition of 1 M Tris-HCl, pH 8.5, to yield solution PS.
Step 4. Alkaline Ammonium Sulfate Fractionation. Fraction PS is
brought to 0.47 saturation by the addition of 0.89 volume of saturated
(NH4)2S04 solution adjusted to pH 7.7 with concentrated NH40H at
0-4 ° (pH 6.5-6.8 for a 1:20 dilution). The precipitate is removed by
centrifugation, and the supernatant is brought to 0.53 saturation by the
further addition of 0.13 volume of the saturated alkaline (NH4)...SO,
solution. The precipitate is collected by centrifugation and dissolved in
[29] ISOCITRATE LVASE 167
TEM. The protein concentration is adjusted to 5-7 mg/ml with TEM,

and the solution is dialyzed overnight as before to yield LP.
Step 5. Chromatography on DEAE-Cellulose. A portion of fraction
LP containing no more than 200 mg of protein is added to a 2 X 32 cm
column containing 14 g of DEAE-cellulose (35-100 mesh), which has
been washed previously with 1 M NaOH, water, 0.05 M Tris-HC1, pH
7.7, and TEM, respectively. A gradient elution is accomplished by using
600 ml beakers as the mixing chamber and reservoir. The two beakers are
mounted at the same level and connected by tubing to maintain hydro-
static equilibrium. To the mixing chamber is added 400 ml of 0.05 M
NaC1 containing 0.05M Tris-HC1, pH 7.9, 1 mM EDTA, and 1 mM
fl-mercaptoethanol; and to the reservoir, 400 ml of 0.3 M NaC1 contain-
ing the same concentrations of other components. Elution is conducted at
a rate of 50-60 ml per hour and is followed conveniently by a recording
ultraviolet flow analyzer. The enzyme typically elutes between fractions
40 and 70 (5 ml per fraction) but all protein-containing peaks should be
assayed for activity. The peak fractions based upon specific activity are
pooled (F).
Step 6. Final Purification. The enzyme can generally, although not
always, be crystallized by the following procedure. Fraction F is brought
to 0.45 saturation by the addition of 0.82 volume of the saturated alkaline
(NH4) 2S04 solution. The precipitate is removed by filtration with cheese-
cloth. The filtrate is allowed to stand in a large pctri dish at 2 ° for 3-4
days until 0.55 saturation is reached (desired decrease of weight, in
grams, -=--0.18 X initial volume, in milliliters). Some denaturation at the
liquid surface occurs, but the denatured material can be removed with
the supernatant by careful decantation. The crystalline material is col-
lected by centrifugation and dissolved in enough T E M to give ca. 4-5
mg of isocitrate lyase per milliliter (solution C). It is stored conveniently
in small aliquots in the freezer and diluted immediately prior to assay.
Alternatively, homogeneous noncrystalline isocitrate lyase can be
obtained in higher yields (ca. 80% from F) by first bringing F to 0.60
saturation by the addition of 1.5 volumes of the saturated (NH~)~SO~
solution. The precipitate is collected by centrifugation, dissolved in TEM
to a protein concentration of 2-2.5 mg/ml, and refractionated by collect-
ing only the precipitate from 47-53% saturation employing the saturated
alkaline (NH4)2S04. The precipitate is recovered by centrifugation and
dissolved in T E M to give a solution analogous to C.
The table illustrates results of the purification procedure.
Comments on the Purification. Sometimes the precipitate at 0.45
saturation in the first fractionation with (NH4)2SO~ has some activity
whereas the supernatant solution at 0.55 saturation has little activity in
PURIFICATION OF ISOCITRATE LYASE
Specific
activity
OD~so Protein (units/mg Recovery
Step Fraction OD~so (mg) protein) (~)
High-speed 0.61 2810 2.4 100

supernatant (E)
1. Iteat-treated E 0.60 1110 4.6 76
(SH)
2. First ammonium 0.56 510 5.1 39
sulfate (P)
3. Protaminesulfate 0.59 397 5.4 32
(PS)
4. Alkalineammonium 1.50 172 11.6 30
sulfate (LP)
5. DEAE-cellulose 1.88 31.8 29 14
eluate (F)
6. Crystalline 2.0 21.7 33 11
enzyme (C)
all cases. Often the protamine sulfate treatment removes more nucleic
acid than is shown in the table, as evidenced by a more pronounced in-
crease in the 280:260 ratio. In any case, treatment with protamine sulfate
is essential to obtain satisfactory subsequent fractionation. Efforts to use
streptomycin instead have been unrewarding.
Pooled fractions (F) from the DEAE-cellulose column are occasion-
ally homogeneous, but more often they contain a major and one or more
minor components as revealed by electrophoresis at pH 8 on cellulose
acetate. The final product is homogeneous by this criterion. The final
product is homogeneous in the ultracentrifuge and contains < 1 ~ im-
purity by electrophoretic criteria 11 using acrylamide gel. Occasionally
specific activities as high as 42 are observed, but they decline rapidly to
the usual values upon storage at --20 ° in TEM; as yet there is no
satisfactory explanation for this. A more reproducible final specific
activity is obtained by relating enzyme activity to dry weight determined
by the Folin phenol method (see Vol. III, pp. 448-450) ; it is 20.
Properties
Physical Properties. The enzyme has been purified to homogeneity
only from Pseudomonas indigofera, where it comprises about 7% of the
soluble protein in butyrate-grown cells. The sedimentation coefficient
(S~o,,), diffusion coefficient (D,,o,w), partial specific volume, extinction
coefficient at 280 mt~, molecular weight, and turnover number, respec-
[29] ISOClT~T~, LYASE 169
tively, are: 9.49)< 10-1~ second, 3.87)< 10.7 cm~/second, 0.730, 1710
cm2/g, 2.22 >( 105, and 7300 moles of glyoxylate formed per minute per
mole of enzyme. Its amino acid composition is known. 11 The molecular
weight estimated by sedimentation equilibrium is 206,000.
Stability. The enzyme from P. indigo/era is stable when stored in
TEM at --20 ° in the course of purification. Pure enzyme is stable at
higher protein concentrations ( > 4 mg/ml) in TEM at --20 °.
Complete protection against inactivation of pure enzyme at 45 ° i~
afforded by 2.5 mM MgCI2, MnC12, CoC12, or NiCl.~ and partial protec-
tion by 2.5 mM CaC12, CuS04, and CdCl2.14
Specificity. threo-Ds-Isocitrate is the only substrate known to undergo
cleavage. Citrate, cis-aconitate, threo-Ls-isocitrate, and threo-Ds-isocitric
lactone are inert? °,15,16 erythro-~L-Isocitrate (allo-isocitrate) may be
inhibitory?8 Although the equilibrium is less favorable for study of the
condensation reaction, detailed investigations have revealed marked
specificity for glyoxylate and succinate. 17
Equilibrium and Kinetic Properties. The equilibrium constant has
been measured using partially purified enzyme from P. aeruginosa. ~5 In
the direction of condensation it is 34.3M at pH 7.6 and 27 °, the
AF °' being --2100 ___ 300 cal mole-1. At physiological concentrations of
reactants and products, isocitrate cleavage is favored thermodynamically.
The pH optimum of the pseudomonad enzyme is broad, but maximal
activity is obtained ~5,1s in the range of 7.7-8.5. The Km for isocitrate is
0.45 to 0.82 mM at 30 ° for the pseudomonad enzyme~,~8 and 1.2 mM
for the yeast enzymeTM at 28 ° and pH 6.0. Under the latter conditions, the
Km for Mg ++ is 0.1 mM. Magnesium ion is saturating at 3 mM with
enzyme from P. indigo]era? S The apparent values of Ka at 28 ° for suc-
cinate and glyoxylate are 0.46 mM and 0.11 mM, respectively, for enzyme
from P. aeruginosa? 9 The Arrhenius activation energy ~7 for isocitrate
cleavage catalyzed by enzyme from P. indigo]era is 12.9 kcal mole-1.
It should be emphasized that these kinetic parameters, and especially
ones for thiol activation (not cited), may depend upon the oxidation state
of the enzyme (see next section).
Activators and Inhibitors. Magnesium ion is the most effective metal-
lic ion as an activator. Other bivalent cations which activate are man-
ganese and cobalt ~',~5 and iron. TM A number of cations are inhibitory in
competition with magnesium ion. ~
"I. Shiio, T. Shiio, and B. A. McFadden, Bioehim. Biophys. Acta 96, 123 (1965).
R. A. Smith and I. C. Gunsalus, J. Biol. Chem. 229, 305 (1957).
~J. A. Olson, J. Biol. Chem. 234, 5 (1959).
"G. R. Ran and B. A. McFadden, Arch. Biochem. Biophys. 112, 294 (1965).
~B. A. McFadden and W. V. Howes, J. Biol. Chem. 238, 1737 (1963).
WH. H. Daron, W. J. Rutter, and t.C.C_mmmlus,Biochemi*try 5, 895 (1966).
Thiols or EDTA are generally, but not always, 2°,~1 required for
activation of isocitrate lyase. With fresh enzyme from P. indigolera
EDTA replaces GSH. However, the enzyme becomes inactive--i.e., EDTA
does not replace GSH in the assay--gradually during cold storage or
rapidly through treatment with oxidized glutathione (GSSG).~' Reactiva-
tion by GSH of GSSG-oxidized enzyme is associated with the generation
of 5 sulfhydryls that react with p-hydroxymercuribenzoate ( P M B ) ?-2
Using EDTA for assay, PMB and N-ethylmalcimide are potent inhibi-
tors.14, ~2
Apparent substrate inhibition by isocitrate is lost during purification
of the enzyme from P. indigolera. 1°, ~s Succinate and glyoxylyate are in-
hibitory to cleavage ~5,~s catalyzed by purified enzyme. Glyoxylate ana-
logs such as glycolate, oxalate, and malonate are cbmpetitive inhibitors
of isocitrate cleavage. ~7 Other inhibitors of the enzyme from P. indigo]era
are a-ketoglutarate, DL-malate, phosphoenolpyruvate (PEP), fructose-
1,6-diphosphate and 3-phosphoglycerate, 17,~8 pyruvate, and oxaloacetate.
Itaconate ~7 and meso-tartrate are especially effective inhibitors. Thio-
malate, ~7 DL-lactate, DL-glycerate, and D- and L-tartrate are somewhat
less inhibitory resulting in 40-80~ inhibition at 1 mM. In Escherichia
coli, P E P may regulate the activity of isocitrate lyase. 23
:o R. L. Hcrberling, J. J. Berky, and R. W. Stone, Arch. Biochem. Biophys. 86, 102

(1960).
21I. Shiio, S. Otsuka, and T. Tsunoda, J. Biochem. (Tokyo) 462 1303 (1959).
2: L Shiio, and B. A. McFadden, Bioch~ra. Biophys. Acta 105, 496 (1965).
~J. M. Ashworth and H. L. Kombcrg, Biochim. Biophys. Acta 73, 519 (1963).
[30] The Reductive Carboxylic Acid Cycle

By BOB B. BUCHANAN and DANIEL I. AgNON
The reductive carboxylic acid cycle is a mechanism for COs assimila-
tion which has recently been found I,~ in the photosynthetic bacteria
Chlorobium thiosulfatophilum 1 and Rhodospirillum rubrum. 2 The cycle
provides a pathway for a synthesis from C02 of organic acids which in
turn provide the carbon skeletons for the biosynthesis of amino acids--
1M. C. W. Evans, B. B. Buchanan, and D. I. Arnon, Proc. Natl. Acad. Scl. U.S. 55,
928 (1966).
2B. B. Buchanan, M. C. W. Evans, and D. I. Arnon, Arkiv. Mikrobiol. 59, 32 (1967).
Thiols or EDTA are generally, but not always, 2°,~1 required for
activation of isocitrate lyase. With fresh enzyme from P. indigolera
EDTA replaces GSH. However, the enzyme becomes inactive--i.e., EDTA
does not replace GSH in the assay--gradually during cold storage or
rapidly through treatment with oxidized glutathione (GSSG).~' Reactiva-
tion by GSH of GSSG-oxidized enzyme is associated with the generation
of 5 sulfhydryls that react with p-hydroxymercuribenzoate ( P M B ) ?-2
Using EDTA for assay, PMB and N-ethylmalcimide are potent inhibi-
tors.14, ~2
Apparent substrate inhibition by isocitrate is lost during purification
of the enzyme from P. indigolera. 1°, ~s Succinate and glyoxylyate are in-
hibitory to cleavage ~5,~s catalyzed by purified enzyme. Glyoxylate ana-
logs such as glycolate, oxalate, and malonate are cbmpetitive inhibitors
of isocitrate cleavage. ~7 Other inhibitors of the enzyme from P. indigo]era
are a-ketoglutarate, DL-malate, phosphoenolpyruvate (PEP), fructose-
1,6-diphosphate and 3-phosphoglycerate, 17,~8 pyruvate, and oxaloacetate.
Itaconate ~7 and meso-tartrate are especially effective inhibitors. Thio-
malate, ~7 DL-lactate, DL-glycerate, and D- and L-tartrate are somewhat
less inhibitory resulting in 40-80~ inhibition at 1 mM. In Escherichia
coli, P E P may regulate the activity of isocitrate lyase. 23
:o R. L. Hcrberling, J. J. Berky, and R. W. Stone, Arch. Biochem. Biophys. 86, 102

(1960).
21I. Shiio, S. Otsuka, and T. Tsunoda, J. Biochem. (Tokyo) 462 1303 (1959).
2: L Shiio, and B. A. McFadden, Bioch~ra. Biophys. Acta 105, 496 (1965).
~J. M. Ashworth and H. L. Kombcrg, Biochim. Biophys. Acta 73, 519 (1963).
[30] The Reductive Carboxylic Acid Cycle

By BOB B. BUCHANAN and DANIEL I. AgNON
The reductive carboxylic acid cycle is a mechanism for COs assimila-
tion which has recently been found I,~ in the photosynthetic bacteria
Chlorobium thiosulfatophilum 1 and Rhodospirillum rubrum. 2 The cycle
provides a pathway for a synthesis from C02 of organic acids which in
turn provide the carbon skeletons for the biosynthesis of amino acids--
1M. C. W. Evans, B. B. Buchanan, and D. I. Arnon, Proc. Natl. Acad. Scl. U.S. 55,
928 (1966).
2B. B. Buchanan, M. C. W. Evans, and D. I. Arnon, Arkiv. Mikrobiol. 59, 32 (1967).
[30] REDUCTIVE CARBOXYLIC ACID CYCLE 171
the main products of bacterial photosynthesis3--and other cellular con-

stituents.
The key carboxylation reactions in the reductive carboxylic acid cycle
are the recently discovered pyruvate and a-ketoglutarate synthases. They
each reverse a reaction of the citric acid cycle that is irreversible in
aerobic cells: (i) the decarboxylation of pyruvate to acetyl-CoA and CO.~,
and (ii) the decarboxylation of ~-ketoglutarate to succinyl-CoA and
CO.,. The reversal of thcse two decarboxylations is made possible I)y the
strongly electronegative potential of ferredoxin (approximately equal to
that of hydrogen gas) which overcomes in each case the energy barrier
for carboxylation. In the pyruvate synthase system, the reducing power
of ferredoxin brings about a synthesis of pyruvate from acetyl-CoA and
C02.*-~ In the a-ketoglutarate synthase system the reducing power of
ferredoxin brings about a synthesis of a-ketoglutarate from succinyl-CoA
and CO.,/
In its overall effect, the reductive carboxylic acid cycle (Fig. 1) in-
cludes a reversal of the degradation of pyruvate via the citric acid cycles
and the resultant liberation of CO~. One complete turn of the reductive
carboxylic acid cycle results in the fixation of four molecules of C02 and
the formation of one oxaloacetate (Fig. 1).
This chapter will deal only with the new, ferredoxin-dependent
pyruvate and a-ketoglutarate synthase reactions that are peculiar to the
reductive carboxylic acid cycle. The other enzymes involved in this cycle
(malate dehydrogenase, fumarate hydratase, succinate dehydrogenase,
succinyl-CoA synthase, isocitrate dehydrogenase, aconitate hydratase,
citrate lyase, aceto-CoA synthase, phosphoenolpyruvate synthase,
phosphoenolpyruvate carboxylase) were assayed by methods that have
been described previously9,~° or are discussed elsewhere in this volume.
~M. Losada, A. V. Trebst, S. Ogata, and D. I. Arnon, Nature 186, 753 (1960); R. C.
Fuller, R. M. Smillie, E. C. Sisler, and H. L. Kornberg, J. Biol. Chem. 236, 2140
(1961); D. S. Hoare, Biochem. J. 87, 284 (1963).
~R. Bachofen, B. B. Buchanan, and D. I. Arnon, Proc. Natl. Acad. Sci. U.S. 51,
690 (1964).
~B. B. Buchanan, R. Bachofen, and D. I. Arnon, Proc. Natl. Acad. Sci. U.S. 52,
839 (1964).
*M. C. W. Evans and B. B. Buchanan, Proc. Natl. Acad. Sci. U~. 53, 1420 (1965).
~B. B. Buchanan and M. C. W. Evans, Proc. Natl. Acad. Sci. U.S. 54, 1212 (1965).
s It. A. Krebs and J. M. Lowenstein, in "Metabolic Pathways" (D. M. Greenberg,
ed.), Vol. 1, p. 129. Academic Press, New York, 1960.
Vol. I, p. 699.
1°"Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.). Academic Press, New
York, 1963.
~ SUCCINYL-CoA
SUCCINATE
FUMARATE
a-KETOGLUTARATE H2o, ~
MALATE
OXALOACETA
TE
ISOCITRATE PHOSPHOPYRUVATE
20 PYRUVATE
¢is-ACONiTATE
~
~\
H20
ACE~YL-CoA
~'ERREIX}XlN] red
CITRATE#' c~p
IOXALOACETATEI
FIo. 1. The reductive carboxylic acid cycle of Chloroblum thiosullatophilum
and Rhodospirillum rubrum. One complete turn of the cycle results in the in-
corporation of four molecules of CO= and the net synthesis of one molecule of
oxMoacetate.
Pyruvate Synthase
0 0
[I TPP I[
C H 3 - - C - - C o A + C*O~ + Fd~d , C H s - - C - - C * O O H + Fdo= + CoA
(1)
Assay Method
Principle.The method 4 is based on the measurement of pyruvate-1"C
(as the 2,4-dinitrophenylhydrazone derivative) formed from acetyl-P,
"C02, and reduced ferredoxin in the presence of catalyticconcentrations
of CoA (Eq. 2).
CoA
Acetyl-P + "CO2 + Fd,ed , p y r u v a t e - l ~ + Pi + Fdox (2)
Acetyl-P is converted to acetyl-CoA by phosphotransacetylase (Eq. 3).
Acetyl-P ~- CoA --~ acetyl-CoA ~- P~ (3)

Photosynthetic bacteria reduce ferredoxin photochemically.6 In the assay
it is more convenient to reduce ferredoxin photochemically with isolated
spinach chloroplasts using reduced 2,6-dichlorophenol indophenol
(DPIP) as the electron donor (Eq. 4).
hv
Fdox -~ DPIPred --* Fdred ~ DPIPox (4)
In the presence of reduced ferredoxin, acetyl-CoA is reductively carbox-
ylated by pyruvate synthase (Eq. 1).
Under the given experimental conditions, the synthesis of pyruvate is
linear with time and proportional to the concentration of enzyme.
Reagents
Buffer: potassium phosphate buffer, 1 M, pH 6.2, for C. thiosul-
]atophilurn; HEPES '1 (N-2-hydroxyethylpiperazine-N'-2-ethane-
sulfonic acid) buffer, 0.5 M, pH 7.5, for R. rubrum.
2,6-Dichlorophenol indophenol, 2 mM
Sodium ascorbate, 0.2 M
Acetyl phosphate, lithium salt, 0.5 M
CoA, 5 mM
Bacterial ferredoxin (see below), 1 mg/ml
Semicarbazide TM (neutralized with KOH), 0.5 M (a trapping agent
for pyruvate)
Thiamine pyrophosphate (TPP),I~ 1 mg/ml
Spinach chloroplasts
Sodium bicarbonate-14C, 0.1 M (containing 10~ cpm/ml)
Soluble enzyme fraction (0.5 mg from C. thiosul]atophilum or 3 mg
from R. rubrum)
Phosphotransacetylase13 (crystalline enzyme from Clostridium
kluyveri; available from Boehringer-Mannheim, New York), 5
~g (see also this volume [59] )
11N. E. Good, G. D. Winger, W. Winter, T. N. Connolly, S. Izawa, and R. M. M.

Singh, Biochemistry 5, 467 (1966).
Z~Omitted when assaying C. thiosul]atophilum pyruvate synthase; added with the
R. rubrum assay.
,s Except for the partly purified pyruvate synthase from C. thiosul]alophilum (see
below) which requires added phosphotransacetylase, the soluble fractions of the
bacteria tested contain sufficient phosphotransacetylase to support pyruvate syn-
thesis.
Procedure. The reaction is carried out in Warburg manometer vessels,

with 2 sidearms, at 25 °, in an illuminated, constant temperature bath. The
vessels are kept on ice while the reagents are being added. Bicarbonate-
14C (0.1 ml) is added to one sidearm, fitted with a glass stopper; the
enzyme preparation (0.5 ml) is added to the second sidearm, which is
fitted with a glass vent. H_~O, 0.9 ml; appropriate buffer, 0.3 ml; aliquots,
0.1 ml each, of DPIP, sodium ascorbate, acetyl phosphate, CoA, ferre-
doxin, TPP (when needed), semicarbazide (when needed); and, last,
chloroplast preparation, 0.5 ml, are added to the main compartment.
Since the reduction of ferredoxin by chloroplasts is light-dependent, the
preliminary steps of the assay are carried out with a minimum of light
in the room.
The vessels are gassed with argon for 6 minutes, the vent is closed,
and the additions are made from the sidearms. The light (about 10,000
lux) is turned on, and the incubation is continued for 30 minutes with the
C. thiosulfatophilum preparation o1" for 60 minutes with the R. rubrum
preparation. The reaction is stopped by adding 0.5 ml 12 N HC1, and the
precipitated protein is centrifuged off (5 minutes at 1000 g). One milli-
liter of the supernatant fluid is used for preparing the 2,4-dinitrophenyl-
hydrazone derivative as described by Rabinowitz. 14 The isolated pyru-
vate dinitrophenylhydrazone is dried on planchets and counted with a
thin-window gas flow counter.
When the pyruvate synthasc preparation used in the photochemical
assay also contains hydrogenase, the photoreduced ferredoxin may give
rise to H.~ evolution with a resultant suppression of pyruvate synthesis.
In such a case hydrogenase can be inhibited by using CO to replace argon
in the gas phase. 4
Replacement of Photochemical System for Generating Reduced Ferre-
doxin with H.,-Hydrogenase. In extracts containing an active hydrogenasc,
the reduction of ferredoxin may be accomplished by H_~, independently
of light. Here the chloroplast preparation, DPIP, and ascorbate are
omitted and H2 replaces argon in the gas phase.
Preparation of Enzyme
All the steps are carried out at 4 ° . Protein is measured by the phenol
reagent method of Rabinowitz and Pricer. 15
C. thiosul]atophilum. Ten grams of frozen cells (see below) are
thawed and suspended in 30 ml of 20 mM potassium phosphate buffer,
pH 6.5, with the aid of a Potter-Elvehjem homogenizer. The cell sus-
pension is disrupted by sonic oscillation (6 minutes with a l0 kc Ray-
14j. C. Rabinowitz, I. Biol. Chem. 235, PCS0 (1960).
l~J. C. Rabinowitz and W. E. Pricer, Jr., J. Biol. Chem. 237, 2898 (1962).
theon sonic generator). The sonicate is centrifuged for 10 minutes at

12,000 g and the precipitate, containing cell debris and colloidal sulfur, is
discarded. The supcrnatant fluid [cell-free extract (S,)] is centrifuged
1 hour at 105,000 g in a Spinco Model L preparative ultracentrifuge, and
the residue is discarded. The supernatant fluid [soluble fraction (S~)] is
assayed for pyruvate synthase activity without further tl'eatmcnt.
R. ~-~tbrum. Ten grams of frozen cells ~ee below) are thawed al~d
suspended in 20 ml 0.1 3I Tris buffer, pH 7.5, containing 0.1 M 2-mcr-
captoethanol. The cell suspension is disrupted by sonic oscillation ~1
minute with ,'t 20 kc Branson sonifier, power setting 3). The sonicate is
centrifuged for 20 minutes at 36,000 g and the precipitate is discarded.
The supernatant fluid [cell-free extract (S,) ] is centrifuged for 1 hour at
105,000 g and the new supernatant fluid [soluble fraction (S~) ] is assayed
for pyruvate synthasc without further treatment.
Removal of Ferredoxin from Cell-Free Extracts. Before the soluble
fractions of C. thiosulfatoph[lum and R. rubrvm show a ferredoxin re-
quirement for pyruvate synthesis, they must be treated to remove ferre-
doxin. The cell-free extract (S~) froln 10 g of cells is passed through a
2 X 3 cm DEAE-cellulose column, which has previously been equilibrated
with the same buffer that was used to disrupt cells. The C. th:osulfato-
philum and R. rubrum fractions which contain pyruv'~te synthase arc
accompanied hy green or red pigments, respectively. The column is
eluted with the preparative buffer until the volume of the eluate collected
is equal to that of the cell-free extract applied. C. thiosulfatophib,m
ferredoxin remains as a dark brown band at the top of the column;
R. rubrum ferredoxin does not appear as a discrete band (at this stage of
purification) but is dispersed throughout the column.
Purification of the Enzyme from C. thiosulfatophilum. All steps of
the purification procedure are carried out at 4 °. The cell-free extract
($1) from 40 g of cells (grown on acetate and CO.~ and suspended in 80 ml
of 20 m M potassium phosphate buffer, pH 6.5) is applied to a 2 X 3 cm
DEAE-ceIlulose column, equilibrated with the ~ame buffer. Collection of
the effluent fraction begins when the green color first appears and is
continued until about 70 ml is collected. The protein content of the
effluent fraction is estimated ~ and the fraction is diluted with 0.02 M
potassium phosphate buffer, pH 6.5, to give a final protein concentration
of 5 mg/ml.
Ammonium Sulfate Fractionation. This step and the ethanol fraction-
ation described below are carried out in Erlenmeyer flasks with the aid of
a magnetic stirrer and with a small stream of hydrogen gas passing over
the surface of the liquid.
Solid ammonium sulfate is added to the DEAE-treated extract to
176 REACTIONS LEADING TO AND FROM TtIE CYCLE [30]
50% saturation. The heavy green precipitate is discarded after a centrif-

ugation for 10 minutes at 39,000 g. (The same centrifugation is used in
all subsequent centrifugation steps.) Solid ammonium sulfate is added to
the supernatant fluid to 70% saturation and the precipitate is collected
by centrifugation. The supernatant fluid is discarded and the precipitate
is dissolved in 37 ml 0 . 0 2 M potassium phosphate buffer, containing
0.1 M 2-mercaptoethanol.
Ethanol Fractionation. Twenty-two milliliters of ethanol at --20 °
(corresponding to 0.6 volume of the 50-70% ammonium sulfate fraction)
is added dropwise. The light green precipitate is removed by centrifuga-
tion and discarded; 37 ml of ethanol (equal to the original volume of the
50-70% ammonium sulfate fraction) is added dropwise to the super-
natant fraction. The amber-colored precipitate is collected, and the
supernatant fluid is discarded. The precipitate is dissolved in 11 ml of
20 m M potassium phosphate buffer containing 0.1 M 2-mercaptoethanol,
and centrifuged again to remove the insoluble residue. The remaining
fraction, containing about 1 mg protein/ml, has the pyruvate synthase,
purified as summarized in the table.
PURIFICATION OF PYRUVATE SYNTHASE FROM Chlorobium thiostdfatophilum

Volume protein activity Yield activity
Step (ml) (rag) (units") (%) (units/mg)
Cell-free extract 68 950 86,300 100 91

DEAE-cellulose filtrate 170 850 35,800 42 42
50-70% (NH~)~SO4fraction 37 124 22,500 26 180
37-61% Ethanol fraction 13 11 7,800 9 710
A unit of enzyme activity is defined as millimicromoles of pyruvate formed in 30
minutes under the conditions of the assay.
The partly purified pyruvate synthase from C. thiosul]atophilum is

free of ferredoxin, a-ketoglutarate synthase, and phosphotransacetylase
activities. However, the enzyme was not stable after storage and was
freshly prepared each day. About 50% of the activity was lost on over-
night storage in argon at 4 ° .
Properties
Ef]ect o] TPP. The soluble fraction of R. rubrum ($2) shows a re-
quirement for T P P for pyruvate synthesis. P y r u v a t e synthase from C.
thiosul]atophilum shows no response to added T P P unless dialyzed
overnight against 80% ammonium sulfate, pH 8, containing 0.1 M

2-mercaptoethanol. ~6
Ef]ect of Ferredoxins ]rom Dif]erent Sources. Native ferredoxins are
ao more effective with the pyruvate synthase from C. thiosul]atophilum
and tile soluble fraction (S,) from R. rubrum than ferredoxins obtained
fl'om C. pasteurianum and Chromatium. However, spinach ferredoxin was
appreciably less effective.
pH Optima. Partly purified pyruvate synthase from C. thiosuI]ato-
philum shows a pH optimum at 6.2; at pH 5.9 and 6.5 the activity is
about three-fourths that observed at. p H 6.2. The R. rubrum soluble frac-
tion has a pH optimum range of pH 7.0-7.5.
CO...-Pgruvate Exchange. All preparations of pyruvate synthase ex-
amined so far catalyze an exchange between C02 and the carboxyl group
of pyruvate. Like pyruvate synthesis, the CO._,-pyruvate exchange reac-
tion requires CoA, but at lower levels. The exchange reaction also re-
quires T P P . With the partly purified pyruvate synthase from C. thio-
sul]atophilum, pyruvate synthesis and CO~-pyruvate exchange have
different pH optima; the optimal pH for pyruvate synthesis is 6.2,
whereas C02-pyruvate exchange is most rapid at pH 7.2.~
Occurrence. P y r u v a t e synthase has been found in all photosynthetic
bacteria tested so far 2,5,G and in certain anaerobic bacteria *'~7-~9 that
contain ferredoxin. An apparently similar enzyme, named pyruvate-ferre-
doxin oxidoreductase, -"° which catalyzes the synthesis of pyruvate, has
been purified from Clostridium acidi-urici.
a-Ketoglutarate Synthase
O
[I TPP
H O O C - - C t t 2 - - C H 2 - - C - - C o A + C*O~ + Fd~d ,
O
H O O C - - C H 2 - - C H 2 - - C - - C * O O H + Fdo, + CoA (5)

"~B. B. Buchanan, M. C. W. Evans, and D. I. Amen, in "Non-Heme Iron Proteins"
(A. San Pietro, ed.), p. 175. Antioch Press, Yellow Springs, Ohio, 1965.
1'I. G. Andrew and J. G. Morris, Biochim. Biophys. Acta 97, 176 (1965).
,aj. R. Stern, in "Non-Heme Iron Proteins: Role in Energy Conversion" (A. San
Pietro, ed.), p. 199. Antioch Press, Yellow Springs, Ohio, 1965.
'*E. tteer and R. Bachofen, Arkiv. Mikrobiol. 54, 1 (1966).
2oS. Raeburn and J. C. Rabinowitz, Biochem. Biophys. Res. Commun. 18, 303 (1965);
S. Raeburn and J. C. Rabinowitz, in "Non-Heine Iron Proteins: Role in Energy
Conversion" (A. San Pietro, ed.), p. 189. Antioch Pre~, Yellow Springs, Ohio, 1965;
K. Uyeda and J. C. Rabinowitz, Federation Prec. 26, 561 (1967).
Assay Method
Principle. The method ~ is based on the measurement of a-ketogluta-
rate-14C (as the 2,4-dinitrophenylhydrazone derivative) formed from
14C0~, succinatc, reduced ferredoxin, and ATP in the presence of MnC1._,
and catalytic concentrations of CoA (Eq. 6).
CoA
14('O,,., + succinate + I:dr~d + ATP 1,
TPP
a-ketoglu~arate-*4C + Fdo~ + ADP + Pi (6)
Succinate is converted to succinyl-CoA in the presence of ATP and
MnC12 by the suecinyl-CoA synthase present in the extract (Eq. 7).
MnC12
Succinate + ATP + CoA , succinyl-CoA + ADP + P~ (7)
Ferredoxin is reduced photochemically by isolated spinach chloroplasts
as described for pyruvatc synthase. The succinyl-CoA, in the presence of
reduced ferredoxin is reductively carboxylated by a-ketoglutarate syn-
thase (Eq. 5).
Under the given experimental conditions, the synthesis of a-keto-
glutarate is linear with time and proportional to the concentration of
enzyme.
Reage~ts
Buffer [potassium phosphate buffer, 0.5M, pH 6.5, for C. thio-
sul]atophilum; HEPES buffer (N-2-hydroxyethylpiperazine-N'-
2-ethanesulfonic acid),~ 0.5 M, pH 7.5, for R. rubrum]
Dichlorophenol indophenol, 0.5 mM
Sodium ascorbate, 0.2 M
MnCl_~, 30 mM
Potassium succinate, 0.1 M
ATP, 50 mM
CoA, 5 mM
Bacterial ferredoxin (see below), 1 mg/ml
Semicarbazide "-~ (neutralized with KOH), 0.SM (as a trapping
agent for a-ketoglutarate)
Thiamine pyrophosphate (TPP),-"~ 4 mg/ml
Spinach chloroplasts
Sodium bicarbonatc-14C, 0.1 M (containing 10T cpm/ml)
Soluble enzyme fraction (1.0 mg from C. thiosul]atophilum, 6 mg
from R. rubrum)
~1Omitted for C. thiosul]atophil~on but added for the R. rubrlm~ a~ay.

Procedure. The assay is carried out in Warburg lnanoineter vessels

with two sidearms, as described for pyruvate synthase. Biearbonate-~4C
(0.1 ml) is added to one sidearm, fitted with a glass stopper; the enzyme
preparation (0.5 ml) is added to the second sidearm, which is fitted with
a glass vent. H:O, 0.7 ml; appropriate buffer, 0.3 ml; aliquots, 0.1 ml
each, of DPIP, sodium ascorbate, MnCI:, suecinate, ATP, CoA, ferre-
doxin, semicarbazide (when needed), T P P (when needed); and last, 0.5
ml of the chloroplast preparation are added to the inain compartment of
the vessel. The vessel is gassed with argon. The reaction is carried out
as described for pyruvate synthase. The 2,4-dinitrophenylhydrazone of
a-ketoglutarate is isolated as described for the pyruvate hydrazone.
C. thiosulfatophilum. The soluble fraction ($2) is prepared from
frozen cells of C. thiosulfatophilum as described for pyruvate synthase,
except that 0.1M potassium phosphate, pH 7.4, rather than 20 mM
potassium phosphate, pH 6.5, is used as the preparative buffer. Prior to
the assay, the soluble fraction is dialyzed 2 hours against 20 mM potas-
sium phosphate buffer, pH 6.5, under an atmosphere of argon.
R. rubrum. The soluble fraction (S.~) is prepared from frozen cells of
R. rubrum as described for pyruvate synthase. Prior to the assay, the
soluble fraction is filtered through Sephadex G-25 to remove interfering
endogenous substrates.
Of the R. rubrum soluble fraction ($2), 13 ml is filtered through a
3 )< 17 em Sephadcx G-25 (coarse grade) column previously equilibrated
with 0.1 M Tris buffer, pH 7.5, containing 0.1 M 2-Inercaptoethanol.
Approximately 13 ml of the protein fraction (characterized by its red
color) is collected and concentrated by dialysis against Carbowax to give
a protein concentration equal at least to that of the original cell-free
extract.
Removal o] Ferredoxin ]rom Cell-Free Extracts. To observe a require-
ment for ferredoxin in a-ketoglutarate synthesis, ferredoxin should be
removed from the cell-free extracts, as described for pyruvate synthase.
Properties
Effect of TPP. A requirement for T P P ill the synthesis of a-keto-
glutarate has been observed only with the soluble fraction of R. rubrumfi
the addition of T P P had no effect on the synthesis of a-ketoglutarate
by the soluble fraction of C. thiosuIfatophilum.
Effect of Ferredoxins from Different Sources. Native fcrredoxins were
no more effective with their respective a-ketoglutarate synthase systems
than ferredoxins obtained from C. pasteurianum and Chromatium. No

comparison was made with ferredoxin from spinach.
pH Optima. For C. thiosul]atophilum the optimal pH for a-keto-
glutarate synthase was 6.5 and for R. rubrum, 7.5.
CO~-a-Ketoglutarate Exchange. Like the pyruvate synthase prepara-
tions, a-ketoglutarate synthase preparations catalyze an exchange reac-
tion between 14C02 and a-ketoglutarate. Cofactors of the exchange reac-
tion, other than CoA, have so far not been identified.
Growth of Microorganisms
C. thiosul]atophilum (strain Tassajara) is grown on Pfennig's me-
dium 22,~ with C02 as the sole source of carbon. For purifying pyruvate
synthase, the cells were grown on the same medium, but supplemented
with 0.05% acetate. R. rubrum is grown in medium S of Lascelles,~3
modified by omitting malate and glutamate and adding 0.1% succinate
and Pfennig's heavy metal solution. Illumination (about 10,000 lux) is
supplied by a bank of incandescent lamps. A mixture of 95% H2-5% C02
was constantly bubbled through the growth medium. Harvested cells
(unwashed) of both C. thiosul]atophilum and R. rubrum stored at --20 °
for at least 1 month yield viable enzyme preparations.
Preparation of Ferredoxin
The methods for obtaining stable, highly purified preparations of
ferredoxin from photosynthetic bacteria have not yet been perfected to
the same degree as those from certain clostridial species or green leaves.
The ferredoxin of C. thiosul]atophilum, although unstable, has been
obtained in relatively pure form s by the procedure of Buchanan et al. 24
The ferredoxin of R. rubrum has been isolated by the same procedure, but
it has not been purified. The absorption spectra of the C. thiosul]ato-
philum and R. rubrum ferredoxins resemble those of Chromatium ~5 and
of anaerobic nonphotosynthetic bacteriaY 6
Because of its abundance and ease of purification,27 ferredoxin from
the anaerobic bacterium Clostridium pasteuria~um is used routinely in
assaying for pyruvate and a-ketoglutarate synthases.
22N. Pfennig, Naturwissenscha]ten 48, 136 (1961); N. Pfennig, Arkiv. Mikrobiol.
42, 90 (1962).
2,j. Lascelles, Biochem. J. 62, 78 (1956).
~ B. B. Buchanan, W. Lovenberg, and J. C. Rabinowitz, Proc. Natl. Acad. Sci. U.S.
49, 345 (1963).
2,R. Bachofen and D. I. Arnon, Biochim. Biophys. Acta 120, 259 (1966).
W. Lovenberg, B. B. Buchanan, and J. C. Rabinowitz, d. Biol. Chem. 238, 3899
(1963).
2,L. E. Mortenson, Biochim. Biophys. Acla 81, 71 (1964).
[31] PROPIONYL-COA CARBOXYLASE FROM PIG HEART 181
Preparation of Chloroplasts
Washed spinach chloroplast particles (PI~) are prepared in isotonic
sodium chloride media according to Whatley and Arnon 28 or in isotonic
sorbitol media according to Kalberer et al. 29 Chlorophyll is estimated as
described by Arnon2 ° The ferredoxin-dependent C02 fixing enzymes are
sensitive to oxygen. Accordingly, the chloroplast preparation is heated
for 5 minutes at 55 ° just before use to destroy its capacity for oxygen
evolution) 1 Ascorbate-DPIP, 3-~rather than water, is used as the electron
donor by heated chloroplasts for photoreducing ferredoxin. The unheated
chloroplast preparation may be stored at --20 ° and remains stable for at
least one month.
2sF. R. Whatley and D. I. Arnon, Vol. VI, p. 308.
-~P. P. Kalberer, B. B. Buchanan, and D. I. Arnon, Proc. Natl. Acad. Sci. U.S. 57,
1542 (1967).
~D. I. Arnon, Plant Physiol. 24, 1 (1949).
31D. I. Arnon and F. R. Whatley, Arch. Bioche,m. Biophys. 23, 141 (1949).
3'L. P. Vernon and W. S. Zaugg, J. Biol. Chem. 235, 2728 (1960).
[31] Crystalline Propionyl-CoA Carboxylase from Pig Heart

[EC 6.4.1.3 Propionyl-CoA: carbon dioxide ligase (ADP) ]
B y YOSHITO KAZmO
Propionyl-CoA + A T P q- HC03-,-~- Ds-methyhnalonyl-CoA + ADP Jr P,
Assay Methods
Principle. Tietz and Ochoa 1,2 have described two methods for the
assay of propionyl-CoA carboxylase. In one, the rate of fixation of
bicarbonate-14C to propionyl-CoA is followed by measuring the increase
in acid-insoluble radioactivity. In the other, the rate of formation of A D P
is measured spectrophotometrically by coupling the propionyl-CoA car-
boxylase reaction with reactions catalyzed by pyruvate kinase 3 and
lactate dehydrogenase. 4 With an excess of pyruvate kinase and lactate
dehydrogenase, the rate of oxidation of N A D H is proportional to the rate
of formation of ADP, and therefore to the rate of fixation of HCO~- to
propionyl-CoA to yield Ds-methylmalonyl-CoA.
At early stages of purification, the presence of ATPase necessitates
' S e e Vol. V [77].
A. Tietz and S. Ochoa, J. Biol. Chem. 234, 1394 (1959).
3See Vol. I [66].
' See Vol. I [67].
Preparation of Chloroplasts
Washed spinach chloroplast particles (PI~) are prepared in isotonic
sodium chloride media according to Whatley and Arnon 28 or in isotonic
sorbitol media according to Kalberer et al. 29 Chlorophyll is estimated as
described by Arnon2 ° The ferredoxin-dependent C02 fixing enzymes are
sensitive to oxygen. Accordingly, the chloroplast preparation is heated
for 5 minutes at 55 ° just before use to destroy its capacity for oxygen
evolution) 1 Ascorbate-DPIP, 3-~rather than water, is used as the electron
donor by heated chloroplasts for photoreducing ferredoxin. The unheated
chloroplast preparation may be stored at --20 ° and remains stable for at
least one month.
2sF. R. Whatley and D. I. Arnon, Vol. VI, p. 308.
-~P. P. Kalberer, B. B. Buchanan, and D. I. Arnon, Proc. Natl. Acad. Sci. U.S. 57,
1542 (1967).
~D. I. Arnon, Plant Physiol. 24, 1 (1949).
31D. I. Arnon and F. R. Whatley, Arch. Bioche,m. Biophys. 23, 141 (1949).
3'L. P. Vernon and W. S. Zaugg, J. Biol. Chem. 235, 2728 (1960).
[31] Crystalline Propionyl-CoA Carboxylase from Pig Heart

[EC 6.4.1.3 Propionyl-CoA: carbon dioxide ligase (ADP) ]
B y YOSHITO KAZmO
Propionyl-CoA + A T P q- HC03-,-~- Ds-methyhnalonyl-CoA + ADP Jr P,
Assay Methods
Principle. Tietz and Ochoa 1,2 have described two methods for the
assay of propionyl-CoA carboxylase. In one, the rate of fixation of
bicarbonate-14C to propionyl-CoA is followed by measuring the increase
in acid-insoluble radioactivity. In the other, the rate of formation of A D P
is measured spectrophotometrically by coupling the propionyl-CoA car-
boxylase reaction with reactions catalyzed by pyruvate kinase 3 and
lactate dehydrogenase. 4 With an excess of pyruvate kinase and lactate
dehydrogenase, the rate of oxidation of N A D H is proportional to the rate
of formation of ADP, and therefore to the rate of fixation of HCO~- to
propionyl-CoA to yield Ds-methylmalonyl-CoA.
At early stages of purification, the presence of ATPase necessitates
' S e e Vol. V [77].
A. Tietz and S. Ochoa, J. Biol. Chem. 234, 1394 (1959).
3See Vol. I [66].
' See Vol. I [67].
182 R E A C T I O N S L E A D I N G TO AND FROM T H E CYCLE [31]
the use of H~4CO~- fixation assay. With partially purified preparations,

however, the more rapid spectrophotometric assay is preferable.
H14C0~- Fixation Assay
Reagents

MgCl~, 60 mM
Reduced glutathione, 20 mM (GSH, neutralized to pH 7.5)
ATP, 30 mM
Propionyl-CoA,5 10 mM
14C-Na2CQ (25,000 cpm per micromole), 50 mM
Enzyme diluted with 20 mM Tris-HC1 buffer, pH 7.5, containing
1 mM EDTA and 0.5 mM GSH
Stock reaction mixture (0.06 RM) is made up by mixing 20 ml of

distilled water with l0 ml each of 1.0 M Tris-HCl buffer, pH 8.0, 60 mM
MgCl_o, 20 mM GSH, and 30 mM ATP; it is kept frozen. This quantity
suffices for 100 assays.
Proced~we. The reaction is carried out at 30 ° in small Thunberg tubes
{1.2 X 5 cm). Place in the main tube 0.6 ml of stock reaction mixture
{0.06 RM) which contains (in micromoles): Tris-HC1 buffer, pH 8.0,
1{~9; MgCl_o, 6; GSH, 2; and ATP, 3. Add 0.1 ml of 10 mM propionyl-
CoA (1.0 micromole) and 0.1 nil of enzyme. In the side bulb, place 10
micromoles of Na_~4CO.~ (250,000 cpm) in a volume of 0.2 ml. After a
short equilibration period, start the reaction by tipping the carbonate
solution into the main tube. Allow incubation to continue for l0 minutes.
Stop the reaction by adding 0.2 ml of 20% trichloroacetic acid; remove
the precipitated protein by centrifugation. Place aliquots (0.2 ml) of the
protein-free supernatant on stainless-steel planchets and dry under an
infrared lamp. Determine the radioactivity with a windowless gas-flow
counter.
Units. One unit of enzyme is defined as the amount of protein
catalyzing the fixation of 1 micromole of H~4CO3- per minute under the
conditions of the assay. Specific activity is expressed in units per milli-
gram of protein. Protein is determined spectrophotometrically by measur-
'Prepared according to the method of E. J. Simon and D. Shemin, J. Am. Chem.

Soc. 75, 2520 (1953). The concentration of propionyl-CoA was determined by
measuring alkali-labile thiol group according to R. R. Grunert and P. H. Phillips.
Arch. Biochem. Biophys. 30, 217 (1951).
[311 PROPIONYL-COA CARBOXYLASE FI~.OM PIG UEART 183
ing the light absorption at wavelength 280 m/~ with a correction for the
nucleic acid content. ''
Optical Assag
Reaget~ts
MgCI~, 40 mM
GSH, 20 mM
ATP, 20 mM
K H C Q , 1.0 M
KC1, 1.0 M
Phosphoenolpyruvatc, 20 mM
Pyruvate kinasc, 25 units'/ml
Lactate dchydrogenase, 35 unitsT/ml
NADH, 5 mM
Propionyl-CoA, l0 mM
The stock reaction mixture (0.85 RM) for optical assay is prepared
by mixing 2 ml of distilled water with l0 ml of 1.0 M Tris-HC1 buffer,
pH 8.0, 10 ml of 40 mM MgCl.,, l0 ml of 20 mM GSH, 10 ml of 20 mM
ATP, 5 ml of 1.0M KHCOa, 10 ml of 1.0M KCI, 5 ml of 20 mM
phosphocnolpyruv'ttc, 10 ml of 25 units/ml pyruvate kinase, 10 ml of
35 units/ml lactate dehydrogenase, and 3 ml of 5 mM NADH. This
solution can be kept frozen for 4-5 weeks and yields 100 optical assays.
Procedure. The reaction is carried out at 25 ° in quartz cell (d, = 1.0
cm) in a Beckman spectrophotometcr. Each cell receives 0.85 ml of the
reaction mixture (0.85 RM) which contains: (in micromoles) Tris-HCl
buffer, pH 8.0, 100; MgCI_~, 4; GSH, 2; ATP, 2; KHC03, 50; KC1, 100;
phosphocnolpyruvate, 1; NADH, 0.15; and also pyruvate kinasc, 2.5
milts; and lactate dchydrogenase, 3.5 units. Propionyl-CoA carboxylasc,
0.1 ml, is added and the optical density at 340 mu is measured. If there
is A rapid decrease in the optical density, the enzyme preparation
probably contains ATPase as a contaminant and should be assayed by
H'~CO:~- fixation assay. The reaction is started by addition of 0.05 ml of
10 m M propionyl-CoA (0.5 micromole), and the rate of decrease at
wavelength 340 m~ is followed.
Units. Since 1 mole of ADP is produced per mole of NADH oxidized,
one unit of enzyme is taken as the amount of protein catalyzing the
formation of 1 micromole of ADP per minute.
SSee Vol. III [73].
7One unit is referred to as the amount of enzyme which transforms 1 micromole of
substrate per minute. Pyruvate kinase and lactate dehydrogenase are obtained from
C. F. Boehringer and Soehne, Mannheim, Germany.
184 REACTIONS LEADING TO AND FROM TIlE CYCLE [31]
Purification Procedure s
Propionyl-CoA carboxylase is purified from pig heart. All operations

are carried out at 0 ° to 3 °.
Step 1. Extraction. Thirty fresh pig hearts are packed in ice, trimmed
of fat, blood clots, and connective tissue, and passed through an electric
meat grinder. The mince (5 kg) is divided into l-kg portions, and each
portion is mixed thoroughly with 1.2 liters of 50 mM Tris buffer, pH 7.5,
containing 1 mM EDTA. The mixture is stirred occasionally over a
period of 30 minutes and then filtered through four layers of cheesecloth,
and the mince is squeezed as dry as possible. The supernatant wash
fluid is discarded, and the mince is extracted in a large capacity (I
gallon) Waring blendor at top speed for I minute with 2 liters of the
above buffer. The mixture is allowed to stand for I0 minutes, then it is
centrifuged in Lourdes model LRA and Servall model RC-I refrigerated
centrifuges at 8000 g for 30 minutes; the sediment is discarded. The
supernatant extracts from the various I kg portions of mince are
mixed, and GSH is added to a final concentration of 0.5 mM. About 7.8
liters of extract with 127 g of protein is obtained from thirty pig hearts.
Step 2. Ammonium Sulfate Fractionation. Finely powdered solid
ammonium sulfate is added slowly to the extract, with mechanical
stirring, in the proportion of 216 g/liter. The mixture is stirred for an
additional 30 minutes, then the precipitate is collected by centrifugation
at 8000 g for 60 minutes and discarded. After the further addition of 136 g
of ammonium sulfate per liter to the supernatant solution, the sus-
pension is kept overnight at 4 °. The next morning, the supernatant is
siphoned off, the precipitate is collected by centrifugation, and the clear
supernatant fluid is discarded. The precipitate is then dissolved in
20 mM Tris-HC1 buffer, pH 7.5, containing 1 mM EDTA and 0.5 mM
GSH and is stored frozen. This procedure is repeated until the am-
monium sulfate fractions from 500 pig hearts have been accumulated.
At this stage, the activity of the enzyme remains unchanged during
storage of the ammonium sulfate fractions at --18 ° for 3 to 4 months.
Step 2a. Dialysis. The ammonium sulfate fractions are thawed,
pooled (volume, 6.7 liters; 80 mg of protein per milliliter), and divided
into portions of approximately 1 liter. Just before the next step, each
portion is dialyzed in the cold room for 24 hours against 10 liters of
33 mM succinate buffer, pH 6.3, containing 1 mM EDTA and 0.5 mM
GSH; the dialysis solution is changed every 8 hours. The whole dialyzed
enzyme solution is diluted with the same buffer to a volume of 27 liters
and a protein concentration of 20 mg/ml.
Step 3. Ethanol Fractionation. The enzyme is (livided into 3-liter
s y . Kaziro, A. Grossman, and S. Ochoa, J. Biol. Chem. 240, 64 (1965).
portions. After cooling each 3-liter portion to 0 °, ethanol (cooled to 0 °)

is added in the proportion of 42 ml per liter with mechanical stirring
over a period of approximately 30 minutes; the temperature is kept at
0 °. The precipitate is removed by centrifugation at 15,000 g at 0 ° for
20 minutes and discarded. Ethanol, cooled previously to --15 °, is added
in the proportion of 143 ml per liter to the clear supernatant fluid. The
teinperature is lowered gradually from 0 ° to --6 °, and stirring is
continued for another 30 minutes after the addition of ethanol. The
precipitate is collected by centrifugation as before for 40 minutes at --6 °
and dissolved in 20 mM Tris-HCl buffer, pH 7.5; the buffer contained
1 mM EDTA and 0.5 mM GSH.
Step 4. Protamine Fractionation. Adjust the enzyme solution from
step 3 (about 5 liters) to pH 6.5 with 0.5M acetic acid. The protein
concentration is 20.5 mg/ml. Add 100 ml of a freshly prepared 1%
protamine solution. Discard the precipitate after centrifugation. Add to
the supernatant 250 ml of 1% protamine. Then collect the precipitate by
centrifugation, wash it with 1 liter of distilled water, and dissolve it in 1
liter of 50 mM potassium phosphate buffer, pH 6.5, containing 1 mM
EDTA and 0.5 mM GSH. Centrifuge the insoluble material and discard
it.
Step 5. Calcium Phosphate Gel Adsorption and Elutiaa. Dilute the
clear supernatant solution (1000 ml) to 1670 ml with the above buffer to
give a protein concentration of 15 mg/ml. Add calcium phosphate gel
(dry weight, 31 mg/ml), 400 ml, with mechanical stirring. After 30
minutes, centrifuge the gel and discard it. To the supernatant add 1200
ml of gel with mechanical stirring. Collect the gel by centrifugation and
elute it three times with 800 ml of 0.2 M potassium phosphate buffer, pH
6.5, containing 1 mM EDTA and 0.5 mM GSH.
Step 6. Second Ammonium Sul]ate Fractionation. Add solid am-
monium sulfate (210 g/liter) to the combined eluates {2600 ml). After
stirring for another 30 minutes, centrifuge the precipitate and discard it.
Add ammonium sulfate ( l l 0 g/liter) to the clear supernatant. Collect
the precipitate by centrifugation and dissolve it in 20 mM potassium
phosphate buffer, pH 6.5, containing 1 mM EDTA and 0.5 mM GSH.
The final volume is 375 ml.
Step 7. Crystallization. Cool the enzyme solution step 6 to 0°;
slowly stir in finely powdered ammonium sulfate, 35 g per 100 ml (55%
saturation). Collect the precipitate by centrifugation and dissolve it in
the minimal volume of the phosphate-EDTA-GSH buffer, pH 6.5. Cau-
tiously add solid ammonium sulfate to this solution until a faint,
permanent turbidity appears. The precipitate, which has little activity,
is removed by centrifugation and discarded. Add saturated ammonium
sulfate to the supernatant solution dropwise with stirring to incipient
18 6 REACTIONS LEADING TO AND FROM T i l e CYCLE [31]
turbidity, and place the mixture in the refrigerator. Crystallization begins
after 12 hours. After 2 (lays, harvest the crystals by centrifugation and
dissoh'e them in a minimal volume of potassimn phosphate buffer. They
have a specific activity of ahout 13. el, recry~tallization, the specific
activity increases to 16.4.
Recrystallization is speeded up by the addition of seine "seed"
crystals and starts after 4 hours. H a r v e s t the crystals after 12 hours.
The enzyine crystallized six times ill this way has a specific activity of
17 (about 25 at 30 °) 2 The results of purification are summarized in tlle
table.
PURIFICATION OF PROPIONYL-CoA CARBOXYLASE FROM PIG HEART a
Specific
activityb
Volume Protein (units/mg Yield
Step (ml) Units (g) protein) (~.)
I. Extract c 130,000 42,500 2,120 0.02 100

?. First (NH,)2S(), 6,700 38,200 533 0.07 90
fractionation
3. Ethanol fractionation 5,000 33,900 103 0.3 80
4. Protamine fractionation 1,000 29,600 24.8 1.2 70
5. Adsorption and elution 2,600 20,300 10.3 2.0 48
from Ca3(PO,)2 gel
6. Second (NH,)2SO, 375 16,700 4.84 3.5 39
fractionation
7. First crystallization 37 11,200 0.85 13.2 26
8. Recrystallization 26 9,500 0.58 16.4 24
a y. Kaziro, A. Grossman, and S. Ochoa, J. Biol. Chem. 240, 64 (1965).
h At 25°. Values at 30° are approximately 1.5 times higher. [Y. Kaziro, S. Ochoa,
R. C. Warner, and Jo-Yun Chen, J. Biol. Chem. 236, 1917 (1961).]
c Minced tissue, 80 kg, from 500 pig hearts was used.
Under the above conditions the enzyme crystallizes as hexagonal

prisms with p y r a m i d a l bases (Fig. 1). Their size and shape varies with
time and conditions of crystallization. The prisms are often elongated
and small (Fig. 1D) but occasionally they are shorter and larger (Fig.
1C). Crystallization at room temperature with ammonium sulfate yields
consistently larger crystals in the form of short hexagonal prisms (Fig.
1A and B).
The crystals are kept as suspension in 20 m M phosphate buffer, p H
6.5, containing 1 m M E D T A , 0.5 m M GSH, and 60% saturated am-
~Y. Kaziro, S. Ochoa, R. C. Warner, and Jo-Yun Chcn, J. Biol. Chem. 236, 1917
(1961).
monium sulfate either at 3-4 ° or at --18 °. When required, the enzyme

is sedimented by centrifugation of aliquots of the stock suspension and
dissolved in 20 mM phosphate buffer, pH 6.5, with 1 mM E D T A and
0.5 mM GSH or other suitable solvent.
When it is difficult to crystallize the enzyme directly from step 6
(specific activity, 3.5), an extra purification step by DEAE-cellulose
column chromatography" can be inserted. Wash the DEAE-cellulose with
1.0 N NaOH until the washings are colorless, and then wash with distilled
water. Remove the wash fluids by decantation. Then stir the cellulose
FiG. 1. Crystalline enzyme [Y. Kaziro, S. Ochoa, R. C. Warner, and Jo-Yun

Chen. J. Biol. Chem. 236, 1917 (1961)]. A and B. Room temperature crystals. C and
D. Two variants of crystals obtained at 0°. Magnifications: A, X800; B, X350;
C, × 700; D, × 950.
with 0.5 N HC1; immediately filter the suspension with suction on a
large, sintered glass funnel, and wash the cellulose with distilled water
until the filtrate is free of chloride. Suspend the cellulose evenly in 20 mM
potassium buffer, pH 6.5, and adiust the pH of the suspension to 6.5 with
2.0N NaOH. Heavy particles sedimenting within 2 minutes and light
particles that do not settle within 1 hour, are removed by decantation.
Then wash the cellulose three or four times with 20 mM potassium
phosphate buffer, pH 6.5, and pack it into columns; apply pressure
during packing with a Saxon aquarium air pump.
Dialyze the enzyme solution from step 6 against 20 mM phosphate
buffer, pH 6.5, containing 1 mM E D T A and 0.5 mM GSH, and pass it
through the column in a cold-room without applying pressure. Wash the
188 R E A C T I O NLEADING
S TO AND FROM THE CYCLE [31]
column with 70 mM potassium phosphate buffer, pH 6.5, containing 1

mM GSH, to remove most of the inactive protein; then elute the enzyme
with 0.12 M phosphate buffer, pH 6.5, containing 1 mM GSH, at a rate
of 0.5 ml per minute. Combine the fractions containing enzyme of specific
activity 6 to 8 and concentrate them by ammonium sulfate precipitation.
Properties of Enzyme
Molecular Weight and Biotin Content. The enzyme is homogeneous

in ultracentrifugation and electrophoresis2 It has a molecular weight of
700,000, and its sedimentation coefficient is S~o,,----19.72_ 0.09. The
enzyme contains 1.395 ~g of biotin per milligram of protein and 1 mole
of bound biotin per 1751000 g or 4 moles per mole of protein2 The biotin
is linked covalently to the ~-amino group of lysine residues of the pro-
tein2, lo The isoelectric point is in the vicinity of pH 6.1. The amino acid
composition of the enzyme has been reported2
pH Optimum. With the H14COs- fixation assay the optimal pH is
8.0-8.2, whereas with the optical assay it is 8.5. In each case there is a
sharp decrease in activity on either side of the optimum2
Substrate Specificity. The nucleoside 5'-triphosphate specificity of pig
heart propionyl-CoA carboxylase is restricted to ATP. 2 Neither GTP,
UTP, CTP, nor ITP can replace ATP. Regarding its fatty acyl-CoA
specificity, the enzyme is active, although to a much lesser extent, with
butyryl-, acetyl-, and crotonyl-CoA besides propionyl-CoA2 Relative
rates, with that of propionyl-CoA being taken as 1001 are approximately
6, 1, and 3, respectively. The absolute configuration of the product of
propionyl-CoA carboxylase reaction is established as D,-methylmalonyl-
CoA.11,12 The latter is converted to its optical antipode L-methylmalonyl-
CoA, the substrate for methylmalonyl-CoA mutase, 13 through the reac-
tion of methylmalonyl-CoA racemaseJ 4
Kinetic Constants. The turnover number (Vm,~) at 30 ° and saturating
concentrations of propionyl-CoA are calculated to be 21,600 moles per
minute per mole of enzyme at pH 8.5, or 5000 per mole of enzyme-bound
biotin. The corresponding value for butyryl-CoA is approximately 2500
moles per minute per mole of enzyme2 The following K~ values are
calculated from Lineweaver-Burk plots using optical assay: K~ (ATP),
'°D. P. Kosow and M. D. Lane, Biochem. Biophys. Res. Commun. 7, 439 (1962).
11M. Sprecher, M. d. Clark, and D. B. Sprinson, J. Biol. Chem. ~11, 872 (1966).
l~j. RStey and F. Lynen, Biochem. Z. 342, 256 (1965).
ISSee this volume [34].
"See this volume [32] ; R. Mazumder, T. Sasakawa, Y. Kaziro, and S. Ochoa, J. Biol.
Chem. 237, 3065 (1962).
80 /dr/; Km (HCO3-), 2.5 raM; Km (propionyl-CoA), 0.2 r M. K~ for

butyryl-CoA is 1.5 mM.
The apparent equilibrium constant of the propionyl-CoA carboxylase
reaction is determined. 8 K' has an average value of 5.7 at pH 8.1 and
28 ° . The calculated free energy change (F ° = - - R T In K) is F3ol =
--1028 calories per mole. The reaction is therefore readily reversible, the
equilibrimn position slightly favoring the carboxylation of propionyl-
CoA.
K' = [ADP][Pi][Ds-methyhnalonyl-CoA]
[ATP][HCO~-][propionyl-CoA]
Reversibility. The reversibility of the reaction is demonstrated by net
formation of ATP from ADP and P~ in the presence of methylmalonyl-
CoA when propionyl-CoA carboxylase is coupled with the reactions
catalyzed by hexokinase and glucose-6-phosphate dehydrogenase. 2
Activator and Inhibitor. The enzyme requires the presence of Mg *+
ions for its activity. It is inhibited by a stoichiometric amount of avidin ;'~
prior addition of excess d-biotin will prevent this inhibition. It is also
inhibited strongly by pCMB, indicating that integrity of thiol groups is
essential for activity.
Reaction Mechanisms. The reaction involves carboxylation and de-
carboxylation of the enzyme according to the reactions below: ~
Mg ~+
ATP + HCO~- + biotin-enzyme ~-~ carboxy ~-~ biotin-enzyme
+ ADP + Pi
Carboxy~biotin-enzyme + propionyl-CoA
D.-methylmalonyl-CoA + biotin-enzyme
The enzyme is carboxylated either by ATP and bicarbonate-a4C, or by
methylmalonyl-CoA-a4C. The l~C-labeled carboxy~biotin-enzyme is
isolated, and it was demonstrated that 1 mole of bicarbonate-~C is taken
up per mole of protein-bound biotin. The carboxy~biotin-enzyme either
transferred its carboxyl group to propionyl-CoA to form methylmalonyl-
CoA-a~C or decarboxylated with concomitant yielding of ATP from ADP
and pi.~6
The carboxy~biotin-enzyme is very unstable at room temperature,
its half-life being of the order of several minutes, but it can be stabilized
by methylation with diazomethane? 7 l'-N-Carbomethoxybiocytin (bio-
~ Y. Kaziro, E. Leone, and S. Ochoa, Proc. Natl. Acad. Sci. U.S. 46, 1319 (1960).
l~y. Kaziro and S. Ochoa, J. Biol. Chem. 236, 3131 (1961).
17F. Lynen, J. Knappe, E. Lorch, G. Jutting, and E. Ringelmann, Angew. Chem.
71, 481 (1959).
cytin = c-N-d-biotinyl-L-lysine) has been isolated from pronase digests

of carboxylated propionyl-CoA carboxylase of bovine liver2 ~
By means of 1~O, it has been shown that ATP is cleaved during the
propionyl-CoA carboxylase reaction between the terminal phosphorus
atom and the bridge oxygen, with transfer of a bicarbonate oxygen
atom to the liberated orthophosphate.'" Two other oxygen atoms of
bicarbonate appear in the free carboxyl of D~-methylmalonyl-CoA. This
indicates that the reacting species of ~*CO., in this reaction is bicarbonate
ion and that the first step of the reaction, the formation of carboxy~
biotin-enzyme, may proceed through a concerted mechanism.
Other properties of the enzyme, including those of other biotin-
enzymes, are described elsewhere3 °,~ Propionyl-CoA carboxylase was
also purified from acetone powder of bovine liver mitochondria, 2z and
was found also in Rhodospirillum rubrum 2"~ and Mycobacterium smeg-
marls.°-4
'~ M. D. L'me and F. l,ynen, Proc. Natl. Acad. Sci. U.S. 49, 379 (1963).
'~ Y. Kaziro, L. F. Hass, P. D. Boyer, and S. Ochoa, J. Biol. Chem. "237, 1460 (1962).
"~"Y. Kaziro and S. O(.hoa, Advan. Enzymol. 26, 283 (1964).
2, S. Ochoa and Y. Kaziro, in "Comprehensive Biochemistry" (M. Florkin and E. tI.
Stotz, eds.), Vol. 16, p. 210, Elsevi(,r, Amsterdam, 1965.
~:See Vol. V [78].
-'3M. Knight, Biochem. J. 84, 170 (1962).
'~ R. L. Stjernholm, R. E. Noble, and D. Koch-Weser, Biochim. Biophys. Acta 64,
174 (1962).
[32] Methylmalonyl-CoA Racemase from Sheep Liver

[EC 5.1.99.1 Met,hylmMonyl-CoA racemase]
B y RAJARSHI MAZUMDER
D-Methylmalonyl-CoA ~- L-methylmalonyl-CoA
Assay Method
Principle. Propionyl-CoA carboxylase, methylmalonyl-CoA mutase,
and methylmalonyl-CoA racemase are incubated with propionyl-CoA,
H1~C03- and ATP. D-Methylmalonyl-CoA formed by the enzymatic
carboxylation of propionyl-CoA is converted by raccmase to L-methyl-
malonyl-CoA. The latter is isomerized to succinyl-CoA by mutase. Since
succinate is resistant to permanganate treatment whereas methyhnalonate
is not, the radioactivity remaining after oxidation of the deproteinized
reaction mixture with potassium permanganate is a measure of the
cytin = c-N-d-biotinyl-L-lysine) has been isolated from pronase digests

of carboxylated propionyl-CoA carboxylase of bovine liver2 ~
By means of 1~O, it has been shown that ATP is cleaved during the
propionyl-CoA carboxylase reaction between the terminal phosphorus
atom and the bridge oxygen, with transfer of a bicarbonate oxygen
atom to the liberated orthophosphate.'" Two other oxygen atoms of
bicarbonate appear in the free carboxyl of D~-methylmalonyl-CoA. This
indicates that the reacting species of ~*CO., in this reaction is bicarbonate
ion and that the first step of the reaction, the formation of carboxy~
biotin-enzyme, may proceed through a concerted mechanism.
Other properties of the enzyme, including those of other biotin-
enzymes, are described elsewhere3 °,~ Propionyl-CoA carboxylase was
also purified from acetone powder of bovine liver mitochondria, 2z and
was found also in Rhodospirillum rubrum 2"~ and Mycobacterium smeg-
marls.°-4
'~ M. D. L'me and F. l,ynen, Proc. Natl. Acad. Sci. U.S. 49, 379 (1963).
'~ Y. Kaziro, L. F. Hass, P. D. Boyer, and S. Ochoa, J. Biol. Chem. "237, 1460 (1962).
"~"Y. Kaziro and S. O(.hoa, Advan. Enzymol. 26, 283 (1964).
2, S. Ochoa and Y. Kaziro, in "Comprehensive Biochemistry" (M. Florkin and E. tI.
Stotz, eds.), Vol. 16, p. 210, Elsevi(,r, Amsterdam, 1965.
~:See Vol. V [78].
-'3M. Knight, Biochem. J. 84, 170 (1962).
'~ R. L. Stjernholm, R. E. Noble, and D. Koch-Weser, Biochim. Biophys. Acta 64,
174 (1962).
[32] Methylmalonyl-CoA Racemase from Sheep Liver

[EC 5.1.99.1 Met,hylmMonyl-CoA racemase]
B y RAJARSHI MAZUMDER
D-Methylmalonyl-CoA ~- L-methylmalonyl-CoA
Assay Method
Principle. Propionyl-CoA carboxylase, methylmalonyl-CoA mutase,
and methylmalonyl-CoA racemase are incubated with propionyl-CoA,
H1~C03- and ATP. D-Methylmalonyl-CoA formed by the enzymatic
carboxylation of propionyl-CoA is converted by raccmase to L-methyl-
malonyl-CoA. The latter is isomerized to succinyl-CoA by mutase. Since
succinate is resistant to permanganate treatment whereas methyhnalonate
is not, the radioactivity remaining after oxidation of the deproteinized
reaction mixture with potassium permanganate is a measure of the
[32] METtlYLMALONYL-COA RACEMASE FROM SttEEP 1,IVER 191
succinyl-CoA formed.' The raeemase is assayed by u~ing this enzyme in

limiting amounts in the presence of an excess of c:trboxyl'tse and mutase.
Reagc,ds
Tris-HC1 buffer, 500 raM, pH 7.5

MgCI_,, 120 mM
(;lutathionc (GStt), 40 nlJ/
ATP, 30 m3I
Na~'~C():, (174,840 Cl)m/microlnole), 50 in3!
Propionyl-CoA, ~ 10 mM
Propionyl-CoA carboxylas& (specific activity, 3.0-4.0)
Methyhnalonyl-CoA nmtase: 0.3 saturated ammonium sulfate
extract of protamine precil)itatc (preparation is described below
under the lmrification procedure)
Perchloric acid, 2.0 N
Procedure. The reaction mixture contained in a final volume of 1.0 ml

the following coml)onents (in micromoles) : Tris-HC1 buffer, pH 7.5, 100;
MgCI:, 6; GSH, 2; ATP, 1.5; Na~'~C():~ 5; propionyl CoA, 0.5. The
mixture contained al.~o pvopionyl-CoA carboxylase, 0.4-0.5 unit; methyl-
malonyl-CoA llltltase, 0.8 rag; racemase, up to 0.008 unit. Prol)ionyl-CoA
is added last. After incubation in stol)t)ered tubes for 10 minutes at 30 °,
0.2 ml of 2.0 N perchloric acid is added. The mixture is kept at 100 ° for
3 mimltes, then centrifuged.
Perchloric "~cid, 2.0 N, 0.1 ml, and 4% potassium permanganate, 1.0
lnl, are added to 0.5 ml aliquots of the supernatant solution. The mixture
is kept in a boiling water bath for 14 nmmtes, cooled in ice, and cen-
trifuged; 0.2 ml aliquots of the clear supernatant solution arc plated on
stainless steel planehets, and their radioactivity is measured in a window-
less gas-flow counter. The permanganate-stable radioactivity in the
s'tmple containing no raeemase is used as a control and subtracted from
that in the complete saml)les. Under these conditions, the succinatc
formed is prol)ortional to the concentration of racelnase.
Units. One unit of enzyme is defined as the amount of protein catalyz-
ing the formation of 1.0 micromole of succinate per minute at 30 ° under
the conditions of the assay. Specific activity is expressed as units pc,'
milligram protoin. Protein is determined spcctrophotometrically. ~
'M, Flavin and S. Ochoa, J. Biol. Cl~cm. 229, 965 (1957).

2Prepared according to the method given in reference eiled in footnote 1.
~Pr(q)ared re'cording to the method of Y. Kaziro, S. Ochoa, R. C. Warnrr, and J.-Y.
Chen, J. Biol. Chem. 236, 1917 (1961).
'See Vol. III 173].
192 REACTIONS LEANING TO AND FROM THE C Y C L E [32]
All operations are performed at 0-3 ° unless stated otherwise.
Step 1. Extraction. Fresh sheep liver is minced in an electric meat
grinder chilled previously. The mince is homogenized with 50 mM Tris-
HC1 buffer, pH 7.4 (2 ml of buffer for 1 g of mince) for 3 minutes in a
Waring hlendor at tol / speed. After centrifugation, tile turbid supernatant
solution is passed through cheesecloth.
Step 2. Ammonium Sulfate Fractio~ation. The extract is diluted to
20 mg of protein per milliliter with 50 mM Tris-HC1 buffer, pH 7.4; finely
powdered ammonium sulfate (29.1 g/100 ml) is added slowly with
mechanical stirring. The stirring is continued for a further 30-40 minutes,
and the mixture is then centrifuged. The precipitate is discarded. More
ammonium sulfate (12.5 g/100 ml) is added to the supernatant solution
as before. The suspension is centrifuged; the precipitate is dissolved in a
small volume of 20 mM Tris-HC1 buffer, pH 7.4, and frozen until used.
Step 3. Protamine Treatment and Ammonium Sul]ate Fractionation.
The solution from the step 2 is dialyzed against 100 volumes of 20 mM
Tris-HC1 buffer, pH 7.3, for approximately 15 hours and centrifuged. The
supernatant solution is diluted to approximately 9 mg of protein per
milliliter with 20 mM Tris-HCl buffer, pH 7.3. A 1% solution of prot-
amine sulfate, freshly prepared (at room temperature), is then added
(12.4 mg/100 mg of protein) with stirring. After further stirring for 1
hour, the suspension is kept at 4 ° overnight and centrifuged. The precipi-
tate contains methylmalonyl-CoA mutase; the supernatant contains the
racemase. Ammonium sulfate, 32.6 g, is added to every 100 ml of prot-
amine supernatant, stirred, and centrifuged. The precipitate is discarded.
Ammonium sulfate (9.3 g/100 ml) is again added to the supernatant
solution, stirred, and centrifuged. The precipitate containing the racemase
is dissolved in 20 mM Tris-HC1 buffer, pH 7.3.
•The mutase is extracted, by means of a glass homogenizer, from the
protamine precipitate with 0.1M Tris-HC1 buffer, pH 7.4, containing
ammonium sulfate at 30% saturation. The mixture is centrifuged, and
the insoluble material is discarded.
Step 4. Adsorption and Elution on Calcium Phosphate Gel. The
racemase-containing solution from step 3 is dialyzed against 100 volumes
of 20 mM Tris-HCl buffer, pH 7.4, for approximately 15 hours and
adjusted to a protein concentration of 10 mg/ml and a Tris-HC1 con-
centration of 10 mM. The diluted solution is adjusted to pH 5.8 by the
dropwise addition of 1.0 N acetic acid with vigorous mechanical stirring.
Calcium phosphate gel (5 mg/ml) is then added in the proportion of
12.75 mg of gel per 100 mg of protein, and the mixture is stirred for 15
minutes. The gel (gel 1) is removed by centrifugation and discarded ~el.
[32] METHYLMALONYL-COA RACEMASE FROM SHEEP LIVER 193
25.5 mg/100 mg of protein, in the original diluted solution is added to the

supernatant solution, stirred as before, and centrifuged (gel 2). The
latter procedure is repeated once more (gel 3). Gels 2 and 3 are washed
separately with water and then eluted individually and successively with
20 mM, 50 mM, 0.1 M, and 0.2 M potassium phosphate buffer, p H 7.3.
The 0.2 M phosphate buffer eluates from gels 2 and 3 are pooled.
PURIFICATION OF ~ETHYLMALCNYL-CoA RACEMASE FROM SHEEP LIVEtl a
Specific
activity
Volume Activity Protein (units/rag Yield
Step (ml) (units b) (mg) protein) (%)
1. Extract 153 34 18,054 0.002 100

2. (NH4)2SO4(0.5-0.7 saturation) 36 22 1,707 0.013 65
and dialysis
3. Precipitation with protamine 18 13 661 0.02 38
and (NH4)2SO4fractionation
(0.55-0.7 saturation) of
supernatant
4. Ca3(PO4)2gel eluate 30 10 28 0.35 29
From 80 g of sheep liver mince.
bOne unit of enzyme is the amount of protein catalyzing the (ormation of 1.0 micro-
mole of succinate per minute at 30° under the assay conditions.
Properties
Stability. The purified enzyme is fairly stable when stored frozen
(--15°). However, it loses activity on repeated freezing and thawing.
Enzymatic Purity. The calcium phosphate gel eluate fraction is
essentially free of methylmalonyl-CoA mutase.
Reversibility. Racemase can also catalyze the conversion of L-methyl-
malonyl-CoA to D-methylmalonyl-CoA. Net formation of 14C0~ can be
demonstrated when carboxyl-labcled succinyl-CoA is converted to pro-
pionyl-CoA by reversal of the reactions catalyzed by mutase, racemase,
and carboxylase in the presence of hexokinase and glucose. 4
Nonenzgmatic Racemization. D-Methylmalonyl-CoA is converted to
the nL-racemic mixture by heating at 100 ° for 2-3 minutes. Slow spon-
taneous racemization also occurs at 30 °.
Mechanism o] Racemization. 5 Racemization of methylmalonyl-CoA
does not occur either by an intermolecular transfer of its CoA moiety to
methylmalonic acid or by an intramolecular CoA transfer from one
R. Mazumder, T. Sasakawa, Y. Kaziro, and S. O(.hoa, J. Biol. Chem. 237, 3065
(1962).
194 REACTIONS
LEADING TO AND FROM THE CYCLE [33]
carboxyl group to the other. However, when chemically synthesized

methylmalonyl-CoA is kept at 30 ° in the presence of tritium-enriched
water, there is a small incorporation of tritium into methylmalonyl-CoA.
This incorporation is increased greatly by either racemase or brief heat-
ing at 100 °. I t appears that both the nonenzymatic and enzymatic
racemization of methyhnalonyl-CoA proceed by a mechanism involving
loss of the a-hydrogen atom with subsequent incorporation of a proton
from the medium.
[33] Methylmalonyl-CoA Racemase from

Propionibacteriurn shermanii 1
[EC 5.1.99.1 Methylmalonyl-CoA racemase]
By S. H. G. ALLEN, Ft. ~V. KELLERMEYER,and HARLAND G. ~VOOD
C~oA .H H~,,. ~.COSCoA
C~ C~
C~ ~CH. COOH CH s
(S)-Methylmalonyl-CoA (R)-Methylmalonyl-CoA
Assay Method
The assay is based on tlle conversion of (R)-methyhnalonyl-CoA to
the (S) form by the racemase. The rate of conversion of (R) to (S) can
be assayed spectrophotometrically by means of a series of coupled
reactions. 'a
CoA transferase
Acetyl-CoA + succinate ~ succinyl-CoA + acetate (1)
methylmalonyl-CoA mutase
Sueeinyl-CoA ~ (R)-methylmalonyl-CoA (2)
DBC 2
raeemase
(R)-Methyhnalonyl-CoA ~--- (S)-nmthyhnalonyl-CoA (3)
oxaloacetate
t ranscarboxylase
(S)-Methylmalonyl-CoA + pyruvate
propionyl-CoA q- oxaloacetate (4)
malate dehydrogenase
Oxaloacetate + N A D H ~ malate + NAD (5)
'This work was assisted by grant GM 11839 from the National Institutes of Health,
United States Public Health Service, Bethesda, MaD'land.
" S. H. G. Allen, R. Kellermeyer, R. Stjernholm, B. Jacobson, and H. G. Wood,
J. Biol. Chem. 238, 1637 (1963).
: Abbreviation : DBC-(5,6-dimethylbenzimidazol3 l)Co-5 deox3adenosine cobamide.
194 REACTIONS
carboxyl group to the other. However, when chemically synthesized

methylmalonyl-CoA is kept at 30 ° in the presence of tritium-enriched
water, there is a small incorporation of tritium into methylmalonyl-CoA.
This incorporation is increased greatly by either racemase or brief heat-
ing at 100 °. I t appears that both the nonenzymatic and enzymatic
racemization of methyhnalonyl-CoA proceed by a mechanism involving
loss of the a-hydrogen atom with subsequent incorporation of a proton
from the medium.
[33] Methylmalonyl-CoA Racemase from

Propionibacteriurn shermanii 1
[EC 5.1.99.1 Methylmalonyl-CoA racemase]
By S. H. G. ALLEN, Ft. ~V. KELLERMEYER,and HARLAND G. ~VOOD
C~oA .H H~,,. ~.COSCoA
C~ C~
C~ ~CH. COOH CH s
(S)-Methylmalonyl-CoA (R)-Methylmalonyl-CoA
Assay Method
The assay is based on tlle conversion of (R)-methyhnalonyl-CoA to
the (S) form by the racemase. The rate of conversion of (R) to (S) can
be assayed spectrophotometrically by means of a series of coupled
reactions. 'a
CoA transferase
Acetyl-CoA + succinate ~ succinyl-CoA + acetate (1)
methylmalonyl-CoA mutase
Sueeinyl-CoA ~ (R)-methylmalonyl-CoA (2)
DBC 2
raeemase
(R)-Methyhnalonyl-CoA ~--- (S)-nmthyhnalonyl-CoA (3)
oxaloacetate
t ranscarboxylase
(S)-Methylmalonyl-CoA + pyruvate
propionyl-CoA q- oxaloacetate (4)
Oxaloacetate + N A D H ~ malate + NAD (5)
'This work was assisted by grant GM 11839 from the National Institutes of Health,
United States Public Health Service, Bethesda, MaD'land.
" S. H. G. Allen, R. Kellermeyer, R. Stjernholm, B. Jacobson, and H. G. Wood,
J. Biol. Chem. 238, 1637 (1963).
: Abbreviation : DBC-(5,6-dimethylbenzimidazol3 l)Co-5 deox3adenosine cobamide.
[33] METHYLMA.LONYL-COA
RACEMASE FROM P . shermanii 195
In the assay, (R)-methylmalonyl-CoA is generated from suceinyl-

CoA formed via Eqs. (1) and (2). The CoA thioesters are prepared by a
modification of the method reported by Simon and Shemin2 ,4 The (S)-
methyhnalonyl-CoA formed as a result of racemase action is converted
to propionyl-CoA and oxaloacetate through the oxaloacetate transcar-
boxylase reaction (Eq. 4). The oxaloacetate is converted to malate with
malate dehydrogenase (Eq. 5) and the x-elocity of the reaction is
determined from the decrease in absorbancy of the N A D H at 340 m~.
CoA transferase, transcarboxylasc, and methyhnalonyl-CoA mutase are
obtained from propionibacteria. ~,~ The malate dehydrogenase is obtained
commercially (Boehringer) since the malate dehydrogenase purified from
propionibacteria contains excessive racemase even in its purest form.
All the enzymes used in the assay arc free of raeemase except the mutase,
which has a specific activity for racemase of 0.01. Consequently, mutase
cannot be added in great excess. In practice, two concentrations of th(,
racemase preparation and a control with no addition of mutase are
assayed simultaneously. The rate of N A D H oxidation after subtraction
of the activity attributed to the racemase in the mutase is dependent on
and proportional to the amount of racemase added. Direct spectro-
photometric assay is permissible in crude extracts because the nonspecific
N A D H oxidase and lactate dehydrogenase activities were considerably
lower than the racemase. One unit of enzyme is defined as that amount
catalyzing the oxidation of 1 micromole of N A D H per minute.
Reagents
(1) 50 ml glutathione, 0.02 ml (Sigma)

(2) 0.2 M, pH 7.8, Tris-HC1 buffer, 0.02 ml
(3) 0.1 M sodium pyruvate, 0.02 ml (Sigma)
(4) 0.1 M sodium succinate, 0.02 ml
(5) Malate dehydrogenase, 0.01 ml containing 0.1 unit
(Boehringer)
(6) 2 m M N A D H , 0.02 ml
(7) Water, 0.1 ml
(8) Transcarboxylase, 0.02 ml containing 0.1 unit
(9) Methyhnalonyl-CoA mutase, 0.02 ml containing 0.005 unit of
mutase and less than 2.5 }( 10-4 units of racemase
3E. J. Simon and D. Shemin, J. Am. Chem. Soc. 75, 2520 (1953).
4R. W. Swick and H. G. Wood, Proc. Nall. Acad. Sci. U.S. 46, 28 (1960).
R. W. Kellermeyer, S. H. G. Allen, R. Stjernholm, and H. G. Wood, J. Biol. Chem.
239, 2562 (1964).
6S. H. G. Alien, R. W. Kellermeyer, I(. Stjernhohn, and H. G. Wood, J. Bacleriol.
87, 171 (1964).
196 •EACTIONS L E A D I N G TO AND FROM T H E CYCLE [33]
(10) 25 mM acetyl-CoA, 0.02 ml

(11) CoA-transferase, 0.02 ml containing 0.1 unit
(12) Methylmalonyl-CoA racemase, 0.02 ml
(13) 10 ~M DBC, 0.01 ml (gift of Karl Folkers, Merck, Sharp &
Dohme Company; reagent must be stored in light-proof con-
tainer; not available commercially)
Procedure. The reagents listed above are added to a 0.5 ml spectro-

photometric cell (1 cm light path) in the order and amounts listed.
If any of the reagents are deleted, additional water is added to
complete the final volume to 0.32 ml. Usually larger but proportional
volumes of reagents 1 through 7 were combined to form a mixture that
could be added as a single volume of 0.21 ml. All the reactants but DBC
are combined in the cell and mixed by inversion, the cell is placed in
a water bath the same temperature as the spectrophotometer cell cham-
ber for 5 minutes prior to adding the DBC. The DBC is added in dim
light and immediately after mixing, the cells are placed in the spectro-
photometer. Control reactions are prepared by eliminating acetyl-CoA,
DBC, or methylmalonyl-CoA mutase. The N A D H oxidation in the
absence of any of these reagents is less than 0.0001 micromole per
minute. The reaction velocity is maximal immediately after the DBC is
added and the reactants are mixed.
The enzyme described in this report is prepared from Propionibac-

terium shermanii (52W), but it is also found in mammalian tissue. 7
The growth of the bacteria and the initial steps (steps 1, 2, and 3)
are identical to those described for the isolation of the oxaloacetate
transcarboxylase, s The recovery of the racemase from step 2 was 75%
of the activity in the crude bacterial extract.
Step 3. Cellulose Phosphate Column. The racemase was eluted from
cellulose phosphate with 0.15M potassium phosphate buffer, pH 6.8. 8a
There is little transcarboxylase acti;rity in this eluate, and none of the
other enzymes used in the assay for the racemase is present. The protein
in this eluate is precipitated by 90% ammonium sulfate and sedimented
at 23,000 g at 4 °. The specific activity of this fraction is 8-10. There is a
~R. Mazumder, S. Sasakawa, T. Kaziro, and S. Ochoa, J. Biol. Chem. 237, 3065
(1962).
s See this volume [36].
sa See Fig. 1 in this volume [36].
[33] METHYLMALONYL-COA RACEMASE FROM tO. shermanii 197
large loss of activity at this step since only 10-15% of the total activity
in the crude extract is recovered.
Step 4. Ammonium Sul]ate Fractionation. The sedimented protein is
dissolved in 0.1 M phosphate buffer, pH 7.4, to a concentration of 20 mg/
ml, and the ammonium sulfate is determined as described, s The solution
is brought to 60% ammonium sulfate with saturated ammonium sulfate;
the resulting precipitate sedimented at 32,000 g at 4 °. The precipitate is
discarded and the supernatant brought to 75% ammonium sulfate with
solid ammonium sulfate. The 60-75% sediment contains racemase that is
approximately twice the specific activity of that in the eluate from the
cellulose phosphate column. The 75-90% ammonium sulfate fraction,
made by adding solid ammonium sulfate to the 60-75% supernatant
fluid and sedimenting the precipitate at 32,000 g at 4 °, contains 75% of
the activity with a specific activity of 30-35. There is no transcarbox-
ylase activity in the 60-75% or the 75-90% fractions. The preparation
with the highest specific activity gives a single symmetrical peak in the
ultracentrifuge sedimentation analysis and represents an overall yield of
2.5% of the activity in the crude bacterial extract.
Properties
Electrophoretic and Sedimentation Patterns. The S2o,w sedimentation

value is 2.95. The electrophoretic mobility (t~) is 8.4 X 10.5 cm 2 per
second per volt at pH 7.4.
Molecular Weight. The molecular weight is 29,000___ 2700 by the
method of Archibald, assuming a partial specific volume of 0.75.
Equilibrium. The conversion of (S)-methylmalonyl-CoA to the (R)
form is reversible in the presence of the racemase. At equilibrium the
racemic mixture is composed of equal amounts of the two.
Acid Stability. Racemase is resistant to 1.0 M perchloric acid for 30
minutes. This property should facilitate the elimination of other enzymes
during the purification process. 6
Heat Stabilitg. Methylmalonyl-CoA racemase is unusually resistant
to heat. Compared to an unheated control, 67% of the activity remained
after exposure to boiling water for 1 minute and 50% remained after 5
minutes. ~ This property could also be used to advantage in the purifica-
tion procedure.
Mechanism
The epimerization does not involve transfer of the coenzyme moiety
between the two carboxyl groups. T It apparently attacks and destroys the
asymmetry of carbon 2. The proton can then enter from either side,
permitting raccmization. ;, 9
Retey and Lynen TM and Sprecher et al., '~ have demonstrated that the
absolute configuration of the methylmalonyl-CoA formed by either
transcarboxylase or 1)ropionyl-CoA carboxylase has the (S) configura-
tion.
P. Overath, G. M. Kellerman, F. Lynen, H. P. Fritz, and H. J. Keller Biochem. Z.
335, 500 (1962).
,oj. Retey and F. Lynen, Biochem. Z. 342, 256 (1965).
'~ M. Sprecher, M. J. Clark, and D. B. Sprinson, J. Biol. Chem. 241, 872 (1966).
[34] Methylmalonyl-CoA Mutase from Sheep Liver

[EC 5.4.99.2 Methylmalonyl-CoA CoA-earbonylmutase]
B y I~AJARSHI •AZUMDER and SEVERO OCHOA
L-Methylmalonyl-CoA ~ succinyl CoA

Assay Method
Several methods are available for the assay of methylmalonyl-CoA

mutase.l,2 The method used routinely for purification of the enzyme from
sheep liver is described.
Principle. It is the same as that described for the assay of methyl-
malonyl-CoA racemase from sheep liver, 3 except that propionyl-CoA car-
boxylase and racemase are present in excess and methylmalonyl-CoA
mutase is limiting.
Reagents
MgC1..,, 120 m M
Glutathione (GSH), 40 m M
ATP, 30 m M
Na214CO~ (174,840 cpm/micromole), 50 m M
Propionyl-CoA, ~ 10 m M
'See Vol. V [79].

~J. J. B. Cannata, A. Foeesi, Jr., R. Mazumder, R. C. Warner, and S. Ochoa, J. Biol.
Chem. 240, 3249 (1965).
~See this volume [32].
~Prepared according to the method described by M. Flavin and S. Ochoa, J. Biol.
Chem. 229, 965 (1957).
asymmetry of carbon 2. The proton can then enter from either side,
permitting raccmization. ;, 9
Retey and Lynen TM and Sprecher et al., '~ have demonstrated that the
absolute configuration of the methylmalonyl-CoA formed by either
transcarboxylase or 1)ropionyl-CoA carboxylase has the (S) configura-
tion.
P. Overath, G. M. Kellerman, F. Lynen, H. P. Fritz, and H. J. Keller Biochem. Z.
335, 500 (1962).
,oj. Retey and F. Lynen, Biochem. Z. 342, 256 (1965).
'~ M. Sprecher, M. J. Clark, and D. B. Sprinson, J. Biol. Chem. 241, 872 (1966).
[34] Methylmalonyl-CoA Mutase from Sheep Liver

[EC 5.4.99.2 Methylmalonyl-CoA CoA-earbonylmutase]
B y I~AJARSHI •AZUMDER and SEVERO OCHOA
L-Methylmalonyl-CoA ~ succinyl CoA

Assay Method
Several methods are available for the assay of methylmalonyl-CoA

mutase.l,2 The method used routinely for purification of the enzyme from
sheep liver is described.
Principle. It is the same as that described for the assay of methyl-
malonyl-CoA racemase from sheep liver, 3 except that propionyl-CoA car-
boxylase and racemase are present in excess and methylmalonyl-CoA
mutase is limiting.
Reagents
MgC1..,, 120 m M
Glutathione (GSH), 40 m M
ATP, 30 m M
Na214CO~ (174,840 cpm/micromole), 50 m M
Propionyl-CoA, ~ 10 m M
'See Vol. V [79].

~J. J. B. Cannata, A. Foeesi, Jr., R. Mazumder, R. C. Warner, and S. Ochoa, J. Biol.
Chem. 240, 3249 (1965).
~See this volume [32].
~Prepared according to the method described by M. Flavin and S. Ochoa, J. Biol.
Chem. 229, 965 (1957).
[34] METHYLMALONYL-COA MUTASE FROM SHEEP LIVER 199
Propionyl-CoA carboxylase:' (specific activity, 3-4)

Methylmalonyl-CoA raeemase 3 (specific activity, 0.3-0.35, essen-
tially free of mutase)
Perchloric acid, 2.0 N
Procedure. The reaction mixture contained in a final volume of 1.0
ml the following components (in micromoles): Tris-HC1 buffer, pH 7.5,
100; MgCI.,, 6; GSH, 2; ATP, 1.5; Na..~4C03, 5; propionyl-CoA, 0.5. The
mixture contained also propionyl-CoA carboxylase, 0.3-0.4 unit; methyl-
malonyl-CoA raeemase, 0.04 unit; and mutase up to 0.006 unit. Ttw
reaction is started by the addition of propionyl-CoA. After incubation in
sealed tubes for 10 minutes at 30 °, the reaction is stopped by the addition
of 0.2 ml of 2.0 N perehlorie acid. The radioactivity resistant to per-
manganate oxidation is then determined in exactly the same manner
described in the procedure for the assay of methylmalonyl-CoA raeemas('
from sheep liverJ
The permanganate-stable radioactivity present in a sample contain-
ing no mut'tse is used as a control and subtracted from that present in the
complete samples. Under these conditions, the suceinate formed is pro-
portional to the concentration of mutase.
To determine the activity of the mutase in the presence of excess
dimethylbenzimidazolyl cobamide (DBC) eoenzyme, holo- and atm-
mutase fractions are preineubated with 1.4 t,3[ coenzyme for 5-10
mimm,s at 30 °, immediately prior to assay.
~:~its. One unit of enzyme is defined as the amount of protein
cat:dyzing the formation of 1.0 micromole of suecinate per minute at
30 ° under the conditions of the assay. Specific activity is expressed as
units per milligram of protein. Protein is usually determined spec-
trophotometrieally.'; However, because of the high absorption of the
enzyme-bound cobamide coenzyme at 280 m/,, the sl)ectrophotometric
method gives erroneously high protein values for highly purified prepara-
tions of the holoenzyme. For this reason, protein concentration of the
purified enzyme has also been determined refractometrieally.'-' The factor
for conversion of sl)ectrophotometrie to refr',etometric values, as de-
termined with ultracentrifugally homogeneous holoenzyme, is 0.73.
Purification Procedure for Holoenzyme

All operations are carried out at 0-4 ° unless otherwise stated. The
protein values given in connection with the purification procedure are
spectrol)hotometric values.
~Prqmred according to the method of Y. Kaziro, S. Ochoa, R. C. Warn,r. aim J.-Y.
Chen, J. Biol. Chem. '136, 1917 (1961).
~'See Vol. III [731.
S TO AND FROM TIlE CYCLE [34]
Method A 7
Steps 1 and 2. Extraction and Ammonium Sulfate Fractionation.
These steps are conducted as described for the purification of methyl-
malonyl-CoA racemase from sheep liver? Batches of approximately 900 g
of sheep liver mince are prepared daily to a total of 6.2 kg. The solutions
of the ammonium sulfate fractions are stored at --18 ° until used. The
total yield of protein is 195 g.
Step 3. Dialysis. Half of the protein of step 2 is put through steps 3
and 4; the same is then done with the remainder, and the solutions are
pooled for step 5.
Solutions of the ammonium sulfate precipitate from step 2 are
thawed, pooled to give about 98 g of protein, and dialyzed against 15
volumes of 0.02 M Tris-HCl buffer, pH 7.3, for 20 hours. During this
time the buffer is changed twice, first after 3 hours of dialysis and then
after 15 hours. Insoluble residue is removed by centrifugation and dis-
carded.
Step ~. Precipitation with Protamine and Ammonium SulJate Frac-
tionation. The supernatant solution from step 3 is made to a protein
concentration of 9 mg/ml by the addition of 20 mM Tris-HC1 buffer, pH
7.3. A 1% solution of protamine sulfate, freshly prepared at room
temperature, is then added (12.4 mg per 100 mg of protein) with stirring.
After further stirring for 1 hour, the suspension is kept at 4 ° overnight
and centrifuged. The precipitate is extracted once with 20 mM and twice
with 50 mM potassium phosphate buffer, pH 7.3, with Servall Omni-
Mixer. The volumes of buffer used for the extraction are approximately
8.5, 8.5, and 5.9 ml per gram of protein, respectively. The suspensions are
centrifuged after each extraction, and the resulting supernatant solutions
are pooled. Solid, finely powdered ammonium sulfate (36.1 g/100 ml) is
then added slowly with stirring. Stirring is continued for an additional
30 minutes, and the mixture is centrifuged. The precipitate is discarded.
More ammonium sulfate (12.9 g/100 ml) is added to the supernatant
solution as above. The precipitate is collected by eentrifugation and dis-
solved in a small volume of 20 mM potassium phosphate buffer, pH 7.3.
The remaining protein from step 2 is put through steps 3 and 4 in
the same way. The protein content of the combined solutions (40 ml) is
1.8 g.
Step 5. Adsorption on Calcium Phosphate Gel and Elution. The com-
bined solutions from step 4 (40 ml) are dialyzed against 4 liters of 10
mM potassium phosphate buffer, pH 7.3, for 15 hours, centrifuged, and
diluted to a protein concentration of 5 mg/ml with the same buffer. This
' R . Mazumder, T. Sasakawa, and S. Ochoa, J. Biol. Chem. 238, 50 (1963).

solution is adjusted to pH 6.5 by the dropwise addition of 0.1 N acetic

acid with vigorous mechanical stirring. Successive additions of calcium
phosphate gel are then made to the solution, each time with stirring for
10 minutes before centrifugation, as follows (in milligrams of Caa(PO,)2
per 100 mg of protein in the original diluted solution) : first addition, 10;
second, 10.35; third, 10.35; fourth, 10.25; fifth, 20.7; sixth, 41.4; and
seventh, 62.1. The sixth and seventh portions of gel are washed with
distilled water and individually and successively eluted with 20 mM,
50 mM, 0.1 M, and 0.2 M potassium phosphate buffer, pH 7.3. The eluates
(gel 6, 20 mM; gel 7, 0.1 M and 0.2M) containing mutase of highest
specific activity are combined to yield 93 ml of enzyme solution with
185.2 mg of protein.
Step 6. Chromatography on DEAE-Cellulose. Before use, DEAE-
cellulose is washed with the six following solutions in the order given:
(1) 0.1 N NaOH until the washings are colorless; (2) water; (3) 0.1 N
HC1; (4) water; (5) 0.1 M potassium phosphate buffer, pH 7.3; and (6)
10 mM potassium phosphate buffer, pH 7.3. The cellulose is then packed
into a column (1.9 X 32 cm) with the aid of a Saxon aquarium pump.
The column is equilibrated overnight against 10 mM potassium phos-
phate buffer, pH 7.3. The enzyme solution from step 5 is dialyzed against
4 liters of the same buffer for 7 hours with change of buffer after 2 and
4 hours. The dialyzed solution is passed through the column at the rate
of 1 ml per minute, and the column is washed stepwise with 170 ml each
of 10 mM and 50 mM potassium phosphate buffer, pH 7.3. The enzyme
is eluted by increasing the phosphate concentration to 0.1 M. Fractions
(each about 23 ml) are collected at a rate of 2 ml per minute. The
elution of protein is followed spectrophotometrically by measuring the
absorbancy at a wavelength of 280 m t~. Fractions containing mutase of
specific activity 0.68-1.27 are combined to yield 115 ml of enzyme
solution with 32.2 mg of protein.
Step 7. Chromatography on Hydroxylapatite2 The solution from step
6 is dialyzed against 3.5 liters of 25 mM potassium phosphate buffer, pH
7.3, for 3 hours with change of buffer every hour. The dialyzed solution is
passed through a hydroxylapatite column (1.6 X 15 cm) equilibrated
previously against the same buffer. At this stage, a narrow pink zone
near the top of the column is readily observable. The column is washed
with 180 ml of 0.1 M potassium phosphate buffer, pH 7.3, with collection
of 6 ml fractions, and the pink zone containing the mutase is then eluted
with 60 ml of 0.2 M potassium phosphate buffer pH 7.3, with collection
of 3 ml fractions. Pressure is applied to obtain a flow rate of 0.35 ml
SPrepared as described by A. Tiselius, S. Hjert6n, and 0. Levin, Arch. Biochem.
Biophys. 65, 132 (1966).
per minute. The elution of protein is followed spectrophotometrically as
before. Fractions of specific activity above 4.6 are pooled to yield
fraction 1.
Fractions of specific activity 1.8 to 3.9 are pooled, diluted to a phos-
phate concentration of 50 mM, and rechromatographed on a hydroxyl-
apatite column (0.9 X 6 cm) equilibrated previously against 50 m M
potassium phosphate buffer, p H 7.3. The column is washed stepwise with
about 8 ml each of 0.125 M and 0.15 M potassimn phosphate buffer, pH
7.3. The enzyme is eluted by increasing the phosphate concentration to
0 . 2 M and 0.8 ml fractions are collected. Fractions of an approximate
specific activity of 6 are pooled to yield fraction 2. Fractions 1 and 2 are
then combined to yield 21 ml of enzyme solution with 2.8 mg of protein.
TABLE I
PURIFICATION OF SHEEP LIVER METHYLMALONYL-CoA MUTASE a
Specific
activityd
Volume Activity (units/rag Yield
Step (ml) (units b) Proteinc protein) (%)
I. Extract 12,432 2010 1516 g 0.001 100

2. (NH4)2SO4 1,962 1900 195 g 0.01 94
fractionation
3. Dialysis 2,578 1757 176 g 0.01 87
4. Protamine and 40 213 1.8 g 0.12 i0.6
(NH4)2SO4
fractionation
5. Cas(PO4)2 gel eluate 93 67 185.2 mg 0.36 3.3
6. Chromatography on 115 35 32.2 mg 1.1 1.8
DEAE-cellulose
7. Chromatography on 21 15.5 2.8 mg 5.5 0.8
hydroxylapatite
" From 6.2 kg of mince.
b One unit of enzyme is the amount of protein catalyzing the formation of 1.0 micro-
mole of succinate per minute at 30° under the assay conditions.
c Spectrophotometric value.
No DBC coenzyme added.
A s u m m a r y of the purification is given in Table I. The procedure

gives a 5000-fold purification from the original extract with about 1%
overall yield.
Co~centration o] Enzyme Solution. The enzyme solution from step 7
is concentrated with use of a hydroxylapatite column. 8 The solution is
dialyzed against 550 ml of 20 m M potassium phosphate buffer, p H 7.3,
for 4 hours with change of buffer after 1 hour and after 21/2 hours. The
dialyzed solution is passed through a small (8 X 9 ram) hydroxylapatite
column equilibrated previously against the same buffer. The pink protein
is eluted as a narrow band with 0.4 M potassium phosphate buffer, pH
7.3, and the drops with the strongest pink color are collected into one
tube. This yields 0.45 ml of pink solution with 1.8 mg of protein
(refractometric). Specific activity is 5.63 without addition of coenzyme.
Ultracentrifugation of this concentrated enzyme solution shows two
sharp peaks. The mutase, associated with the slow-moving component,
represents approximately 70% of the protein. As judged by activity
assays in the absence and presence of added DBC coenzyme, the prepa-
ration contains 85% holoenzyme and 15% apoenzyme.
Method B'
Only the main deviations from Method A are indicated.
Steps 1-4. These are the same as for Method A, except that the total
amount of fresh sheep liver mince processed is about 90 kg. The average
specific activity of step 4 enzyme is around 0.03, or about one-fourth of
the value indicated in Table I. This is probably because of long periods
of frozen storage (up to 6 months) of step 4 fractions, and of other
fractions at earlier steps of purification, necessitated by the very large
scale at which the purification is undertaken.
Step 5. Adsorption on Calcium Phosphate Gel and Elution. The
dialyzed enzyme from step 4 is diluted to 10 mg of protein per milliliter.
The adsorption is carried out without any prior adjustment of pH
(i.e., pH 7.3). The gel is added in successive amounts of 43, 20, 41,
and 61 mg of Ca3(P04)2 per 100 mg of protein in the original diluted
solution. The enzyme is eluted from the third and fourth gels, each
eluate is assayed, and the eluates containing mutase of highest specific
activity are pooled. The enzyme is then concentrated by precipitation
with solid ammonium sulfate (85% saturation), the precipitate dissolved
in a small volume of 20 mM potassium phosphate buffer, pH 7.3, and
kept frozen until ready for the next step.
Step 6. Chromatographg on Triethylaminoethyl (TEAE)-CeUulose.
TEAE-cellulose is substituted for DEAE-cellulose. The resin is washed
successively with 1.0N NaOH, water, 1.0N HC1, water, and 0.5M,
0.1 M and 0.01 M potassium phosphate buffer, pH 7.3. It is then packed
into a column (4.5 X 30 cm) without applying pressure. The enzyme
solution from step 5 is dialyzed against 10 mM potassium phosphate
Imffer, pH 7.3, adjusted to a protein concentration of 36 mg per milliliter
and passed through the column. The enzyme is eluted as in Method A.
The eluate is then concentrated by ammonium sulfate precipitation
(85~ saturation) and solution in a small volume of 20 mM potassium

phosphate buffer, pH 7.3, and stored frozen.
Step 7. Chromatography on Hydroxylapatite. s The enzyme solution
from step 6 (about 30 mg of protein per milliliter) is dialyzed for 22
hours against 4 liters of 10 mM potassium phosphate buffer, pH 7.3, with
a change of buffer after 12 hours. A hydroxylapatite column (3 X 30 cm)
is used. Elution is performed as in Method A. Chromatography discloses
two distinct, well-separated colored bands, a faster moving, intensely
yellow one and a slower moving, deep pink band corresponding to the
mutase. The former is eluted with 0.1 to 0.15M potassium phosphate
buffer, pH 7.3; its nature is unknown. The latter is eluted with 0.175
to 0.2 M buffer. The fractions with highest specific mutase activity are
pooled, and the enzyme is concentrated by precipitation with solid am-
monium sulfate (80% saturation) and solution in a small volume of
10 mM potassium phosphate buffer, pH 7.3. After dialysis against the
same buffer, the enzyme is rechromatographed on a hydroxylapatite col-
umn (1.4 X 12.5 cm) and eluted as above. The pink color and the
absorbance ratio 280 m~:520 m/~ help to follow the elution of the
enzyme. The fractions of highest specific activity are pooled to give 7.0
ml of intensely pink enzyme solution. The ratio of A_.so to A26o is 1.53;
A2so:A52o is 13.4. The enzyme is precipitated with ammonium sulfate as
before and dissolved in 0.6 ml of 0.2M potassium phosphate, pH 7.3.
Based on the refractometric determination of protein, 90 kg of sheep
liver mince, prepared over a period of 8 months, yields 24 mg of con-
centrated step 7 enzyme of specific activity 7.2. This preparation is
homogeneous in the ultracentrifuge. Since the specific activity of the
initial extract is of the order of 0.001, the overall purification is about
7000-fold. The purified holoenzyme preparation consists of 88% holo-
enzyme and 12% apoenzyme.
Preparation of Apoenzyme from Purified Holoenzyme

Resolution of the purified holoenzyme is carried out as described for
crude mutase holoenzyme from sheep kidney cortex2 All operations are
performed at 0-4 °. A typical preparation is given in Table II. Holo-
enzyme (concentrated step 7 enzyme, Method B, stored frozen for 10
months), 0.2 ml with 27.8 mg of protein per milliliter (refractometric) is
diluted with 1.8 ml of l0 mM Tris-HC1 buffer, pH 7.5. To the diluted
solution are added 2.2 ml of saturated ammonium sulfate, with vigorous
stirring, followed by 0.5 ml of 0.004 N HC1. The pH is then adjusted to
3.5 (measured with a glass electrode) with 1.0N HC1. The mixture is
pp. Lengyel, :R. Mazumder, and S. Ochoa, Proc. Natl. Acad. Sci. U.S. 46, 1312 (1960).
[34] METHYLMALONYL-COA
MUTASE FROM SHEEP LIVER 205
TABLE II
PREPARATION OF APOENZYME
Specific activity
(units/mg protein)
With
No DBC excess With
coenzyme DBC No DBC excess Yield (%)
added coenzyme Protein eoenzyme DBC
Mutase (units) (units) (rag) a d d e d coenzyme Units~ Protein
ttoloenzyme 28.1 31.8 5.56 5.05 5.72 100 100
Apoenzyme, 3.5 24.8 4.42 0.8 5.62 78 79.5
before chro-
matography
Apoenzyme, 1.9 11.8 1.59 1.2 7.4 37 29
after chro-
matography
a With excessDBC coenzyme.
centrifuged immediately in a Servall centrifuge at 6000 rpm for 7 minutes.

The protein precipitate is taken up at once in 2.0 ml of 75% saturated
ammonium sulfate (adjusted with N H , O H to pH 9.4), dispersed
thoroughly with a glass rod, and centrifuged as above. This washing is
repeated once more, and then the precipitate is dissolved in 0.5 ml of
0.5 M potassium phosphate buffer, pH 7.5. A small amount of insoluble
material is removed by centrifugation. Ultracentrifugation of the resolved
enzyme shows two components, a fast (about 2 1 ~ ) and a slow (about
79~) sedimenting component. The fast component is removed by
chromatography on hydroxylapatite, as in step 7 of the holoenzyme
purification procedure, with 0.1 to 0.15 M phosphate buffers containing
10 mM GSH to prevent inactivation of the apoenzyme. Elution with
0.18M potassium phosphate buffer, pH 7.3, containing 10 mM GSH,
followed by precipitation with ammonium sulfate (80~b saturation) and
solution in 0.2 ml of 0.2 M potassium phosphate buffer-10 mM GSH,
pH 7.3, yields an ultracentrifugally homogeneous preparation of the
apoenzyme which consists of 7 5 ~ apoenzyme and 2 5 ~ holoenzyme.
A higher degree of resolution is obtained by decreasing the pH to
slightly below 3.5 during the acid treatment of the holoenzyme; however,
this decreases the yield of undenatured apoenzyme.
Resolution of the holoenzyme by the above procedure does not result
in release of the coenzyme from the protein. The ammonium sulfate
supernatant solution is colorless after resolution whereas the precipitate
and its solutions have a strong pink color.
Properties 2
Holoenzyme
Absorption Spectrum. The region between 450 and 600 m# has a
maximum around 520 m~. This suggests that the prosthetic group of thc
sheep liver mutase is either DBC or benzimidazolyl cobamide coenzyme.
Sedimentation, Molecular Weight, and Coenzyme Content. The sedi-
mentation coefficient (S2o,w) of the enzyme is 7.7 S. The molecular weight,
averaged from two short column equilibrium runs, is 165,000 ± 3000. The
coenzyme content is determined spectrophotometrically 7 by measuring
the absorbancy of the enzyme at 520 ms. Based on the refractometric
determination of protein and a content of 88% holocnzyme and 12%
apoenzyme, the holoenzyme is found to contain approximately 1 mole of
cobamide coenzyme per 75,000 g of protein. This assumes that the bound
coenzyme is DBC coenzyme and that there is no change in its absorbance
due to binding by the protein.
pH Optimum. The pH optimum of the holomutase is at 7.0.
Stability. The enzyme is fairly stable. However, on storage at --12 °
for 10 months there is a loss of activity of about 30%.
Michaelis Constants. The apparent K~ Values for L-methylmalonyl-
CoA and succinyl-CoA are 0.24 mM and 62 ~M respectively. Separate
experiments run with DL- and L-mcthylmalonyl-CoA show that the
inactive D enantiomorph is inhibitory.
Equilibrium. The average value for the apparent equilibrium constant
K ~ succinyl-CoA/L-methylmalonyl-CoA is 18.6.
Ef]ect o] Charcoal Treatment and Illumination. The treatment of
holomutase with Nuchar C or illumination of the enzyme solution do not
reduce the specific activity.
Ef]ect o] -----SH-Binding Reagents. The holoenzyme is moderately
sensitive to ---SH binding reagents. It is inhibited to the extent of 41, 24,
and 34%, respectively by 10 ~ p-hydroxymercuribenzoate (HMB),
0.1 mM iodoacetamide and 0.1 mM N-ethylmaleinimide.
Apoenzyme
Stability. Contrary to the holoenzyme, the apomutase is very unstable
in solutions of low ionic strength. It is, however, stabilized in the presence
of GSH or DBC coenzyme.
Sedimentation and Molecular Weight. The sedimentation coefficient,
$2o,,, of apomutase is 7.8 S. The molecular weight based on a single
determination is 144,000, which is only slightly lower than that of the
holoenzyme.
Ef]ect o] --SH-Binding Reagents. As compared to the holoenzyme,
[35] MUTASE FROM P. shern~anii
METHYLMALONYL-COA 207
the apoenzyme is very sensitive to ---SH-binding reagents. HMB (10

~M), iodoacetamide (0.1 mM), and N-ethylmaleinimide (10 ~M) in-
hibits the apomutase 82, 65, and 59%, respectively. Preincubation of the
apomutase with DBC coenzyme prior to addition of the above reagents
has a marked protective effect. The results suggest that resolution of the
holoenzyme leads to exposure of essential - - S H groups.
Absorption Spectrum. The absorption spectrum of apomutase solu-
tions is similar to that of the photoinactivated cobamide coenzymes with
peaks at 354, 405, 505, and 535 m~.
Effect of Illumination. The reconstituted holoenzyme, i.e., the active
enzyme formed on incubation of apomutase with an excess of DBC
coenzyme, shows considerable resistance to illumination, approaching that
of the native holocnzyme.
[ 3 5 ] 2 - M e t h y l m a l o n y l - C o A M u t a s e f r o m Propionibacterium
shermanii ( M e t h y l m a l o n y l - C o A I s o m e r a s e ) 1
[EC 5.4.99.2 Methylmalonyl-CoA CoA-carbonylmutase]
By R. W. KELLERMEYERand HARLANDG. WOOD
CH3--CH--COOH ~ CH~--CH,.--COOH
COSCoA COSCoA
(R)-Methyhnalonyl-CoA Succinyl-CoA
Assay Method
Principle. When the enzyme is prepared free of NADH oxidase,
lactate dehydrogenase, and succinyl-CoA deacylase, it can be assayed
spectrophotometrically by a series of coupled reactions shown below? a
The rate of N A D H oxidation in this assay is directly proportional to the
amount of mutase added. One unit of enzyme is defined as that amount
CoA
transferase
Acetyl-CoA q- succinate . acetate -b succinyl-CoA (1)
mutase
Succinyl-CoA " . (R) -methylmalonyl-CoA (2)
DBC~
1This work was assisted by grant AT-(30-1)-1320 from Atomic Energy Commission.
1" R. W. Kellermeyer, S. H. G. Allen, R. Stjernholm, and H. G. Wood, J. Biol. Chem.
239, 2562 (1964).
2Abbreviation: DBC-(5,6-dimethylbenzimidazolyl)Co-5'-deoxyadenosinecobamide.
[35] MUTASE FROM P. shern~anii
the apoenzyme is very sensitive to ---SH-binding reagents. HMB (10

~M), iodoacetamide (0.1 mM), and N-ethylmaleinimide (10 ~M) in-
hibits the apomutase 82, 65, and 59%, respectively. Preincubation of the
apomutase with DBC coenzyme prior to addition of the above reagents
has a marked protective effect. The results suggest that resolution of the
holoenzyme leads to exposure of essential - - S H groups.
Absorption Spectrum. The absorption spectrum of apomutase solu-
tions is similar to that of the photoinactivated cobamide coenzymes with
peaks at 354, 405, 505, and 535 m~.
Effect of Illumination. The reconstituted holoenzyme, i.e., the active
enzyme formed on incubation of apomutase with an excess of DBC
coenzyme, shows considerable resistance to illumination, approaching that
of the native holocnzyme.
[ 3 5 ] 2 - M e t h y l m a l o n y l - C o A M u t a s e f r o m Propionibacterium
shermanii ( M e t h y l m a l o n y l - C o A I s o m e r a s e ) 1
[EC 5.4.99.2 Methylmalonyl-CoA CoA-carbonylmutase]
By R. W. KELLERMEYERand HARLANDG. WOOD
CH3--CH--COOH ~ CH~--CH,.--COOH
COSCoA COSCoA
(R)-Methyhnalonyl-CoA Succinyl-CoA
Assay Method
Principle. When the enzyme is prepared free of NADH oxidase,
lactate dehydrogenase, and succinyl-CoA deacylase, it can be assayed
spectrophotometrically by a series of coupled reactions shown below? a
The rate of N A D H oxidation in this assay is directly proportional to the
amount of mutase added. One unit of enzyme is defined as that amount
CoA
transferase
Acetyl-CoA q- succinate . acetate -b succinyl-CoA (1)
mutase
Succinyl-CoA " . (R) -methylmalonyl-CoA (2)
DBC~
1This work was assisted by grant AT-(30-1)-1320 from Atomic Energy Commission.
1" R. W. Kellermeyer, S. H. G. Allen, R. Stjernholm, and H. G. Wood, J. Biol. Chem.
239, 2562 (1964).
2Abbreviation: DBC-(5,6-dimethylbenzimidazolyl)Co-5'-deoxyadenosinecobamide.
~08 REACTIONS LEADING TO AND FROM THE CYCLE [35]
methylmalonyl-CoA
racemase
(R)-Methylmalonyl-CoA " , (S)-methylmalonyl-CoA (3)
oxaloacetate
transearboxylase
(S)-Methylmalonyl-CoA -[- pyruvate
oxaloacetate W propionyl-CoA (4)
malate
dehydrogenase
Oxaloacetate -[- NADH " NAD -t- malate (5)
The assay is based on the rate of conversion of suceinyl-CoA to (R)-
methylmalonyl-CoA. The succinyl-CoA is generated in the reaction
mixture using CoA-transferase and either acetyl-CoA or propionyl-CoA
as a CoA donor, since the latter two CoA derivatives are stable during
storage at --20 ° while synthetic succinyl-CoA is relatively unstable. If
desired, succinyl-CoA may be used for the assay. The methylmalonyl-
CoA racemase, 3 CoA-transferase,4 oxaloacetate transcarboxylase 5 and
malate dehydrogenase4 may be obtained from propionibacteria free of
mutase. The acetyl-CoA, propionyl-CoA, and succinyl-CoA are prepared
by a modification of the method described by Simon and Shemin.6
When the NADH oxidase or lactate dehydrogenase are present in
excessive amounts, and frequently they are in the crude extracts, the
reaction is done in two steps. In the first step [Eqs. (2, 3, and 4)] 10
micromoles of sodium pyruvate, 75 micromoles of Tris-HC1, pit 7.5,
2.5 micromoles of reduced glutathione, 0.25 unit of oxaloacetate trans-
carboxylase, 2 X 10-4 micromoles of DBC, 2 2.5 micromoles of succinyl-
CoA, and the sample to be tested for mutase activity are combined in a
final volume of 0.6 ml. The mutase sample is omitted for the blank.
The reaction mixtures are incubated 5 minutes at 37 °. The reaction is
stopped by adding 0.4 ml of 10~ trichloroacetic acid (w/v), mixing, and
placing the tube in a 1° bath for 5 minutes. The precipitate is removed
by centrifugation and the supernatant fluid is assayed for oxaloacetate.
Oxaloacetate content is determined by adding 0.1 ml of the supernatant
to 50 micromoles of Tris to neutralize the trichloroacetic acid. To this
mixture the following reactants are added to make a final volume of
0.64 ml: 100 micromoles of Tris-HC1, pH 7.5; 0.25 micromoles of
s S. H. G. Allen, R. Kellermeyer, R. Stjernholm, B. Jacobson, and H. G. Wood, J.

Biol. Chem. 238, 1637 (1963); also see this volume [33].
S. H. G. Alien, R. W. Kellermeyer, R. Stjernholm, and H. G. Wood, J. Baeleriol.
87, 171 (1964).
H. G, Wood, S. H. G. Allen, R. Stjemholm, and B. Jacobson, J. Biol. Chem. 238,
547 (1963); also see this volume [36].
*E. J. Simon and D. Shemin, J. Am. Chem. Soc. 75, 2520 (1953).
[35] METHYLMALONYL-COAMUTASE FROM P . shermanii 209
NADH; 0.1 unit of malate dehydrogenase (Eq. 5). The oxaloacetate

content is calculated from the NADH oxidized using the E34o 6.22 X 106
cm2/mole.
One-Step Assay
Reagents
{1) 0.05 M glutathione, reduced, 0.02 ml (Sigma)
(2) 0.2 M potassium phosphate buffer, pH 7.5, 0.02 ml
(3) 0.1 M sodium pyruvate, 0.02 ml (Sigma)
(4) 0.1 M sodium succinate, 0.02 ml
(5) Malate dehydrogenase, 0.01 ml containing 0.05 unit
(6) 2 mM NADH, 0.02 ml
(7) Water, 0.10 ml
(8) Oxaloacetate transcarboxylase, 0.02 ml containing 0.1 unit
(9) Methylmalonyl-CoA racemase, 0.02 ml containing 0.1 unit
(10) 25 mM aeetyl-CoA, 0.02 ml
(11) CoA-traDsferase, 0.02 ml containing 0.1 unit
(12) Methylmalonyl-CoA mutase, 0.02 ml
(13) 10 ~M DBC, 0.01 ml (gift of Karl Folkers, Merck, Sharp &
Dohme Company; reagent must be stored in light-proof con-
tainer. Benzimidazolylcobamide or adenylcobamide may also be
used as coenzymes; none of these are available commercially.)
The malate dehydrogenase stock is diluted in 1% bovine albumin.
The remaining enzymes were diluted in 50 mM phosphate, pH 7.4.
Procedure. The reagents are added to a 0.5 ml spectrophotometric cell
(1 cm light path) in the order and amounts listed above.
If any of the reagents are deleted, additional water is added to
complete the final volume of 0.32 ml. Usually larger but proportional
volumes of reagents 1 through 7 are combined to form a mixture that
could be added as a single volume of 0.21 ml. All the reactants but DBC
were combined in the cell and mixed by inversion; the cell was placed
in a water bath with the same temperature as the spectrophotometric
cell chamber prior to addition of the DBC to permit temperature equil-
ibration. The DBC is added in dim light and, after mixing, the cells
are placed immediately in the spectrophotometer.
The enzyme described in this report is prepared from Propionibac-
terium shermanii (52W) but it is also found in mammalian tissues and
other bacteria3
7W. S. Beck, M. Flavin, and S. Ochoa, J. Biol. Chem. 229, 997 (1957).
210 REACTIONS LEADING TO AND FROM THE CYCLE [3S]
The bacteria are grown, harvested, and extracted as described in step

1 of the purification procedure for transcarboxylase. Steps 2 and 3 for
the isolation of the mutase are also the same as those described for
isolation of oxaloacetate transcarboxylase. 7a
Step 3. Cellulose Phosphate Column. The initial effluent of this col-
umn and the eluate with 50 mM phosphate buffer, pH 6.8, contains
essentially all the mutase with more than a 2-fold increase in specific
activity. The oxaloacetate transcarboxylase and most of the methyl-
malonyl-CoA racemase remain adsorbed on the cellulose phosphate.
Step ~. TEAE-Cellulbse Column 1. TEAE-cellulose (Brown Com-
pany, 0.8 meq/g) is washed with 0.1 N NaOH, 0.1 N HC1, distilled water,
0.1 M phosphate buffer, pH 6.8, and finally with 50 mM phosphate
buffer, pH. 6.8. Usually about 3.5-4 g of enzyme protein from the
previous step is placed on a 4.5 X 21 cm TEAE-cellulose column equili-
brated previously with 50 mM phosphate buffer, pH 6.8, at 4 °. The
protein is eluted successively with 600 ml each of 0.1 and 0.15M
potassium phosphate buffer, pH 6.9, and the mutase is eluted with 0.15 M
phosphate buffer. The fractions containing the mutase are brought to
90% saturation with ammonium sulfate, and the precipitate is collected
by centrifugation at 25,000 g at 4 °.
Step 5. TEAE-Cellulose Column 2. The precipitate is taken up in
50 mM phosphate buffer, pH 6.9, and the solution is dialyzed against
50 mM phosphate buffer, pH 6.9. The dialyzed product is placed on a
second TEAE-cellulose column, prepared as before, and eluted with a
gradient phosphate buffer (500 ml of 75 mM phosphate buffer, pH 6.9,
in the mixing bottle with addition of 0.3 M phosphate buffer, pH 6.9).
The mutase in a protein peak obtained from this column is precipitated
with ammonium sulfate (90% saturaton) and redissolved in 50 mM
phosphate buffer, pH 6.9. The solution (approximately 20 mg of protein
per milliliter), is fractionated by addition of saturated ammonium
sulfate to 67~ and then solid ammonium sulfate was added to 90%; 70%
of the activity is precipitated between 67 and 90% ammonium sulfate
saturation. Analytical ultracentrifugal studies of this preparation shows
5-10% of a slowly sedimenting impurity (by comparison of the areas of
the schlieren photograph).
Step 6. Sucrose Gradient. An aliquot of the preparation from step 5
was subjected to a final purification step consisting of centrifugation in
a 10-37% sucrose gradient s and the sucrose column is fractionated ac-
cording to the method of Martin and Ames2 The protein is prepared for
T, See this volume [36].
8G. M. Edelman, It. G. Kunkel, and E. C. Franklin, J. Exptl. Med. 108, 105 (1958).
R. G. Martin and B. N. Ames, J. Biol. Chem. 236, 1372 (1961).
[35] MUTASE FROM P. shermanii
this step by dialysis against 50 mM phosphate buffer, pH 6.9, and 0.5

ml of this preparation containing 15 mg protein is placed on top of the
sucrose column in a 2 X 0.5-inch tube and spun for 15 hours at 0 ° and
30,000 rpm in a SW 39 rotor with a Spinco Model L ultracentrifuge.
This purification permits the removal of a slowly sedimenting and faintly
yellow contaminating protein but does not significantly alter the specific
activity. The mutase protein is now homogeneous in the ultracentrifuge.
No attempt is made to shield the enzyme preparation from light
(luring any of the purification procedures. Consequently, none of the
preparations are active without additional DBC. The purified prepara-
tions are usually pink with a maximum specific activity of approximately
9.0. One preparation was white and had a specific activity of 14.4 (see
the table).
The overall yield of the purified mutase is approximately 50% of the
total units in the crude material. The percentage of the total units recov-
ered at each step (see the table), however, is probably not accurate since
the increase in specific activity during the purification procedure is a
reflection of: (1) removal of contaminating proteins, and (2) the cleav-
age of light-inactivated coenzyme from the apoenzyme. The latter un-
covers potential enzyme activity present in the crude extract but not
detectable by the assay (see below).
1)I'RIFICATION OF ~IETHYLMALONYI,-CoA~UTASE
Total Specific activity

Step recovered (%) (apprexima(e range)
1. Bacterial extract 100 0.01-0.03

2. DEAE-cellulose, 0.3 M buffer 100 0.05-0.09
3. Cellulose-phosphate, 50 mM buffer 110 0.15-0.21
4. TEAE-cellulose, 0.15 M buffer 120 2.5-2.9
5. TEAE-cellulose, gradient buffer 74 37
6. (NH4)~SO4, 0.60-0.90 65 9.7-12.9
7. (NH4)2SO4, 0.67-0.90 50 14.4
Purified preparations of the mutase have variable amounts of the

inactivated coenzyme attached to the apoenzyme. The amount of in-
activated coenzyme in a preparation could be determined spectrophoto-
metrically since the pink enzymes have absorption peaks at 352, 407,
505, and 535 m~. This spectrum is nearly identical to light-inactivated
benzimidazolyl, dimethylbenzimidazolyl-, and adenylcobamide coen-
zymes. One enzyme preparation was whitc and was assumed to be
composed for the most part of apoenzyme since little coenzyme could be
detected as determined speetrophotometrically. It appears that the apo-

enzyme (white) is separated from the coenzyme (pink) to a variable
degree by the purification process and that the residual attached co-
enzyme is inactivated by light since there is no activity without the
addition of active DBC. Both the highly purified white and pink
preparations formed homogeneous single peak patterns in the analytical
ultracentrifuge, yet the pink preparations never attained the higher
specific activity of the white preparation. It is presumed that the pink
preparations have a lower specific activity because the firmly attached in-
active coenzyme does not dissociate in the assay mixture allowing the
resulting apoenzyme to combine with the exogenous, active DBC. The
activity that does develop with the addition of coenzyme is derived from
the free apoenzyme in the preparation. Thus as the inactive coenzymc
is cleaved, the enzyme becomes less pink and the specific activity in-
creases. Calculations based on molecular weight and cobamide content
determined spectrophotometrically suggest there is one enzyme molecule
attached to one apoenzyme in the unaltered state of the bacterial en-
zyme. 1 The conditions required to obtain the apoenzyme free of the
inactivated coenzyme have not been determined, and preliminary at-
tempts to separate them have not been successful. The apoenzyme and
the coenzyme of the mutase purified from sheep liver also are not
separated readily, but the mammalian and the bacterial preparations
differ in that the attached coenzyme of the mammalian enzyme does not
appear as sensitive to light inactivation. 1°
Properties
Equilibrium. The equilibrium constant is determined to be 23.5
favoring succinyl-CoA.
pH Optimum. The pH optimum is approximately 7.6 but there is little
decrease in activity until the pH is lower than 6.0 or higher than 8.0.
Stability. The enzyme is relatively stable in the purified state since
when stored at --20 ° as an ammonium sulfate precipitate there is a 40%
loss in activity after 1 year. When the enzyme is diluted in 50 mM
phosphate buffer pit 7.0, the activity decreases 27% after 10 minutes at
42 ° and is completely destroyed after 10 minutes at 55 °.
Molecular Weight, Sedimentation, Coefficient, and Electrophoretic
Mobility. As determined on enzyme preparations with specific activities
of 9.7 or 14.4, these were as follows: molecular weight, 56,000 ± 3000;
S~o,w ---- 7.0; electrophoretic mobility 14.6 X 10-5 cm2/sec/volt.
K,n Values. The K,, for succinyl-CoA is 34.5 t~M and for methyl-
'" P~. Mazumder, T. Sasakawa, anti 8. Ochoa, J. Biol. Chem. 238, 50 (1963).
[35] METHYLMALONYL-COA MUTASE FROM P. shermanii 213
malonyl-CoA it is 80 t,.M.la,~1 The K,n for DBC is 0.035/rill? a,~ These

values are obtained using a mutase preparation free of racemase and the
assay system described above.
Requirements. Although no metal requirement has been noted for
methylmalonyl-CoA mutase, the dioldehydrase, another B12 dependent
enzyme, is stimulated by K* and NH~÷ ions? 2 Reaction rates with the
purified mutase reported here showed no stimulation of K ÷ ions and NH4 ÷
ions up to a final concentration of 0.33 M. Further, when EDTA is in-
cubated with the enzyme at concentration of 80 mM for 10 minutes, no
alteration in the specific activity of the isomerase is noted.
The enzyme does not appear to be dependent on sulfhydryl groups
since incubation of a purified preparation with either N-ethylmaleimide
or p-hydroxymercurobenzoate has no detectable effect on the activity.
Mechanism
Although considerable data pertaining to the mechanism of isomeri-
zation have been obtained, the exact manner in which the substrate, the
coenzyme, and the apoenzyme interact is unknown.
Eggerer et al.,~s were able to demonstrate that methylmalonyl-CoA-2-
~4C is converted by enzymatic isomerization to succinyl-CoA-3-1~C.
H H
H3C--C*--COOH ~- H2C--C*--COOH
i I H
COSCoA COSCoA
Swick did similar experiments with methyl-labeled methylmalonyl-
CoA. 14 These data prove that the reaction involves not transcarboxyla-
tion, but rather a movement of the carboxylthioester group from the 2 to
the 3 position of the propionic acid moiety. These results do not indicate
whether the transfer occured intermolecularly or intramolecularly.
Kellermeyer and W o o d ~5 and Hegre et al.,IG using mass spectrometric
techniques, demonstrated that the movement of the thioester group oc-
curred intramolecularly. Neither of these findings defines the role of
the B~2 in the reaction.
In the mutase reaction it is likely that the B ~ coenzyme accepts
a hydrogen from a carbon adjacent to the methyl carbon of methyl-
U H. G. Wood, R. W. Kellermeyer, R. Stjernholm, and S. H. G. Allen, Ann. N.Y.
Acad. Sci. 112, 661 (1964).
i~R. H. Ables and B. Zagalak, J. Biol. Chem. ~AI, 1246 (1966).
H. Eggerer, P. Overath, F. Lynen~ and E. R. Stadtman, J. Am. Chem. ,%c. 82,
2643 (1960).
I'R. W. Swick, Prec. Natl. Acad. Scl. U.S. 48, 288 (1962).
R. F. Kellermeyer and H. G. Wood, Biochemistry 1, 1124 (1962).
=C. S. Hegre, S. J. Miller, and M. D. Lane, Biochim. Biophys. Acta 56, 538 (1962).
malonyl-CoA thus permitting a partial bonding of the CoA group to the

adjacent or number 2 carbon:
COSCoA E,OSCoA
H\ / IH_ H \ ,' ", / H
/ C- C~--H
\
COOH ~¢,/H
~:oenzyme ~ I~I f r o m eoenzyme
.COSCoA
H \ /H /
/ C--C--H
\
COOH H
Rearrangement resulting in the formation of succinyl-CoA is completed
when the hydrogen is returned from the B12 coenzyme to the second
carbon of the methylmalonyl-CoA. Current data suggest a uniform role
for the B12 coenzyme in the methylmalonyl-CoA mutase, dioldehydrase,
and methyl aspartate mutase catalyzed reactions. 12,17,1s
Using mass spectrometric techniques and ~S0-labeled substrates,
Retey e t al., 17 demonstrated that an oxygen atom is transferred from C-2
to C-1 in the conversion of propane-l,2-diol to propionaldehyde.~7
Based on this study the following mechanism was proposed.
HO. OH H. O .OH
+H
/
~"~ Coenzyme
ILjC~ .OH
H H "OH
Studies done on the mechanism of the dioldehydrase reaction indicate
that one hydrogen atom is removed from the C-1 of a diol (propanediol,
17j. Retey, A. Umani-Ronchi, J. Seibl, and D. Arigoni, Experientia 22, 502 (1966).
18j. Retey and D. Arigoni, Experientia 22, 783 (1966).
[36] OXALOACETATE TRANSCARBOXYLASE 215
glycol) during the conversion to the corresponding aldehyde.12 This

hydrogen is accepted by the C-5' of the adenosyl moiety of the B12
coenzyme (DBC). From the C-5' of the adenosyl moiety, a hydrogen
atom is then transferred into the C-2 position of the substrate. Moreover,
it is not necessarily the same hydrogen molecule that replaces the
hydroxyl group on C-2 of the diol. In order for this mechanism to occur,
it is necessary for the carbon-cobalt bond to be disrupted to permit the
C-5' to accept the hydrogen. The hydrogens on the C-5' then become
equivalent, permitting the donation of either of them to the substrate
prior to reestablishing the carbon-cobalt bond. If the hydrogens are
equivalent, then the donation of either of them completes an intra-
molecular reaction.
That this mechanism may also be operative in the methylmalonyl
mutase reaction is supported by an observation of Retey and Arigoni.
They demonstrated that tritium transferred from propanediol to the BI.,
coenzyme by dioldehydrase then can be transferred from the coenzyme
to succinyl-CoA by methylmalonyl-CoA mutase, is
[36] O x a l o a c e t a t e T r a n s c a r b o x y l a s e f r o m Propionibacterium 1
[EC 2.1.3.1 Methylmalonyl-CoA:pyruvate carboxyltransferase]
By HARLAND G. WOOD, BIRGIT JACOBSON,
BRENDA I. GERWIN, and DEXTER B. 'NORTHROP
(S)-Methylmalonyl-CoA + pyruvate ,~- propionyl-CoA + oxaloacetate
Assay Methods
Principle. The enzyme activity is assayed spectrophotometrically by
determining oxaloacetate formation through coupling with malate dehy-
drogenase. This assay is used routinely, except when lactate dehydro-
genase or DPNH oxidase is present. If the latter are present in small
amounts, a control value is determined by omission of methylmalonyl-
CoA and is subtracted from the value obtained with the complete assay
mixture. When these contaminants are excessive the reaction is carried
out without the addition of malate dehydrogenase and DPNH, and after
a suitable interval the mixture is deproteinized with trichloroacetic acid
and the oxaloacetate is determined in the neutralized solution using
malate dehydrogenase. The direct assay with correction for the control
1This work was assisted by grant GM 11839 from the National Institutes of Health,
United States Public Health Service, Bethesda, Maryland.
glycol) during the conversion to the corresponding aldehyde.12 This

hydrogen is accepted by the C-5' of the adenosyl moiety of the B12
coenzyme (DBC). From the C-5' of the adenosyl moiety, a hydrogen
atom is then transferred into the C-2 position of the substrate. Moreover,
it is not necessarily the same hydrogen molecule that replaces the
hydroxyl group on C-2 of the diol. In order for this mechanism to occur,
it is necessary for the carbon-cobalt bond to be disrupted to permit the
C-5' to accept the hydrogen. The hydrogens on the C-5' then become
equivalent, permitting the donation of either of them to the substrate
prior to reestablishing the carbon-cobalt bond. If the hydrogens are
equivalent, then the donation of either of them completes an intra-
molecular reaction.
That this mechanism may also be operative in the methylmalonyl
mutase reaction is supported by an observation of Retey and Arigoni.
They demonstrated that tritium transferred from propanediol to the BI.,
coenzyme by dioldehydrase then can be transferred from the coenzyme
to succinyl-CoA by methylmalonyl-CoA mutase, is
[36] O x a l o a c e t a t e T r a n s c a r b o x y l a s e f r o m Propionibacterium 1
[EC 2.1.3.1 Methylmalonyl-CoA:pyruvate carboxyltransferase]
By HARLAND G. WOOD, BIRGIT JACOBSON,
BRENDA I. GERWIN, and DEXTER B. 'NORTHROP
(S)-Methylmalonyl-CoA + pyruvate ,~- propionyl-CoA + oxaloacetate
Assay Methods
Principle. The enzyme activity is assayed spectrophotometrically by
determining oxaloacetate formation through coupling with malate dehy-
drogenase. This assay is used routinely, except when lactate dehydro-
genase or DPNH oxidase is present. If the latter are present in small
amounts, a control value is determined by omission of methylmalonyl-
CoA and is subtracted from the value obtained with the complete assay
mixture. When these contaminants are excessive the reaction is carried
out without the addition of malate dehydrogenase and DPNH, and after
a suitable interval the mixture is deproteinized with trichloroacetic acid
and the oxaloacetate is determined in the neutralized solution using
malate dehydrogenase. The direct assay with correction for the control
1This work was assisted by grant GM 11839 from the National Institutes of Health,
United States Public Health Service, Bethesda, Maryland.
usually can be used for step 2 of the purification described below and
thereafter the control value is so small that it may be neglected.
Reagents
Malate dehydrogenase (Calbiochem) or that prepared from propi-
onibacteria TM
Sodium pyruvate (Sigma) DPNH (Sigma)
Methylmalonyl-CoA is prepared by the mixed anhydride method of
Beck et al? from methylmalonic acid and ethylchloroformate.
The methylmalonyl-CoA is stored frozen in a water solution at
pH 6.0 and is stable for several months. Methylmalonie acid
is obtained from K and K Labs and the CoA from Pabst. The
amount of the CoA-ester is determined by the hydroxamate
method of Lipmann and Turtle 3 using succinic anhydride as the
standard. The optimum concentration to be used in the assay is
determined for each new preparation of methylmalonyl-CoA be-
cause the hydroxamate method does not always correlate with
the enzymatically reactive material.
Enzyme: Dilutions of the enzyme are made with solutions con-
taining 5 mM glutathione and 0.25 M phosphate buffer, pH 6.8.
Transcarboxylase (10.0 #g/ml) is quite stable in this solution at
0 ° ; it loses 16~ of its activity in 7 days and 52~ in 30 days. The
high concentration of polyvalent anions (phosphate or sulfate)
and the glutathione are beneficial in maintaining activity. The
glutathione is not added to concentrated enzyme solutions which
are to be stored for long periods of time or to dilutions which will
not be stored longer than 8 hours.
Spectrophotometric Assay. The assay system contains in micromoles
per milliliter: pyruvate, 10; phosphate, 350; DPNH, 0.1; mcthylmalonyl
CoA, 0.2; and in units per milliliter: malate dehydrogenase, 2.0. A
mixture (SA mix) containing 2.0 times the required strength of some of
the reagents is prepared and is stable for 2 or 3 days. It contains the
following: 0.1M sodium pyruvate, 2.0 ml; 1.0M potassium phosphate
(pH 6.8), 7.0 ml; malate dehydrogenase (40 units/ml), 1.0 ml.
The assays are done in a euvette with 10 mm light path and 2 mm
,RS. H. G. Allen, R. W. Kellermeyer, R. L. Stjemholm, and H. G. Wood, J. Bacteriol.
87, 171 (1964).
J W. S. Beck, M. Flavin, and S. Ochoa, J. Biol. Chem. 229, 997 (1957) ; see also Vol.
VI [77], p. 538.
*F. Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945); see also E. R. Stadt-
man, Vol. I I I [39], p. 231.
width containing the following: SA mix, 0.15 ml; methylmalonyl-CoA

( ~ 3 raM), 0.02 ml; D P N H (3 raM), 0.01 ml; transcarboxylase (in
diluent), 0.01-0.04 ml; H20 to give a volume of 0.30 ml. All solutions
are at 25 ° except those of methylmalonyl-CoA, D P N H , and transcar-
boxylase, which are kept cold. The reaction is started by addition of
the transcarboxylase and is conducted at 25 °. The decrease in absorbance
is linear with time for at least 4 minutes and with an enzyme concentra-
tion in the assay below 0.02 unit/ml.
For full activity a high concentration of phosphate or sulfate is re-
quired in the assay. The above mixture with phosphate buffer has been
found most convenient for general use.
Units. Units are expressed as micromoles of oxaloacetate produced
per minute at 25 ° , and specific activities are expressed as units per milli-
gram of protein. Protein is measured spectrophotometrically 4 in purified
preparation and by the biuret procedure 5 in the crude extract. The value
given by the spectrophotometric method was 93% ~ of that determined by
refractive index increment.
Purification of Transcarboxylase
Source. Thus far oxaloacetate transcarboxylase has been reported
only from Propionibacterium shermanii. Transcarboxylase may be iso-
lated from the bacteria grown in lactate, glucose, or glycerol. The yields
of cells and transcarboxylase are quite similar from glucose or glycerol
media ;~ glycerol has the advantage that it does not cause caramelization
when sterilized with other constituents of the medium, and contamination
by other bacteria is less likely with this substrate.
The optimal medium 7 contains Na2C03, 80 raM; KH2P04, 90 raM;
glycerol, 0.35 M; and the following in milligrams per liter: yeast extract,
3500 (Yeast Products Inc.); C o ( N 0 3 ) ' 6 H20, 10; calcium pantothenate,
1; thiamine hydrochloride, 1; biotin, 1. The carbonate and phosphate
solutions are sterilized separately and the remaining components in com-
bination. The three solutions are mixed just before inoculation. The
fermentation is at 30 ° usually with 18 liters of medium in 20-liter carboys
with cotton plugs, and the flasks are shaken by hand once each day for a
few minutes. A liter of a vigorously fermenting culture of P. shermanii
(19W) growing on the same medium is used as the inoculum. It is neces-
4See Vol. I I I [73], p. 454. Factors 1.45 X A~0-0.74 X A~o a r e u s e d .

~See Vol. I I I [73], p. 450.
' H. G. Wood, S. H. G. Allen, R. Stjernholm, and B. Jacobson, J. Biol. Chem. 238,
547 (1963) ~
' H. Lochmi~ller, H. G. Wood, and J. J. Davis, J. Biol. Chem. 241, 5678 (1966); s e e
this volume [47].
218 REACTION'S LEADING TO AND FROM THE CYCLE [361
sary to inoculate with bacteria that have been transferred four or five
times in this medium to obtain a culture which grows rapidly. The pH of
the medium is about 7.8 at the beginning and drops to about pH 5.8 after
prolonged fermentation. The cells are harvested after 5 to 24 days in a
Sharpies centrifuge, and the yield of cells is 3-5 g per liter.
Reagents and Equipment

Pyrex beads, 100 tL in diameter (Minnesota Mining and Manufac-
turing Corporation) washed with concentrated HCI, water, and
dried
DEAE-cellulose (Type 40, capacity 0.9 meq/g, Brown Company),
washed successively with 0.1 N NaOH, water, 0.1 N HC1, water,
and finally 50 mM phosphate buffer, pH 6.8. The fine particles
are removed by repeated centrifugation at 1000 rpm for 5 min-
utes in an International Centrifuge Model PR-2.
Cellulose phosphate (reagent grade, capacity 0.8 meq/g, Brown
Company), washed as described for'DEAE-eellulose
TEAE-cellulose (0.8 meq/g, Brown Company), prepared as de-
scribed for DEAE-cellulose
Sepharose 2B (Pharmaeia)
Barnstead purity meter (Still and Sterilizer Company, Boston,
Massachusetts). The conductivity of 1.0, 2.0, and 3.0M
(NH4)2SO~ at a 1:50,000 dilution in distilled water is determined
to establish a linear plot of conductivity against concentration.
The salt concentration of an unknown is determined using a
1:50,000 dilution if it contains 0.4 M or greater of (NH4)2S04 or
a 1.'5,000 dilution if the concentration is less than 0.4 M. In the
latter case the concentration of (NH4)2S04 as read from the
standard plot is divided by 10.
Eppenbach Mill (Gilford-Wood Company, Hudson, :New York)
Fraction collector and test tubes
Sorvall refrigerated centrifuge, RC-2
Bfichner funnel, 15 cm in diameter, and suction flask
Whatman :No. 4 filter paper, 15 cm
Magnetic stirrer
(NH,)2S04 (special enzyme grade, Mann Research Laboratories)
used without further purification
Potassium phosphate buffer, 0.3M, pH 6.8, to be diluted for
various purposes. Just prior to use for elution of the enzyme
from columns, the diluted buffer is made 1 mM with respect to
mercaptoethanol
Cysteine-HC1
Dialysis tubing, 2 cm
Columns, 7 X 25 cm and 3 X 40 cm, with coarse sintered-glass
disks; 2.5 X 100 and 0.9 X 100 cm columns suitable for use with
Sepharose 2B
fl-Mereaptoethanol
Step 1. Preparation o] the Crude Extract. The extract may be ob-

tained by sonic rupture, 8 by grinding with glass beads in a Waring
blendor, s with a Nossal shaker, 9 a Schlossman shaker, or French press.
The most convenient apparatus for large quantities of cells is the Eppen-
bach Mill2 The following is a typical example: 300 g of Pyrex beads is
added to 300 g of packed cells as obtained from the Sharples centrifuge
together with 150 ml of 0.2M K.~HP04 containing 10 mg of cys-
teine.HC1. The mixture is ground at top speed for 30 minutes. The mill
is cooled by circulating a refrigerated solution of glycol antifreeze at
--5 ° through the mill. If during the grinding the temperature of the mix
reaches 15 °, the grinding is stopped until the temperature falls to 5 °.
The resulting mixture is centrifuged for 30 minutes at 16,000 g at 0 °,
giving a supernatant solution containing the enzyme. The sedimented
beads and cellular material are resuspended in 300 ml of 0.2 M phosphate
buffer, pH 6.8 and the grinding is repeated. The resulting material is
centrifuged for 30 minutes at 16,000 g at 0 °. The residual beads and
cellular material are suspended in 200 ml of cold distilled water and
centrifuged for 30 minutes at 16,000 g. The three combined supernatant
solutions are centrifuged in a Sorvall centrifuge at 43,000 g for 30 min-
utes. About 600 ml of clear brown solution containing 10-15 g of protein
and 15,000-25,000 units of transcarboxylase is obtained
Alternatively the glass beads are removed before centrifugation by
filtration through "Feon" saran cloth. The beads are washed with 0.2 M
phosphate buffer pH 6.8. The wash is combined with the filtrate and
centrifuged as above. The beads and cellular material are combined as
above and the grinding is repeated. The resulting material is filtered,
washed, and centrifuged as before. The cellular material is combined
and centrifuged at 43,000 q for 30 minutes. All supernatants are then
combined.
Step 2. Adsorption on DEAE-Cellulose and Batch Elution. Trans-
carboxylase is adsorbed by DEAE-cellulose from 50 mM phosphate buf-
fer, pH 6.8, and is eluted with 0.3 M phosphate. The 600 ml of bacterial
extract (step 1) is diluted six times with cold distilled water, and then
about 2000 g of the moist filtered DEAE-cellulose is added to it. The
8H. G. Wood and R. Stjernholm, Proc. Natl. Acad. Sci. U.S. 47, 289 (1961).
R. W. Swick and H. G. Wood, Proc. Natl. Acad. Sci. U.S. 46, 28 (1960).
220 BEACTIONS LEADING TO AND FROM THE CYCLE [361
mixture is stirred at 0 ° for about 1 hour; then a small portion is centri-

fuged and the transcarboxylase activity is determined in the supernatant
solution. A control assay without addition of methylmalonyl-CoA is used
to correct for oxidation of D P N H by lactate dehydrogenase or D P N H
oxidase. Usually most of the transearboxylase is adsorbed; if it is not,
more DEAE-cellulose is added and the process is repeated. When the
enzyme is adsorbed, the mixture is filtered on a Biichner filter at 4 ° using
Whatman No. 4 filter paper.
The DEAE-cellulose is suspended in 3000 ml of 0.1 M phosphate
buffer, pH 6.8, stirred for 20 minutes at 0 °, and again filtered. The
filtrate should contain no transcarboxylase. The transcarboxylase is
eluted from the DEAE-cellulose with 0.3 M phosphate buffer, pH 6.8.
Two washes of 0.3 M phosphate are used; the first is with 3500 ml, and
the mixture is stirred for 1 hour at 0% It is filtered as described previ-
ously, and the washing is repeated for 30 minutes with 2500 ml of
buffer. About 7 5 ~ of the recovered units of transcarboxylase are in the
first wash, and the specific activity of the transcarboxylase is usually
about 2.4. The second wash contains about 25% of the units, and the
specific activity is approximately 1.5. Malate dehydrogenase, ~ acetyl
kinase, 1 CoA transferase, 1 methylmalonyl-CoA mutase, ~° methylmalonyl-
CoA racemase, ~ and carboxytransphosphorylase ~ also are present in
these eluates.
The combined 0.3 M eluates are brought to 90% saturation by the
addition of 62.3 g of ammonium sulfate per 100 ml of solution and the
mixture is stirred for at least 30 minutes at 0 °. The precipitate is centri-
fuged in a Sorvall centrifuge at 16,000 g for 30 minutes. The precipitate is
stored at --10% The recovery in this step is usually near 100%.
Step 3. Chromatography on Cellulose Phosphate. Transcarboxylase is
adsorbed by cellulose phosphate equilibrated with 0.05 M phosphate buf-
fer, pH 6.8 and is eluted with 0.3 M phosphate, pH 6.8. This and all
subsequent chromatography is performed in a cold room at 4 °, and in
each case the buffers are pH 6.8 and 1 mM in mercaptoethanol, which
is added to the buffer just before use.
A 7 X 20 cm column of cellulose phosphate is prepared in successive
2 cm layers by adding a suspension of cellulose phosphate in 50 mM
phosphate buffer, allowing it to settle and then applying 1 psi of air
pressure until the fluid reaches the surface of the cellulose phosphate.
The process is repeated to obtain 20 cm and the column is equilibrated
~oR. W. Kellermeyer, S. H. G. Allen, R. Stjemholm, and H. G. Wood, Y. Biol. Chem.

~,39, 2567 (1964) ; see also this volume [35].
uS. H. G. Allen, R. W. Kellermeyer, R. Stjernholm, B. Jacobson, and H. G. Wood,
Y. Biol. Chem. 238, 1637 (1963); see also this volume [33].
~6] OXALOACETATE TRANSCARBOXYLASE 221
by washing it with 500 ml of the 50 mM buffer. Protein (3--5 g) from

step 2 is dialyzed for 6 hours in 2 cm tubing against 2 liters of the 0.1 M
phosphate buffer. The buffer is changed at 2-hour intervals. The conduc-
tivity of a 1:5000 dilution is determined using a Barnstead purity meter.
The enzyme solution is then diluted if necessary to give an ionic strength
equivalent to or lower than that of 50 mM (NH4)2SO~. The dialysis
should be for as short a time as possible.
The enzyme solution (~300 ml and containing ,~20,000 units of
enzyme) is placed on the column which then is washed with 50 mM
phosphate. Most of the protein is not retained, but the methylmalonyl-
CoA racemaseTM and transcarboxylase are adsorbed on the cellulose
phosphate. These are eluted with 0.15 M and 0.3 M phosphate buffer,
pH 6.8, with 1 mM mercaptoethanol (see Fig. 1). Fractions containing
the transearboxylase are combined, precipitated with 80% saturated
(NH4) 2S04 (52.6 g/100 ml). The mixture is stirred for about 30 minutes
and kept in the cold for 6-12 hours. The protein is recovered by centrif-
ugation at 16,000 g and is taken up in 0.1 M phosphate buffer, pH 6.8
(15-30 mg of protein per milliliter). When stored at --20 ° it is stable for
at least 6 months.
Attempts to substitute a batch adsorption and elution for this step
have failed, although this appears feasible since most of the protein is
not adsorbed from 50 mM phosphate. Results are given in Fig. 1.
Step 4. Chromatography on TEAE-CelluIose. Transcarboxylase is
adsorbed by TEAE-cellulose equilibrated with 50 mM phosphate buffer
and is eluted with 0.15 M and 0.225 M phosphate buffer. A tightly packed
3 X 32-cm column is prepared by forming successive layers with a
suspension of the TEAE-cellulose. About 5 psi of pressure is used to
pack each successive layer (about 3 cm each). The column is equilibrated
by washing with ~ 5 0 0 ml of 50 mM phosphate buffer, pH 6.8.
After use the column may be regenerated by washing it with 500 ml
each of 0.5 M KC1, 0.5 M phosphate buffer, pH 6.8, containing 0.1 mM
EDTA, and finally 50 mM phosphate buffer, pH 6.8.
Transcarboxylase from step 3 (--- 0.5 g of protein) is dialyzed briefly
against 50 mM phosphate, pH 6.8, or passed through a Sephadex G-50
column to reduce the (NH4)~S04 concentration. The salt concentration
is determined with a Barnstead purity meter, and the solution is diluted
if necessary to reduce the salt concentration to the equivalent of 50 mM
(NH~)2SO~. The sample is then added to the column, which is washed
with 50 mM phosphate buffer until the protein concentration falls to
0.10 mg/ml (see Fig. 2.) Elution is started with 0.15M phosphate
'~ It. G. Wood, H. Lo~'hmiillcr, C. Riepertinger, and F. Lynch, BiocltenL. Z. 337, 247
(1963).
buffer. The results are variable at this step; although some transcar-
boxylase may be eluted with 0.15 M phosphate, often very little is eluted
at this stage and all the enzyme is obtained in the 0.225 M eluate. The
fractions containing transcarboxylase are precipitated with 6 5 ~ satu-
rated (NH~)2S0~ (40.4 g/100 ml). The mixture is stirred for at least
30 minutes at 0 ° and is kept cold for 6-12 hours. The precipitate is re-
,tO0
- 3"0 24
-90
.80
,60 ~
Sp.Ac
,40
...i~:~
.0 M Protem/ml
¢¢
'30
.20
,IO
8 12 16 20 28 32 36 40 44
FRACTIONS
Flo. 1. Cellulose phosphate column, step 3. The column was 7 × 19 cm, and
3.8 g protein, specific activity 1.9 in 300 ml, was placed on the column. It was washed
with 340 ml of 50 m M phosphate (pH 6.8, 1 m M mercaptoethanol). Elution with
0.15M phosphate (pH 6.8, 1 m M mercaptoethanol) was for 9 hours (overnight),
fractions 1-28 (65 ml per fraction in 20 minutes). Elution with 0.3 M phosphate
(pH 6.8, 1 m M mercaptoethanol) was for 11 hours, fractions 28-52 (~42 ml per
fraction in 13 minutes). Methylmalonyl-CoA racemase was in fractions 8-16 contain-
ing 2'72 mg of protein. Fractions 40-45 were combined and precipitated with
(NH4)2S04 (80% saturation) and taken up in 0.1 M phosphate buffer, pH 6.8. This
solution contained 282 mg of protein (specific activity ~ 22) and 6418 units represent-
ing a recovery of 61%.
covered by centrifugation and is taken up in 0.10M phosphate buffer

(pH 6.8, without addition of glutathione), to obtain 20-40 mg/ml.
Usually there is a loss of activity following the elution, whether the
protein is concentrated or not. The material obtained in the 0.15M
eluate is more stable initially than that from the 0.225M eluate. After
the initial loss, the activity from either e]uate stabilizes at a specific
activity of about 25 and remains stable for at least 6 months at 0 °

or frozen at --20 ° .
Step 5. Gel Filtration. Additional purification of material from step 3
or step 4 is achieved by gel filtration on Sepharose 2B. Two columns are
packed with resin equilibrated with 0.2 M phosphate buffer, pH 6.8. To
.24
~-2.0 A~
;-. ss:.
50-
40.
g
•l.( ~. Sp.Ac, 30-
c J
o
O- 0.~ 20-
g
18 22 26 30 34 3'8
Fractions
4'2 46 5'o ~4
FIG. 2. TEAE-cellulose column, step 4. The column was 3 × 32 cm and 0.48 g

protein, specific activity 13, 6250 units were placed on the column and then washed
for 8 hours with 350 ml of 0.05M phosphate (pH 6.8, 1 m M mercaptoethanol).
Elution was with 0.15M phosphate (pH 6.8, 1 m M mercaptoethanol) during frac-
tions 1 to 32 (~11 ml per fraction in 30 minutes) and with 0.225 M phosphate (pH
6.8, 1 m M mercaptoethanol) fractions 28 to 56. Fractions 20 to 27 contained 59 mg
protein, specific activity ~-~30. The specific activity was 27 after concentration of
the protein by (Ntt0~SO~ precipitation. Fractions 45 to 54 contained 149 mg protein,
specific activity ~ 32 and a total of 4760 units, The specific activity fell to 25 after
concentration of the protein by (NH0~SO~ precipitation, giving 2760 units. Fractions
55 to 60 wcre precipitated, giving 13.4 mg of protein and with a specific activity of
11 and a total of 147 units. The total recovery after precipitation of the protein was
4550 units or 73%.
the larger column (2.5 X 80 cm) is applied 5-8 ml of solution from the
ammonium sulfate precipitate of step 3 or 4. This solution is made as
concentrated as possible. The protein is eluted with 0.2 M phosphate
buffer, pH 6.8. The first material to be eluted has an A2Go/A28o ratio
greater than 1.0 and contains no transcarboxylase activity. Only one
additional protein peak is obtained but the transcarboxylase activity
precedes this peak. The fractions of specific activity 30 or above are

pooled (pool A) as are all other fractions of specific activity greater than
10 (pool B). Both pools are precipitated with 80% (NIL)2SO~ and col-
lected as in step 3. Pool B is set aside for reproccssing by the same
method while pool A is dissolved in a minimal volume of 0.2 M phosphate
buffer, pH 6.8. This material (1-3 ml) is then applied to a second Seph-
arose 2B column (0.9 X 60 cm). A small initial peak is obtained with a
characteristically high A.-so followed by a protein peak where the bulk
of the material is transcarboxylase of specific activity 40 ± 4. This ma-
terial does not lose activity after elution and is precipitated and stored as
in step 3. Good results have been obtained omitting step 4.
A summary of the purification of transcarboxylase is given in Table
I with the ranges of purification and recovery which m a y be expected at
each step.
TABLE I
PURIFICATION OF TRANSCARBOXYLASEa
Specific activity Recovery

Step (units/mg protein) (%)
1. Crude extract
2. DEAE-cellulose, 0.3 M 1.5-3 N100
3. Cellulose phosphate column 5-30 ~60
4. TEAE-cellulose column, 0.15 M
4. TEAE-cellulose column, 0.225 M
10-40
10-40
f ,~40
5. Gel filtration 35-40 ~30
° The protein from step 4 is nearly pure transcarboxylase but for unknown reasons
it becomes partly inactivated, giving a specific activity of ~25. From 100 liters of
medium approximately 300 g of cells is obtained (wet weight) containing ~18,000
units of transcarboxylase. About 250 mg of transearboxylase of specific activity 25
is recovered in step. 4.
Properties
Sedimentation Pattern. The protein obtained from the 0.15 M eluate
(step 4) usually gives a single peak on sedimentation in the ultracentri-
fuge with an S2o,w = 16 S. 6 The protein of the 0.225M eluate usually
has two partially separated peaks with 82o,w values of 16 and 18 $2
Further purification by ammonium sulfate fraetionation 6 or by reverse
ammonium sulfate extraction (52%) has not yielded significantly more
active preparations or separated the 16 S and 18 S fractions, although it
does remove a small amount of slower sedimenting protein. The protein
of both peaks is active. It seems likely that the transcarboxylase is quite
pure after step 4 and that variations in activity are due to unknown
causes, including formation of subunits and reaggregation.
~6] OX&LOACETATE TRANSCARBOXYLASE 225
Molecular Weight. T h e m o l e c u l a r w e i g h t of t h e 16 S p r o t e i n has been

e s t i m a t e d b y t h e A r c h i b a l d m e t h o d to be 670,000 ± 40,000 a s s u m i n g a
p a r t i a l specific v o l u m e of 0.75.
Electrophoretic Mobility. U s i n g t h e 16 S p r o t e i n t h e r e was no d e t e c t -
a b l e i n h o m o g e n e i t y on e l e c t r o p h o r e s i s a t p H 6.2 or p H 7.3. T h e e l e c t r o -
p h o r e t i c m o b i l i t y ( m i c r o n s ) w a s 7.5 X 10 -~ cm 2 p e r second p e r v o l t a t
p H 6.2 a n d 8.0 X 10 -5 a t p H 7.3. 6
Biotin Content. T h e b i o t i n c o n t e n t a t different s t a g e s of p u r i t y ( p r i o r
to e v i d e n c e of i n a c t i v a t i o n of t h e e n z y m e ) is 0.047 /~g p e r u n i t of en-
z y m e 6,12 or 1.92 X 10 `7 m i l l i m o l e s of b i o t i n p e r u n i t (4.7 X 10 -~ - - 244.3;
t h e m o l e c u l a r w e i g h t of b i o t i n is 244.3). T h e m o s t p u r e p r o t e i n t h u s far
t e s t e d b y m i c r o b i o l o g i c a l a s s a y c o n t a i n e d 1.66 ~g of b i o t i n p e r m i l l i g r a m
of p r o t e i n 1~ or 4.5 m i l l i m o l e s of b i o t i n p e r m i l l i m o l e of e n z y m e of m o l e c -
u l a r w e i g h t 6.7 X 10 ~ ( T a b l e I I ) . T h i s m a t e r i a l is k n o w n to h a v e in-
c l u d e d some i n a c t i v e p r o t e i n . C a l c u l a t i o n s of t h e b i o t i n c o n t e n t b a s e d on
the h i g h e s t o b s e r v e d specific a c t i v i t y or on t h e r a d i o a c t i v i t y of e n z y m e
c o n t a i n i n g ~ H - b i o t i n give higher v a l u e s ( T a b l e I I ) . I t t h u s a p p e a r s t h a t
TABLE II
ESTIMATION OF BIOTIN CONTENT OF TRANSCARBOXYLASE
Calculation:
mol. wt. enzyme = 6.7 X 10~ Moles of
mol. wt. biotin = 244.3 biotin per mole
mmoles biotin/unit enzyme = of enzyme
Method of calculation 1.92 4- 10-~
From biotin content of protein (1.66 X 10-3 X 6.7 X 10s) + 244.3 4.5
(yeast assay); 1.66 ug biotin
per mg protein•
From highest observed 40 X 1.92 X 10-7 X 6.7 X 105 5.1
specific activity of
enzyme (40)
From specific activity (48) 48 X 1.92 X 10-~ X 6.7 X 105 62
estimated from radioactivity
of protein b containing
biotin-SH
The enzyme was from a pool of fractions from step 4 and had an observed specific
activity of 33.5 when first eluted from the TEAF_~cellulose column. The specific
activity fell to 27 after concentration of the protein by (NH4)2SO4 precipitation.
b During the purification and prior to evidence of inactivation the enzyme was
found to contain 48 cpm per unit of enzyme. On further purification there was a
loss of enzymatic activity but an increase in radioactivity per milligram of protein.
Assuming that 48 cpm was equivalent to a unit~ the enzymatic specific activity of
the enzyme was calculated to be 48 if there had been no inactivation (see text
footnote 6). Unfortunately the biotin content was not determined by microbiological
assay.
transcarboxylase contains at least 5, and probably 6, moles of biotin per

mole of enzyme of molecular weight of 670,000.
Subunits. When transcarboxylase is treated with 7 M urea a single
peak is observed on sedimentation with an $2o --- 1.35 $2 On treatment
with 2 M urea a number of peaks are seen; the main peak has an
$2o ~ 6 S and a smaller peak has 12 S (unpublished data). A more
convenient method of obtaining subunits with S.oo.w--~ 6 S is to incubate
the enzyme at an alkaline pH. The enzyme is passed through a Sephadex
G-50 column equilibrated with Tris-sulfate 0.1 M, pH 8.0. The protein
solution (3-5 mg/ml) is incubated at 25 ° for 12 hours. On sedimentation
it yields approximately 75% of 6 S material, 15% of 12 S, and 10% of
16 S. During the 12 hours, the specific activity decreases from ~ 23 to
3. For reaggregation of the protein, the solution is brought to pH 5.2
by addition of 0.5 M acetate, pH 4.4 (0.2 ml to 1.0 ml of enzyme solu-
tion). It is held at 0 ° for 15 hours. Some precipitate forms which is
removed by centrifugation. The specific activity increases to approxi-
mately 22 and on sedimentation about 77% is 25 S material, 8% is 12 S,
and 15% is 6 S (unpublished data).
It seems likely that there are at least four active forms of trans-
carboxylase with $2o,~, values of 12 S, 16 S, 18 S, and 25 S. These are
probably made up of combinations of the inactive subunits which have
an S2o,w value of 6 S. The subunits with an $2o value of 1.35 in 7 M
urea must be a mixture, since they cannot all contain biotin.
Metals. Transcarboxylase does not require the addition of metal ions,
and the presence of 0.1 M EDTA in the assay mixture does not'inhibit
the reaction. However, the enzyme does contain tightly bound cobalt
(Northrop and Wood is) and also zinc (unpublished results). Radioactive
measurements with transcarboxylase from bacteria grown in medium
containing ~°Co*+ or 65Zn++, and determination of the metals by atomic
absorption indicates that there are ~ 2 atoms of cobalt and ~ 4 atoms
of zinc per molecule of enzyme, assuming a molecular weight of 670,000.
The function of these metals is not known; they may serve to bind
subunits together or have a more direct catalytic function. Such a
catalytic function has been proposed I~ for the tightly bound manganese
of pyruvate carboxylase, which likewise is a biotin enzyme of high
molecular weight.
pH Optimum. Transcarboxylase has a broad pH optimum; there is
little change in activity between pH 5.5 and 7.8.9
Stability. Transcarboxylase is quite stable to acid pH. In the crude
lSD. B. Northrop and H. G. Wood, Federation Proc. 26~ 491 (1967).
14A. S. Mildvan, M. C. Scrutton, and M. F. Utter, J. Biol. Chem. 241, 3488 (1966);
see also this volume [38].
extract it is stable at 25 °, pH 5 (acetate buffer 0.25 M) for 5 hours, but

inactivation occurs at pH 4.0 even at 0 °. At pH 8.2 (0.1 M KHC03)
there is 60% inactivation in 45 minutes at 25 ° and 10% inactivation at
0 °. Above pH 8.2 in Tris-HC1 buffer there is a very rapid loss of activity.
The enzyme is stabilized to a considerable extent by polyvalent ions,
such as phosphate or sulfate (0.25M or greater). The partial loss of
activity of highly purified enzyme as described under step 4 may be
due to dissociation to less active subunits, but thus far studies on
nmterial obtained directly from the column and after partial loss of
activity have not given evidence of formation of inactive 6 S units during
the inactivation.
Transcarboxylase is not cold labile, and does not undergo deaggrega-
tion to subunits in the cold as does pyruvate carboxylase. 1~
Inhibitors. Transcarboxylase, a biotin enzyme, is inhibited strongly
by avidin2 ,9 It is not inhibited strongly by - - S H reagents. Incubation
for 20 minutes in 10 tLM p-chloromercuribenzoate at 0 ° caused 24% in-
activation; in 0.1 mM iodoacetate there was 13% inactivation, but 1 mM
N-ethylmaleimide had no effect. ~
The reaction is inhibited strongly by oxalate and also is inhibited by
a-ketobutyrate, CoA, propionyl pantetheine, and fl-methyloxaloacetate
(unpublished data; see Table III).
Specificity. Transcarboxylase appears to be highly specific for the
keto acid component; a-ketobutyrate, a-ketovalerate, a-ketoglutarate,
and fl-ketoglutarate do not replace pyruvate as the acceptor of the
carboxyl group from methylmalonyl-CoA, s Likewise fl-methyloxal-
acetate does not serve as a carboxyl donor to propionyl-CoA (unpub-
lished data).
The specificity for the CoA ester is broad. Acetyl-CoA, butyryl-CoA,
and acetoacetyl-CoA serve as carboxyl acceptors from oxaloacetate. The
rates were 1/2, 1/10, and 1/40, respectively, of that observed with
propionyl-CoA as the acceptor, s Malonyl-CoA 6 and ethylmalonyl-CoA, s
which are the expected products of earboxylation of acetyl-CoA and
butyryl-CoA, serve as carboxyl donors to pyruvate. The rate with
malonyl-CoA is 1/2 and that with ethylmalonyl-CoA is 1/7 the rate
obtained with methylmalonyl-CoA. Propionyl pantetheine does not serve
as a carboxyl acceptor.
The enzyme is specific for the (S) isomer of methylmalonyl-CoA.
This is the isomer which is formed by propionyl carboxylase and the
opposite of the (R) isomer produced by methylmalonyl-CoA mutase. 1°
'~M. C. Scrutton and M. F. Utter, J. Biol. Chem. 240, 1 (1965); see also this
volume [38].
228 REACTIONS LEANING TO AND FROM THE CYCLE [35]
~qubstrate and Inhibitor Affinity Constants. The Km and K~ values

for substrates and inhibitors are shown in Table III.
TABLE III
g~ AND g i VALUES OF SUBSTRATES AND INHIBITORS OF TRANSCARBOXYLASE
K,~ K~~
Substrate ( × 10-~ M) Inhibitors ( × 10-~ M)
Pyruvate 7.6 a a-Ketobutyrate 23

Aeetyl-CoA 5.6 CoA 4.5
Propionyl-CoA 0.30 a Propionyl-CoAb 4
Butyryl-CoA 2.5 Propionyl pantetheine 40
MalonyloCoA 0.35 Oxalate 0.02
Methylmalonyl-CoA 0.044~ ~-Methyloxaloacetate 3
Oxaloacetate 0.57 °
° These are unpublished values obtained in more recent studies. Previous values (see
text footnote 6) were pyruvate, 10; propionyl-CoA, 0.27; methylmalonyl-CoA,
0.08; oxaloacetate, 1.0.
bSubstrate inhibition.
c Unpublished data.
Equilibrium Constant2 The equilibrium constant at p H 6.5 and 30 °
expressed as total analytic concentrations was found to be 1.9 ± 0.1.
However, the transcarboxylase used to measure this equilibrium con-
tained methylmalonyl-CoA racemase. Thus, the (S) isomer of methyl-
malonyl-CoA which was produced in the transcarboxylase reaction was
converted to and was in equilibrium with the (R) isomer of methyl-
malonyl-CoA. The equilibrium for the racemase reaction is 1.11 Therefore
the equilibrium constant given above is for the combined transcarboxyl-
ase and racemase reactions. For the transcarboxylase reaction alone the
constant is half of this value and is calculated as follows:
K',n~l -- [pyruvateT][S-methylmalonyl-CoAT] = 1.0 4- 0.1

[oxaloaeetateT][propionyl COAT]
Since all the species are ionized at p H 6.5 and there was no metal present
which strongly binds the ions, the ionic equilibrium constant for p H 6.5
should be nearly the same, i.e.,
Kioni~ = [pyruvatel-][S-methylmal°nyl'C°A1-] = 1.0 4-0.1

[oxaloacetate~-][propionyl-CoA]
Mechanism o] the Reaction. The biotin is linked through the valeric
acid side chain by an amide linkage to the r-amino group of a lysyl
moiety of the protein. The earboxyl transfer is mediated b y the biotin
residue, which accepts a carboxyl group from the donor. The carboxyl
group is bound to the I ' - N of the ureido group of the biotin. 12 I t then is
transferred to the carboxyl aceeptor.
[35] OXALOACETATE TItANSCARBOXYLASE 229
Transcarboxylation thus involves two partial reactions:

C H 3 - - C H ( C O O - ) - - C O - - C o A -t- E - - b i o t i n
CH3--CH2--CO--CoA + E-biotin--CO0- (la)
E - b i o t i n - - C O O - -t- C H a - - C O - - C O O -
E-biotin ~- - O O C - - C H 2 - - C O - - C O O - (lb)
CH3--CH(COO-)--CO--CoA + CH~--CO--COO-
CHa--CH~--CO--CoA + -OOC--CH2--CO--CO0- (1)
The partial reactions are demonstrated readily. The ~4C-carboxyl-biotin-
enzyme m a y be obtained by carboxylation with ~4C-methylmalonyl-CoA,
followed by filtration on Sephadex gel. The carboxylatcd biotin-enzyme
may then be used to carboxylate pyruvate to form oxaloacet:tte. ~
The carboxylated biotin-enzyme is quite labile, having a half-life of
260 minutes at 0 °, 40 minutes at 10 °, 9.9 minutes at 20 °, or 2.2 minutes
at 30°. ~2 I t m a y be stabilized by treatment with diazomethane to obtain
the methyl ester of the carboxylated biotin-enzyme. This derivative on
digestion with pronase yields l'-N-carbomethoxybiotinyl lysine, thus
proving the structure stated above. ~2
The equilibrium of the partial reaction (la) at p H 7.0 and 2 ° is
35 ± 4 when enzyme-biotin is expressed as moles of biotin.
[E-biotin-COO-][propionyl CoA] = 35
[E-biotin][S-methylmalonyl-CoA]
The AF'~,~z calculated for this equilibrium constant is --1.9 kcaP 2 and
that for cleavage of the carboxyl bond of the carboxylated enzyme to
yield C02 and enzyme is --4.4 kcal. ~e
~The value given previously was --4.7 kcal. The present calculations (see Wood
et al. ~T for definitions and methods) are as follows:
A/"'~nal
a. E-biotin-COO- + propionyl-CoA ~ E-biotin + methylmalonyl-CoA -{-1.9 kcal

b. (S)-methylmalonyl CoA -t- pyruvate ~ oxaloacetate + propionyl CoA 0.0 keal
c. oxaloacetate --~ pyruvate -t- CO~ - 6 . 3 kcal
E-biotin-CO0- -* E-biotin + CO2 - 4 . 4 kcal
The 5F',,,L was previously given for the transcarboxylase reaction (b) as 0.39
kcal,~'12 but it is now known that the equilibrium constant is 1 (see above). Thus
the ~F',..L is 0.0. The ~F value of reaction (c) was given as --7.06 kcal instead of
--6.3. The previous value was for decarboxylation of oxaloacetate to free CO:
and did not include the energy of hydration and ionization accompanying forma-
tion of bicarbonate from CO~. The present vahm takes this hydration and ioniza-
tion into account at pH 7.0 (with no Mg÷÷ present) and has been calculated as
described by Wood et a l l The equilibrium of reaction (a) was measured at 0°
and pH 7 and that of (t,) at 30° and pH 6.5. No adjus1~n~nt h:~s be~,n made f()r
these differences.
'~H. G. Wood, J. J. Davis, and H. Lochmiiller, J. Biol. C h e m . 241, 5692 (1966).
230 REACTIONS
From the above discussion it would be expected that the two half-
reactions would give "ping-pong" kineticsY D. B. Northrop, in unpub-
lished studies, has found this to be the case.
18W. W. Cleland, Biochim. Biophys. Acta 67, 104 (1963).
[37] Malic Enzyme

[EC 1.1.1.40 L-Malate: NADP oxidoreductase (decarboxylating)]
By R. Y. H s u and H. A. LARDY
Mn ++ or Mg++
L-Malate + T P N , ' p y r u v a t e + COs + T P N H (1)
Mn ++ or Mg++
Oxaloacetate , pyruvate + CO~ (2)
Assay Method
Principle. The malic enzyme activity is assayed by measuring T P N H
formation in Eq. (1). The method is essentially that of 0choa 1 with
modifications to meet the requirements of an isolation procedure. The
modifications provide better p H stability and linearity over a wider range
of protein concentrations and time. Production of T P N H is monitored
in any suitable spectrophotometer with cell compartments thermostated
at 26 °. The rate of T P N H formation is proportional to enzyme concen-
tration up to a change of 0.2 optical density units (X 340 mt~) per minute.
Spectrophotometric Assay
Reagents
Triethanolamine buffer, 0.4 M. Dissolve 5.97 g in water. Neutralize
to p H 7.4 with 2 N HC1, and dilute to 100 ml
L-Malate, 30 mM. Dissolve 40.2 mg in water. Neutralize to p H 7.4
with 2 N KOH, and dilute to 10.0 ml
MnC12, 0.12 M. Dissolve 2.38 g of MnCl._,.4 H.,O in water and dilute
to 100 ml
T P N , 3.4 mM. Dissolve 27.2 mg in water and dilute to 10.0 ml
Procedure. Mix 0.5 ml of 0.4 M triethanolamine buffer, 0.05 ml of
0 . 0 3 M L-malate, 0.1 ml of O.12M MnC12-4 H20, 0.2 ml of 3.4 m M
T P N , and an appropriate amount of water in the spectrophotomete,'
cuvette and bring to 26 ° . Add enzyme to start the reaction and to
1S. 0choa, Vol. I, p. 739.
230 REACTIONS
From the above discussion it would be expected that the two half-
reactions would give "ping-pong" kineticsY D. B. Northrop, in unpub-
lished studies, has found this to be the case.
18W. W. Cleland, Biochim. Biophys. Acta 67, 104 (1963).
[37] Malic Enzyme

[EC 1.1.1.40 L-Malate: NADP oxidoreductase (decarboxylating)]
By R. Y. H s u and H. A. LARDY
Mn ++ or Mg++
L-Malate + T P N , ' p y r u v a t e + COs + T P N H (1)
Mn ++ or Mg++
Oxaloacetate , pyruvate + CO~ (2)
Assay Method
Principle. The malic enzyme activity is assayed by measuring T P N H
formation in Eq. (1). The method is essentially that of 0choa 1 with
modifications to meet the requirements of an isolation procedure. The
modifications provide better p H stability and linearity over a wider range
of protein concentrations and time. Production of T P N H is monitored
in any suitable spectrophotometer with cell compartments thermostated
at 26 °. The rate of T P N H formation is proportional to enzyme concen-
tration up to a change of 0.2 optical density units (X 340 mt~) per minute.
Reagents
Triethanolamine buffer, 0.4 M. Dissolve 5.97 g in water. Neutralize
to p H 7.4 with 2 N HC1, and dilute to 100 ml
L-Malate, 30 mM. Dissolve 40.2 mg in water. Neutralize to p H 7.4
with 2 N KOH, and dilute to 10.0 ml
MnC12, 0.12 M. Dissolve 2.38 g of MnCl._,.4 H.,O in water and dilute
to 100 ml
T P N , 3.4 mM. Dissolve 27.2 mg in water and dilute to 10.0 ml
Procedure. Mix 0.5 ml of 0.4 M triethanolamine buffer, 0.05 ml of
0 . 0 3 M L-malate, 0.1 ml of O.12M MnC12-4 H20, 0.2 ml of 3.4 m M
T P N , and an appropriate amount of water in the spectrophotomete,'
cuvette and bring to 26 ° . Add enzyme to start the reaction and to
1S. 0choa, Vol. I, p. 739.
[37] MALIC ENZYME 231
provide a final volume of 3.0 ml. Normally enzyme dilutions are made
with 50 m M Tris-HC1-20 m M magnesium acetate-2 m M 2-mercapto-
ethanol, p H 7. After the zinc step (fraction IV), enzyme solutions are
diluted with 50 m M T r i s - H C l - 1 0 m M E D T A - 0 . 2 M magnesium ace-
rate-2 m M 2-mercaptoethanol, pH 7.4, in order to achieve maximum
activity.
Isolation Procedure'-'
I n all alcohol fractionation steps, 95% ethanol is used. Alcohol con-
centrations, expressed as a percentage, are calculated as percentage of
95% ethanol, assuming no volume changes on mixing.
All purification steps are carried out at 0-5 ° unless otherwise specified.
The initial steps are modified from the method of Rutter and Lardy. "~
The procedure described is that of Hsu and Lardy. 2 A typical protocol
from among m a n y successful preparations is presented in the table.
PURIFICATION OF MALIC ENZYME FROM PIGEON LIVER
Specific
Total Total activity Purifi-
Volume activity protein (units/mg Yield cation
Fraction (ml) (units,) (rag) protein) (%) factor
1. Supernatant frac- 1,060 4,200 20,000 0.21 (100) ---

tion
2. Heat treatment 1,010 4,350 5,840 0.75 103 3.5
3. First ethanol 211 2,960 1,580 1.87 70 8.9
fractionation
4. Second ethanol 41.0 3,080 302 10.2 73 48.5
fractioaation
5. Ammonium sulfate 10.0 2,340 106 22.1 56 105
fractionation
6. DEAE-cellulose 18.4 1,780 60.2 29.6 42 140
chromatography
7. Concentration with 1.05 1,570 54.7 28 ~7 37 --
ammonium sulfate
8. Crystals 1.82 740 27.1 27.3 18 --
a One unit is defined as the amount of enzyme catalyzing the reaction of 1 umole
of substrate/minute under the conditions of the assay.
Step 1. Preparation of Pigeon Liver Supernatant Fraction. Pigeons are

killed by decapitation, and the livers are collected and chilled im-
mediately on ice. After weighing, the livers (347 g from 45 birds) are
washed in 0.25 M sucrose, cut into small pieces, and homogenized with 3
2R. Y. Hsu and H. A. Lardy, J. Biol. Chem. 242, 520 (1967).
3W. J. Rutter and H. A. Lardy, J. Biol. Chem. 233, 374 (1958).
volumes of 0.25M sucrose in a Potter-Elvehjem homogenizer. If the

mitochondria are to he retained for other purposes, the homogenate is
centrifuged at 1000 g for 10 mbmtes. The supernatant fraction is saved,
and the sediment is rehomogenized with l volume of 0.25.]1/" sucrose and
again centrifuged for 10 minutes at 1000 g. The sediment is discarded.
The supernatant fractions from the two centrifugation steps are com-
bined and centrifuged at 13,200 g for 10 minutes to remove the mito-
chondria. The supcl'natant fraction from this step is centrifuged again in
the No. 30 rotor of the Spinco Model L preparative ultracentrifuge for
30 minutes at 30,000 rpm (78,500 g). The activity is almost exclusively
in the 1060 ml of supernatant liquid (fraction I) which is kept in ice
overnight for further purification. If mitochondria are not to be retained
for other purposes, the homogenate may be centrifuged at 78,000 g
directly.
Step 2. Heat Treatment. Fraction I is made 0.1 M in magnesium ace-
tate by the addition of 22.75 g of this compound and acidified to pH 5.5
with ice cold 1 N acetic acid. Portions (200 ml) of the acidified enzyme
solution are heated for 5 minutes with vigorous stirring in a stainless
steel beaker immersed in a constant-temperature water bath at 58 °.
After heating, the flocculent suspension is cooled immediately to 5--10°
in an alcohol bath at --4 °, and centrifuged at 9000 g for 10 minutes;
1010 ml of supernatant solution (fraction II) is obtained.
Step 3. First EthaTwl Fractionatiol~. Fraction II is cooled with stirring
in a --4 ° alcohol bath. Then 302 ml of 95% ethanol (calculated to give
23% of 95% ethanol), chilled to about --70 ° with a mixture of dry ice
and acetone, is added slowly below the liquid surface. The suspension is
stirred slowly for 2 hours and centrifuged at 6000 g for 30 minutes at
--4 °. The precipitate is discarded. The supcrnatant solution is again
brought to --4 °, and 446 ml of chilled 95% ethanol is added to bring the
alcohol concentration to 43%. After the alcohol has been added, the
stainless steel beaker containing enzyme suspension is removed from the
--4 ° alcohol bath, placed in a --15 ° alcohol bath, and stirred slowly
overnight. The suspension is centrifuged at 6000 g for 30 minutes at
--15 °. The precipitate is collected and suspended immediately in 210 m]
of EDTA buffer containing 0.02 M EDTA and 2 mM 2-mercaptoethanol,
pH 6.7, stirred 30 minutes, and centrifuged at 6000 g for 10 minutes.
From this step, 211 ml of supernatant solution (fraction III) are obtained
and used for further purification.
Step ~. Second Ethanol Fractionation. After its protein content is
determined, Fraction III is diluted with 579 ml of the EDTA buffer
described under Step 3 to obtain a protein concentration of 2 mg/ml.
Fifteen milliliters of 0.1 M Na2HPO4-0.02 M EDTA at pH 8.0 is added.
[37] MALIC ENZYME 233
The diluted enzyme solution is equilibrated in the --4 ° bath with stir-
ring, and chilled (--70°), after which 95% ethanol is added to 21% (214
ml). The suspension is stirred for 1 hour, then centrifuged at 6000 g for
30 minutes at --4 °. The supernatant solution is brought to 33% ethanol
by the addition of 172 ml of chilled 95% ethanol. The stirring is con-
tinued overnight in the --15 ° bath. The suspension is centrifuged at
6000 g for 30 minutes at --15 °, and the precipitate is collected and
suspended immediately in 153 ml of 0.1 M zinc acetate-0.1 M glycine
(Tris), pH 7.0. The suspension is stirred for 30 minutes, then centrifuged
at 6000 g for 10 minutes. The precipitate is dissolved in 42 ml of 0.1 M
histidine-2 mM 2-mercaptoethanol, pH 6.8. The cloudy solution is
clarified by centrifugation. The clear supernatant solution, which con-
tains most of the enzyme activity, is dialyzed for.3 hours against 1 liter
of 20 mM Na_~SQ-20 mM EDTA-2 mM 2-mercaptoethanol, pH 7.4,
with one change of dialyzing buffer.
Step 5. Ammonium Sul]ate Fractionation. After the addition of 4.1
ml of 1 M Tris-HC1 buffer, pH 7.0, 14.6 g of solid ammonium sulfate is
added slowly to the dialyzate (fraction IV) with stirring to give 55%
saturation. Stirring is continued for 30 minutes, and the precipitate is
removed by centrifugation (6000 g for 10 minutes). The supernatant
solution is brought to 67% saturation by the addition of 18.3 ml of
saturated ammonium sulfate solution, pH 7.4, stirred for 15 minutes, and
again centrifuged as before. The 55-67% saturated ammonium sulfate
precipitate is dissolved in a small amount of 30 mM Tris-HC1-2 mM
2-mercaptoethanol, pH 7.7 (fraction V), and dialyzed against 500-ml
portions of the same buffer overnight with one change of buffer.
Step 6. DEAE-Cellulose Chromatography. A column (1.0 X 30 em) is
prepared from washed DEAE-cellulose and equilibrated with 1 liter of
30 mM Tris-HC1-2 mM 2-mercaptoethanol buffer, pH 7.7. Fraction V is
added to the column and eluted with the Tris buffer described above.
Protein peaks are detected by recording light absorption at 280 mu. The
first protein peak is inactive and is discarded. When the elution of the
first peak is complete, the enzyme activity is eluted with the equilibra-
tion buffer containing 20 mM magnesium acetate. The second peak
amounts to about 18 ml (fraction VI).
Step 7. Concentration with Ammonium Sul]ate and Crystallization.
To fraction VI, 0.2 ml of 0.1 M dithiothreitol is added. The solution is
brought to 75% saturation by the addition of 8.72 g of solid ammonium
sulfate, stirred for 60 minutes, and centrifuged at 6000 g for 10 minutes.
The pellet contains the purified enzyme and is dissolved carefully in 0.1
ml of 1 M Tris-HC1 buffer, pH 7.0, and a minimum amount of cold
water (0.40 ml). To this solution _(k25 ml of 3.4 mM TPN is added to
make a total volume of 1.05 ml (fraction VII). On the assumption that

the pellet is also 75% saturated with ammonium sulfate and from its
calculated volume of 0.3 ml, fraction VII is calculated to be 21.4%
saturated. To fraction VII is added 0.01 ml of 0.1 M dithiothreitoI and
0.41 ml of saturated, recrystallized ammonium sulfate, containing 0.2 M
EDTA, pH 7, to bring the solution to 36% saturation. The solution is
centrifuged quickly at 6000 g for 2 minutes to remove traces of insoluble
material. It is transferred to a clean tube, stoppered, and kept in ice for
crystallization. Usually a sheen of crystals appears after several hours,
and rod-shaped crystals can be seen under the microscope after a day.
However, occasionally crystals do not appear after a day, and more
saturated ammonium sulfate is then added (up to 44% saturation) to
induce crystallization. Crystallization is also facilitated by the addition
of seed crystals.
The crystals are harvested after a week by centrifugation (6000 g
for 20 minutes). The pellet is usually dissolved in a small volume of 50
mM Tris-HCl, 1 mM dithiothreitol buffer, pH 7.0, and dialyzed against
the same buffer overnight with one change of buffer to yield fraction VIII.
Recrystallization is accomplished readily with seeding; however, no
further purification is achieved. Recrystallization should be accomplished
in 44% saturated ammonium sulfate to minimize loss of enzyme.
Properties
Specific Activity. The specific activity of the crystalline malic enzyme
isolated by this procedure is 27-30 (micromoles ~of-TPNH formed per
minute at 26 ° per milligram of protein).
Stability. Malic enzyme is stable when stored in the crystalline state
at 2-5 ° as a suspension in 36% saturated ammonium sulfate solution.
Howevcr, the dissolved crystals gradually lose activity after several
weeks at --15 ° . The partially inactivated enzyme can be reactivated by
incubation in the presence of 1 mM dithiothreitol. Maximal reactivation
was obtained after 80 minutes of incubation at 26 ° .
Physical Properties. The crystalline enzyme has a $2o.wof 10.0 (extrap-
olated to zero protein concentration). It has an apparent diffusion co-
efficient of 3.17 X 10-7 cm 2 per second and an apparent partial specific
volume of 0.74. Its molecular weight is 2.8 X 105. It has an ultraviolet
absorption maximum at 278 mt~; the extinction coefficient for the enzyme
crystallized in the presence of TPN is 0.92 for a 0.10% protein solution.
Purity. The crystalline enzyme appears homogeneous in gradient
centrifugation, gradient chromatography, and velocity sedimentation. It
is free of heme, flavins, cytochromcs, and other materials absorbing light
in the visual region. The protein appears to be free of several dehydro-
[38] PYRUVATE CARBOXYLASEFROM CHICKEN LIVER 235
genases whose substrates were tested, but it exhibits an intrinsic lactic

dehydrogenase activity specific for TPN. ~ Crystalline enzyme prepara-
tions may contain a trace (less than 0.01%) of lactate dehydrogcnase.
Coenzyme Binding2 The malic enzyme binds TPNH in proportions
of 1 mole per 76,500 g of protein with a site dissociation constant of 0.75
t~M. TPN binds in competition with TPNH, and its site dissociation
constant was calculated to be 0.97 tLM. These data indicate four binding
sites for coenzyme per mole; in agreement with this is the finding that
the enzyme dissociates at pH 12 into subunits apparently one-fourth the
size of the parent protein molecule.~
Kinetic Constants. 6 At pH 7.0, the limiting Michaelis constants for
TPN and L-malate are 1.42 ___0.2 t ~ / and 86 ± 5 t~/, respectively; the
apparent Michaelis constants for bicarbonate, pyruvate, and TPNH are
13 ± 1 mM (pyruvate 3.33 mM, T P N H 53.5 p21/), 6.4 ± 0.5 mM (bicar-
bonate 50 raM, TPNH 0.107 raM), and 2.1 ± 0.1 t~M (bicarbonate 50
mM, pyruvate 5.0 mM), respectively; the dissociation constants for TPN
and TPNH are 0.96 ___0.2/~M and 2-3 ~M, respectively.
R. Y. Hsu and H. A. Lardy, Acta Biochim. Polon. 14, 183 (1967).
"R. Y. Hsu and H. A. Lardy, J. Biol. Chem. 242, 527 (1967).
~R. Y. Hsu, H. A. Lardy, and W. W. Cleland, J. Biol. Chem. 242, 5315 (1967).
[38] P y r u v a t e C a r b o x y l a s e f r o m C h i c k e n L i v e r
[EC 6.4.1.1 Pyruvate: carbon-dioxide ligase (ADP)]
By M. C. SCaUTTON, M. R. OLMSTED,and M. F. UTTER
acetyl-CoA
Mg++
Pyruvate W ATP -{- HCO~- , " oxaloacetate ~- ADP ~ Pi
Assay Methods
Principle. Pyruvate carboxylase activity is assayed spectrophoto-
metrically by measurement of oxaloacetate production with malate dehy-
drogenase. The assay is used routinely with the highly purified enzyme,
but is less satisfactory in the presence of marked contamination with
lactate dehydrogenase or D P N H oxidase, i.e., prior to stage 2 of the
purification procedure described below. In crude systems, such as liver
homogenates, additional interference occurs due to breakdown of acetyl-
CoA and ATP. Under these conditions Henning and Seubert 1 have used
I H. V. Henning and W. Seubert, Biochem. Z. 340, 160 (1964).

[38] PYRUVATE CARBOXYLASEFROM CHICKEN LIVER 235
genases whose substrates were tested, but it exhibits an intrinsic lactic

dehydrogenase activity specific for TPN. ~ Crystalline enzyme prepara-
tions may contain a trace (less than 0.01%) of lactate dehydrogcnase.
Coenzyme Binding2 The malic enzyme binds TPNH in proportions
of 1 mole per 76,500 g of protein with a site dissociation constant of 0.75
t~M. TPN binds in competition with TPNH, and its site dissociation
constant was calculated to be 0.97 tLM. These data indicate four binding
sites for coenzyme per mole; in agreement with this is the finding that
the enzyme dissociates at pH 12 into subunits apparently one-fourth the
size of the parent protein molecule.~
Kinetic Constants. 6 At pH 7.0, the limiting Michaelis constants for
TPN and L-malate are 1.42 ___0.2 t ~ / and 86 ± 5 t~/, respectively; the
apparent Michaelis constants for bicarbonate, pyruvate, and TPNH are
13 ± 1 mM (pyruvate 3.33 mM, T P N H 53.5 p21/), 6.4 ± 0.5 mM (bicar-
bonate 50 raM, TPNH 0.107 raM), and 2.1 ± 0.1 t~M (bicarbonate 50
mM, pyruvate 5.0 mM), respectively; the dissociation constants for TPN
and TPNH are 0.96 ___0.2/~M and 2-3 ~M, respectively.
R. Y. Hsu and H. A. Lardy, Acta Biochim. Polon. 14, 183 (1967).
"R. Y. Hsu and H. A. Lardy, J. Biol. Chem. 242, 527 (1967).
~R. Y. Hsu, H. A. Lardy, and W. W. Cleland, J. Biol. Chem. 242, 5315 (1967).
[38] P y r u v a t e C a r b o x y l a s e f r o m C h i c k e n L i v e r
By M. C. SCaUTTON, M. R. OLMSTED,and M. F. UTTER
acetyl-CoA
Mg++
Pyruvate W ATP -{- HCO~- , " oxaloacetate ~- ADP ~ Pi
Assay Methods
Principle. Pyruvate carboxylase activity is assayed spectrophoto-
metrically by measurement of oxaloacetate production with malate dehy-
drogenase. The assay is used routinely with the highly purified enzyme,
but is less satisfactory in the presence of marked contamination with
lactate dehydrogenase or D P N H oxidase, i.e., prior to stage 2 of the
purification procedure described below. In crude systems, such as liver
homogenates, additional interference occurs due to breakdown of acetyl-
CoA and ATP. Under these conditions Henning and Seubert 1 have used
I H. V. Henning and W. Seubert, Biochem. Z. 340, 160 (1964).

an assay which provides for maintenance of a constant level of acetyl-
CoA, and this method may be used with an ATP-regenerating system.
Studies of the exchange reactions catalyzed by pyruvate earboxylase
have permitted the formulation of the minimal mechanism,2 Eqs. (1)
and (2), as:
acetyl-CoA
Mg+ +
E-biotin A- ATP + HCOs. * E-biotin~C02 + ADP A- P~ (1)
E-biotin ~CO2 + pyruvate ~ E-biotin + oxaloacetate (2)
It has been suggested 2 that the exchange of pyruvate-14C with oxalo-
acetate might provide a simple routine assay for pyruvate earboxylase
activity in crude systems. The only other enzyme known to catalyze
exchange of pyruvate-l'C with oxaloacetate is methylmalonyl-CoA-
oxaloacetate transcarboxylase. Neither this assay nor that described by
Henning and Seubert 1 has been evaluated by us for use in crude systems.
Reagents. Reagents for enzyme preparation and assay are dissolved
in water distilled once from a metal still and subsequently redistilled
from glass. All pH's are measured at 25 ° unless stated otherwise
Tris-HC1, 0.5 M, pH 7.8, prepared from Tris base recrystallized

once at alkaline pH from 85% ethanol containing 10 mM EDTA
as described by Sutherland and Wosiliat s
Tris-pyruvate, 0.5M. Best results are obtained with pyruvic acid
purified by distillation under reduced pressure and diluted to give
a 1 M solution which is stored at --20 °. A small volume is
neutralized to pH 6.8 with 1 M Tris base immediately prior to use
Disodium ATP, 0.05 M (Sigma, Sigma grade) neutralized to pH 7
with Tris base
MgCI2, 0.1 M
KHCOa, 0.3 M
Acetyl-CoA, 0.2 raM, prepared from acetic anhydride and CoASH 4
and assayed spectrophotometrieally as described by 0choa 5
Malate dehydrogenase (EC 1.1.1.37) (Calbiochem) (10 mg/ml)
diluted in 1 ~ bovine serum albumin to give a solution containing
100 units/m]
DPNH, 4.5 raM. For optimal results a fresh solution is prepared
each day.
2 M. C. Scrutton, D. B. Keeeh, and M. F. Utter, J. Biol. Chem. 240, 574 (1965).
SE. W. Sutherland, and W. D. Wosiliat, J. Biol. Chem. 218, 459 (1956).
'See Vol. III [137].
aS. Oehoa, Biochem. Prep. 5, 19, (1957).
[38] PYRUVATE CARBOXYLASE FROM CHICKEN LIVER 237
Enzyme. Dilutions of the enzyme are prepared in 1 M sucrose con-

taining 0.1 M phosphate and 0.06 M (NH~)~S04 to a final con-
centration of 0.1-0.5 mg/ml. Shake the diluent thoroughly before
use to eliminate the layering which develops during storage.
Assay for pyruvate carboxylase and protein should be conducted
immediately, since a loss of 20% of the enzymatic activity occurs
during the first 15 minutes after dilution. Thereafter the diluted
enzyme solutions are stable for at least 4 hours at 23 °.
Spectrophotometric Assay. The assay system contains in 1.0 ml: Tris-
HC1, pH 7.8, 100 micromoles; Tris-pyruvate, pH 6.8, 10 micromoles;
ATP, pH 7, 1 micromole; MgC12, 5 micromoles; KHC08, 15 micromoles;
acetyl-CoA, 0.1 micromole; malate dehydrogenase, 5 units; DPNH, 0.225
micromole; pyruvate carboxylase, 0.01-0.05 units.
After equilibration of the assay system to 25 ° the reaction is started
by addition of either pyruvate carboxylase or MgCl~ and the absorbance
at 340 m~ is measured. The decrease in absorbance is linear with time
for at least 5 minutes with enzyme concentration below 0.05 unit. The
contribution of contaminating enzymes to the rate of DPNtI oxidation
is evaluated by omitting acetyl-CoA or by preincuba~ing the enzyme
with excess avidin for 20 minutes. The latter control is preferred.
The most common causes of difficulty with the assay are malate
dehydrogenase with depressed activity, poor quality pyruvate, D P N H
which is not freshly prepared, or the presence of high levels of salt. A
final concentration of 0.095M (NH~)2S04 or 0.27M NaC1 gives 50%
inhibition of pyruvate carboxylase activity in this assay system.
Units. Units are expressed as micromoles of oxaloacetate produced per
minute at 25°; and specific activities as units per milligram of protein.
Protein is measured spectrophotometrically6 in purified preparations. At
earlier stages of the purification procedure the biuret method is used/
The values given by the spectrophotometric method are 92-95 3 of those
obtained either by the biuret procedure or by measurement of the refrac-
tive index increment.
Purification o] Pyruvate Carboxylase
Preparations of high specific activity from chicken liver mitochondria
require speed and unusual care. The procedures involved are therefore
described in detail.
Source. Best results are obtained with White Rock chickens (7-8-
weeks-old and weighing 2-21/~ pounds) which have been starved for
o See Vol. I I I [73]. T h e f a c t o r s 1.55 × A2~o - - 0.76 × A~o a r e u s e d .

' See Vol. I I I [73].
36-48 hours. These chickens are smaller than the usual commercially
available White Rock chickens of this age and represent the smallest
members of a flock.
Reagents
Sucrose; Mallinckrodt AR grade. Other grades have not always
been satisfactory
(NH,)2S04, Merck AR grade recrystallized twice from 10 mM
EDTA at alkaline pH. The commercial "enzyme-grade" (NH4):
S04 is not satisfacto~T for this preparation. Percentage satura-
tions are based on the saturated solution at 22-25 ° and pH 7.0
EDTA, disodium ethylenediaminetetracetate (Sigma ED 2 SS)
neutralized to pH 7.0 with 1 N NaOH
Cas(PO,)2 gel, prepared by a modification of the procedure of
Keilin and Hartree as described by Singer and Kearney.8 The
gel preparations are stable for at least 1 year when kept at 23 °
Preparation o] Lyophilized Chicken Liver Mitochondria. All proce-

dures in this section are conducted at 0-4 °, and all reagents are prepared
not more than 24 hours prior to use.
Chickens in batches of 15 are killed by decapitation; the livers (300-
400 g) are removed immediately and cooled in excess 0.3 M sucrose. After
removal of connective tissue and blood clots, the livers are washed in
0.3 M sucrose. Individual livers are dried on absorbent paper and minced
using a block which holds 4 razor blades placed 1 cm apart. Aliquots of
100 g each of the liver mince are homogenized in 400 ml of 0.3M
sucrose using a Waring blendor speed--controlled by a Variac. The
Variac settings used are 44 for 30 seconds followed by 70 for 13 seconds
(maximum current at 110-120 V is given by a setting of 120).
The aliquots are combined and centrifuged at 700 g for 20 minutes
to give separation into three layers: a supernatant fraction (broken
mitoehondria, microsomes, and cell soluble fraction), a loosely packed
central layer (intact mitochondria), and a more firmly packed lower
layer (nuclei and unbroken cells). The supernatant fraction is discarded
cautiously and the central layer is collected by decantation. This mito-
chondrial layer is washed with 2000 ml of 0.5 mM EDTA at pH 6.8.
The suspension is stirred for 3 minutes, centrifuged at 14,000 g for 20
minutes, and the supernatant fraction is discarded. The washed mito-
chondria are removed from the centrifuge bottles using 20-30 ml of 0.5
mM EDTA pH 6.8. The resulting suspension is distributed equally
between two 2000 ml round-bottomed flasks, shell-frozen in a --70 °
* T. P. Singer and E. B. Kearney, Arch. Biochem. Biophys. 29, 190 (1950).
(solid COs-ethanol) cooling bath and lyophilized on a Labfreeze Dryer

(Savant Instruments Inc.). 9 The elapsed time from the trimming of
connective tissue from the livcrs to the commencement of lyophilization
should not exceed 2 hours, and the final yield of lyophilized mitochondria
should be in the range of 30--60 g from 300-400 g of liver.
This procedure permits rapid separation of the mitochondria, removes
much water-soluble protein, and allows subsequent extraction of pyruvate
carboxylase from the lyophilized mitochondria under very mild condi-
tions. Mitochondria from pigeon, rat, and calf liver may be prepared by
an identical procedure and give satisfactory yields of pyruvate carboxyl-
ase after lyophilization and extraction. The procedure is not applicable
without modification to some other mammalian species, e.g., rabbit and
guinea pig, since in these cases the mitochondria are not separated from
the supernatant fraction by centrifugation at 700 g.
Maximal yields of pyruvate carboxylase are obtained when the
lyophilized mitochondria are extracted within 24 hours of preparation.
Storage for 3-4 days at --20 ° in vacuo causes a variable (5-25%)
decrease in the activity when extraction is conducted as described below.
Purification of Pyruvate Carboxylase from Lyophilized

Mitocbondria from Chicken Liver
All procedures are carried out at 20-25 ° unless stated otherwise.

Pyruvate carboxylase from chicken liver is not affected by exposure at
room temperature during the extraction (stage 1) and becomes cold-
labile after precipitation with 33% (NH4)2S04 (stage 2). All centrifuga-
tions are carried out at a minimum of 37,000 g unless stated otherwise.
Stage I. Extraction o] the Mitochondria. Lyophilized mitochondria
(30 g) are extracted with 280 ml of 50 mM Tris-acetate, pH 6.5, contain-
ing 5 mM ATP, 5 mM MgS04, and 0.5 mM EDTA for 10 minutes with
slow mechanical stirring. The pH falls during the addition of the mito-
chondria to the extracting medium but is not permitted to drop below
pH 6.3. If necessary, the pH can be adjusted with 1 M Tris base. The
suspension is centrifuged for 20 minutes, the red supernatant fraction is
collected, and the pH is adjusted immediately to 7.2 with 1 M Tris base.
The inclusion of ATP and MgS04 in the extracting buffer improves the
yield of pyruvate carboxylase. TM Other pH's and molarities of Tris-
acetate, e.g., 0.02M or 0.1M, yield pyruvate carboxylase of lower
specific activity. This is also true in extractions carried out with other
~Conventional lyophilization equipment may be used but is less satisfactory

because of the large volumes of liquid involved.
'°B. R. Landau and M. F. Utter, unpublished observations, 1965.
Tris buffers, e.g., Tris-C1, Tris-S0~, and Tris-citrate, or with phosphate

and glycyl-glycine buffers.
Stage 2. Fractionation o] the Extract with (NH4)2SO,. In prepara-
tions where the supernatant fraction from stage 1 is clear, solid (NH~)2
S04 is added to a final concentration of 33% with the pH maintained at
7.0 by addition of 1 M Tris base. After stirring for 20 minutes thc
precipitate is collected by centrifugation for 15 minutes.
If the supernatant fraction from stage 1 is turbid, solid (NH,)2S04
is added to a final concentration of 25% with appropriate adjustment to
pH 7.0. The preparation is stirred for 20 minutes, then the small precipi-
tate is removed by centrifugation for 20 minutes and discarded. The
(NH,)2S04 concentration of the supernatant fraction is increased to 33%
by addition of a solution of saturated (NH4)~S0, which has been adjusted
to pH 7.0 with NH40H, and stirring is continued for 20 minutes. The
precipitate is collected by centrifugation for 20 minutes.
Stage 3. Removal o] Inactive Protein by Denaturation with Heat and
Treatment with Ca3 (P04)2 Gel. The precipitate from stage 2 is dissolved
in 100 ml 0.4 M sucrose containing 5 mM ATP, 5 mM MgS04 and 15
mM KHC08, which is adjusted to pH 6.8 immediately before use. The
presence of these reactants is required to stabilize pyruvate carboxylase
against inactivation at 49 °. After the precipitate has dissolved, the pH
is readjusted to pH 6.8. The resulting clear yellow solution in a 300 ml
Erlenmeyer flask is heated rapidly with swirling to 49 ° in a 100 ° water
bath and is then transferred to a 50 ° water bath for 8 minutes. The solu-
tion is cooled to 25 ° in a --70 ° (dry ice-Ethanol) cooling bath with rapid
swirling. A small precipitate is observed after this step. The pH is
adjusted to 6.3 with 1 M acetic acid, and 4-5 ml of Ca~(P04)2 gel (30
mg/ml) is added with rapid mechanical stirring. After 30 seconds the
suspension is centrifuged for 10 minutes and the gel is discarded. Since
Ca3 (P04)~ gels differ in their absorptive capacities, each gel preparation
is titrated by adding small aliquots to the enzyme preparation until 20-
30% of the enzymatic activity is lost after removal of the gel. Calibrated
aged gels can be used for at least 1 year without further adjustment.
The supernatant fraction from the gel step is immediately adjusted
to pH 7.2 with 1 M Tris base, and solid (NH,)2S0~ is added to a final
concentration of 4'5% with the pH maintained constant. The preparation
is stirred for 20 minutes, then the precipitate is collected by centrifuga-
tion for 20 minutes.
If the supernatant fraction from the gel treatment is turbid, the
(NH4)2S04 concentration of the supcrnatant fraction is raised to 20%
by addition of solid (NH4)~SO, at pH 7.0. The suspension is stirred for
15 minutes, then centrifuged for 15 minutes; the precipitate is discarded.
The (NH,)~SO, concentration of the supernatant fraction is raised to

45% by addition of solid (N-I-I,)2S0, and the precipitate is collected as
described above.
Stage ~. {NH,)~S04 Extraction. The precipitate obtained from stage
3 is serially extracted with (NHs).,S04 solutions of decreasing concen-
tration. The extracting solutions are prepared immediately prior to use
by dilution of saturated (NH,)2S04, pH 7.0, with 0.25M EDTA, pH
7.0. The pH of these mixtures falls to 6.6-6.7 and should not be re-
adjusted. Extraction must be carried out without alteration of, or addi-
tion to the extracting solutions.
Successful extraction is also dependent on protein concentration. A
small precipitate from stage 3 requires a reduction in the recommended
volumes of the extracting solutions, whereas large precipitates require
an increase in the volume used. The first stage of the extraction protocol
is designed to remove contaminant protein from the stage 3 precipitate.
For an average preparation the protocol is:
1. 20 ml 33% (NH,)2S04
2. 20 ml 32% (NH,)2SO,
3. 20 ml 30% (NH4)2S04
4. 20 ml 28% (NH4)2S04
5. 20 ml 28% (NH4)2S04
The stage 3 precipitate is suspended successively in each of the abovc
solutions in the order given. After suspension, it is stirred for 10 minutes
with a magnetic stirrer, and the precipitate is separated from each extrac-
tion solution by centrifugation for 5 minutes. After each centrifugation,
the supernatant is discarded.
The pellet remaining after the second extraction with 28% (NHs)~S04
is then serially extracted, as described previously, with:
1. 10 ml 22% (NHs)2SO,
2. 5 ml 22% (NH4)2S04
Pyruvate carboxylase at maximal specific activity is extracted from the
pellet in these two steps and the supernatant fractions are adjusted to
pH 7.0 immediately after centrifugation and retained. The residual pre-
cipitate after the second extraction with 22% (NH,)2S0~ is discarded.
Note: Losses of material resulting from transfer may be avoided by
conducting the entire series of extractions in a 50 ml polycarbonate
centrifuge tube. The stirring bar may remain in the tube during centrif-
ugation without causing breakage. Extraction of pyruvate carboxylase
without concomitant extraction of glutamate dehydrogenase and other
proteins requires that the 22% extracts are obtained by very gentle
stirring of the suspension. The use of more vigorous stirring or further

extraction with either 22% or 20% (NH,)~SO4 increases the yield of
pyruvate carboxylase but gives preparations of lower specific activity
that are contaminated heavily with glutamate dehydrogenase.
Stage 5. (NH,)2SO, Precipitation. The (NH4)~SO, concentration of
the 2 2 ~ extracts is raised to a final concentration of 33% by addition of
solid (NH4)2S04 with the pH maintained at 7.0 by addition of 1 M
Tris base. The preparation is stirred for 20 minutes, then the precipitate
is collected by centrifugation for 20 minutes and is dissolved in 1.5M
sucrose containing 0.1 M potassium phosphate, pH 7.2 (or 0.1 M Tris-
S0~, pH 6.7) and 0.06 M (NH4)2S0~ to give a final protein concentration
of at least 15-20 mg/ml. The sucrose--phosphate--(NH4)2SO, or sucrose-
Tris--(NH,)2S0, solutions should be mixed thoroughly before use to
eliminate layering. The pH of the final enzyme solution is adjusted to
pH 7.0 by addition of 1 M Tris base. If the sucrose-Tris medium is used,
the final pH should be measured at 4 °.
Stage 5 preparations of pyruvate carboxylase contain a major (14.8
S) and a minor (6.75 S) component. Enzymatic activity is confined to
the 14.8 S component, but several lines of evidence suggest that at least
a portion of the protein present in the 6.75 S fraction is a subunit of
pyruvate carboxylase. Much of the residual 6.75 S material may be
removed from stage 5 preparations by gel filtration on Sephadex G-200
as described in the next section.
Stage 6. Gel Filtration on Sephadex G-~O0. Stage 6 is used only when
preparations are required for studies demanding a high degree of homo-
geneity.
The precipitated protein from stage 5 is used directly or, if it has
been dissolved in the sucrose storage medium, it is precipitated by addi-
tion of an equal volume of saturated (NH~)~S04 followed by centrifuga-
tion at 107,000 g for 30 minutes. The precipitate is dissolved in a minimal
volume of 0.1 M phosphate, pH 7.2, containing 0.06 M (NH~)2S0~ and
10 mM EDTA, and is applied to a 20 X 2.5 cm column of Sephadex
G-200 equilibrated with the same buffer. The column is eluted with this
buffer at a flow rate of 10-15 ml per hour and fractions of 2-3 ml are
collected. The fractions exhibiting maximal specific activity are pooled.
The protein is precipitated by addition of solid (NH4)2S04 to 45%
saturation, collected by centrifugation at 107,000 g for 20 minutes, and
dissolved in the sucrose--phosphate-(NH4)2S04 or sucrose-Tris-(NH4)2
S04 buffers described in stage 5. The 14.8 S and 6.75 S components are
not resolved clearly on Sephadex G-200, and only 60-70% of the enzy-
matic activity applied to the column is recovered in the peak fractions.
Results o] Purification Procedure. The results of a typical purification
TABLE I
PURIFICATION OF PYRUVATE CARBOXYLASE
Pyruvate Specific
Volume carboxylase Protein activity Recovery
Stage (ml) units~ (mg) b (units/mg) (%)
Stage 1 198 2870 3755 0.77 100

Stage 2
0-33% precipitate 104 3320 832 3.98 115
33% supernatant fraction 210 336 2940 0.12
(discarded)
Stage 3
Heat supernatant fraction 104 3265 -- -- 114
Gel supernatant fraction 103 3152 592 5.32 110
20% (NH,)2SO4 109 3120 516 6.05 109
supernatant fraction
Stage 4
33% extract (discarded) 20 - - 1 7 . 6 - - - -
32% extract (discarded) 20 0.4 12.4 0.03 --

30% extract (discarded) 20 4.4 14.4 0.3 - -
28% extract (I) (discarded) 20 15.4 14.0 1.1 --

(II) (discarded) 20 11.0 9.8 1.1 --
22% extract (I) 10 994 36.8 27.0 35
(II) 5 498 18.0 27.7 17.4
20% extract (discarded) 5 548 27.7 19.8 19.1
Residue (discarded) 10 225 250 0.9 7.2
Stage 5
Precipitate from 22% 3.3 1480 57.5 26.2 53
extracts, I and II
Measured by the spectrophotometric assay procedure described above. In all the

fractions prior to the 30% extract of stage 4 controls were employed to correct for
nonspecifie DPNH oxidation. The control rate should become negligible after the
30% extract of stage 4. The control rate at stage 1 was 56% of the observed total
rate. Identical control rates were given by a system lacking acetyl-CoA and by one
in which an aliquot of the enzyme preparation was preincubated with an equal
volume of avidin (3 mg/ml: specific activity 12.0). The pyruvate carboxylase
content measured at stage 1 is approximately 20% low as compared with the more
reliable assay at stage 2.
Measured by the biuret reaction except for the 22 and 20% extracts of stage 4 and
for stage 5 when the spectrophotometric method was used. The residue fraction
from stage 4 is obtained as a suspension in 0.1 M phosphate pH 7.2 and the protein
content reported for this fraction is therefore subject to some uncertainty. The
(NH,)~SO4 supernatant fraction from stage 3 after precipitation of the pyruvate
carboxylase activity contains no significant residual protein.
procedure to stage 5 are s u m m a r i z e d in T a b l e I a n d show a 34-fold
increase in specific a c t i v i t y with a 53% r e c o v e r y of e n z y m a t i c a c t i v i t y
from the m i t o c h o n d r i a l extract. U t t e r a n d K e e c h 11 h a v e shown t h a t the
,1M. F. Utter and D. B. Keech, J. Biol. Chem. 238, 2603 (1963).
specific activity of pyruvate carboxylase in chicken liver homogenates

is in the range of 0.1-0.2 unit per milligram of protein. This procedure
therefore gives approximately a 200-fold overall increase in specific
activity. The maximal specific activity of pyruvate carboxylase from
chicken liver is 35-40 units per milligram of protein under standard assay
conditions. The final yield of active enzyme is variable and the specific
activities obtained at stage 5 in various preparations range from 20-40
units per milligram of protein. The factors responsible for the variation
are not fully understood, they include the size and source of the chickens,
the yield of lyophilized mitochondria, the extent and specificity of extrac-
tion at stages 1 and 4, and the elapsed time required to complete the
procedure to stage 5. We complete the procedure described from stage 1
through stage 5 in 8-10 hours. It should be noted that the enzyme cannot
be stored prior to stage 5 without incurring major losses in enzymatic
activity.
Stability and Storage Properties of the Purified Enzyme (Stage 5)

Pyruvate carboxylase preparations stored in sucrose-buffer-salt solu-
tions as described in stage 5 may he stored for 1-2 months at 4 ° with
a loss of enzymatic activity which varies from 0 to 50~ in different
preparations. Loss of activity is most rapid during the first few days of
storage.
Pyruvate carboxylase is an unstable enzyme. The major defined condi-
tions resulting in instability follow:
1. Low ionic strength: The presence of salt to maintain an ionic
strength of 0.2-0.3 is required. Addition of (NH4)2S04 (0.05-0.1 M) or
KCI (0.2-0.3 M) is satisfactory.
2. Exposure to acid or alkaline pH: Pyruvate carboxylase in 50 mM
phosphate containing 60 mM (NH4)2S0~ is most stable in the pH range
6.8-7.3. At pH values below 6.0 rapid, irreversible inactivation occurs
accompanied by precipitation. The inactivation observed at alkaline pH
(7.5--8.5) is partially reversible on readjustment of the pH to 7.0.
3. Exposure at 2°: Pyruvate carboxylase from chicken liver is a
cold-labile enzyme; on incubation at 2 ° in 10 mM phosphate pH 7.2 con-
taining 0.2M KC], it undergoes a rapid loss of enzymatic activity
accompanied by dissociation of the 14.8 S species to yield a 6.75 S sub-
unit. This inactivation is reversible on rewarming to 23 °, but maximal
regain of active enzyme requires the addition of 5 mM ATP. 12,13 Cold
,2 M. (3. Serutton and M. F. Utter, d. Biol. Chem. 240, I (1965).
Is R. (3. Valentine, N. C~. Wrigley, M. C. Scrutton, J. J. ]rias, and M. F. Utter,
Biochemistry 5, 3111 (1966).
inactivation is prevented by the presence of 1.5 M sucrose, 4 M glycerol,

0.1 M phosphate, or 0.5 mM acetyl-CoA. Pyruvate carboxylase stored at
23 ° in 0.1 M phosphate pH 7.0 containing 60 mM (NH~)~S04 shows a
slow decrease in enzymatic activity.
Presence of Other Enzymes

The only other enzymatic activity detected thus far in stage 5 pyru-
vate carboxylase is glutamate dehydrogenase (less than 2 ~ in the best
preparations). Other enzymes of C02 fixation, other dehydrogenases,
citrate synthase, and certain enzymes of ATP metabolism, e.g., myo-
kinase, nucleoside diphosphokinase, are absent.
Physical Properties and Bound Cofactors

The enzyme purified through stage 6 is essentially homogeneous as
examined by sedimentation and electrophoretic analysis. The active
enzyme has a $2°o., of 14.8 S and the molecular weight is estimated as
660,000 by the approach to equilibrium technique. 12 The electrophoretie
mobility in 50 mM potassium phosphate pH 7.2 containing 60 mM
(NH~) 2S04 and 1 mM EDTA is 1.36 X 10-' cm 2 per second per volt at
24°. 12 The protein contains both bound biotin and bound manganese at
levels approximating to 4 moles per mole of enzyme? TM The bound
manganese is not removed by incubation with 10 mM EDTA and does
not appear to exchange with exogeneous manganese. In the electron
microscope pyruvate carboxylase is defined by negative staining as a
tetrameric molecule with 4 apparently equivalent subunits arranged at
the corners of a square. 13
Specificity
Pyruvate carboxylase is highly specific for both its nucleotide and
a-ketoacid substrates. ATP may be replaced by 2'-deoxy-ATP without
significant change i n / ~ or Vm,x, but no other nucleotides tested thus far
are active. 1' The nucleotides are used as their metal-ATP 2- complexes.
CO~ fixation on a-ketobutyrate is catalyzed at 3% of the rate found for
pyruvate. The product of this reaction is ~-methyloxaloacetate.16
Pyruvate carboxylase requires activation by free divalent metal cation
which is satisfied by the addition of Mg++, Mn ++ or Co ++. Other metal
ions are inactive and certain of them, e.g., Ca ++, Zn++, Cu ++, Cd++ are
1,M. C. Scrutton, A. S. Mildvan, and M. F. Utter, J. Biol. Chem. 241, 3480 (1966).
15M. C. Scrutton and M. F. Utter, Y. Biol. Chem. 240, 3714 (1965).
D. S. Kerr and M. F. Utter, unpublished observations, 1964.
potent inhibitors. The inhibition results either from formation of an

inactive metal ATP ~- complex (Cu +÷, Cd ÷+) o1" from binding to the site
for free metal activator (Ca÷÷). Pyruvate carboxylase has not been
examined for activation by monovalent cations.
Pyruvate carboxylase purified from chicken liver is essentially in-
active in the absence of an acyl-CoA. The most effective activators are
acetyl-, propionyl-, crotoz~yl-, and isobutyryl-CoA, but the enzyme is
also activated less effectively by formyl-, n-butyryl-, and n-valeryl-
CoA. 17,1s The enzyme from chicken liver is activated very weakly by
CoASH is in contrast to pyruvate carboxylase purified from bakers'
yeast2 9
Oxaloacetate D e c a r b o x y l a t i o n a n d E x c h a n g e R e a c t i o n s
Catalyzed by Pyruvate Carboxylase
Pyruvate carboxylase catalyzes oxaloacetate decarboxylation in the

presence of acetyl-CoA, Pi, and ADP, and also several exchange reac-
tions. The properties of these reactions and their rates (relative to the
rate of C02 fixation as 100) are summarized in Table II. The enzyme-
bound intermediate of pyruvate carboxylase has been isolated and
TABLE II
DECARBOXYLATION AND EXCHANGE REACTIONS
CATALYZED BY PYRUVATE CARBOXYLASE a
Other reactants Relative rate

Reaction required (C02 fixation = 100)
Oxaloacetate decarboxylation b ADP, phosphate, 10

Me ++, acyl-CoA
Exchange of ~C-pyruvate with None 50-75
oxaloacetatec
Exchange of np~ with ATP c ADP, HCO,-, Me ++, 0.5-1.0
acyl-CoA
Exchange of ADP-8-~C with ATP d Me ++ 0.2-0.4
° Oxaloacetate decarboxylation is assayed by measuring the rate of pyruvate pro-

duction dependent on the addition of acetyl-CoA in the presence of lactate dehydro-
genase and DPNH. The assays used to estimate the rates and properties of the
exchange reactions are described in the references indicated.
b M. C. Serutton and M. F. Utter, J. Biol. Chem. 240, 1 (1965).
c M. C. Scrutton, D. B. Keech, and M. F. Utter, J. Biol. Chem. 240, 574 (1965).
d M. C. Scrutton and M. F. Utter, J. Biol. Chem. 240, 3714 (1965).
1~D. B. Keech and M. F. Utter, J. Biol. Chem. 238, 2609 (1963).

18M. C. Scrutton aDd M. F. Utter, J. Biol. Chem. 242, 1723 (1967).
M. J. Losada, J. L. Canovas, and M. Ruiz-Amil, Biochem. Z. 340, 60 (1964).
shown to have properties consistent with its identification as F-N-car-

boxybiotin-enzyme. 2
Kinetic and Thermodynamic Parameters

Pyruvate carboxylase exhibits a sharp o p t i m u m a t p H 7.8. '~
The observed Michaelis and activator constants for C02 fixation and
oxaloacetate decarboxylation by pyruvate carboxylase are shown in Table
III. ATP and ADP, HC03- phosphate, oxaloacetate, and the divalent
TABLE III
MICHAELIS, ACTIVATOR, AND DISSOCIATION CONSTANTS FOR
SUBSTRATES AND COFACTORS OF PYRUVATE CARBOXYLASE
Reacl ant K,,/~ K A K dd
ATP 5.8 >< 10 -5 M -- 7.5 >< 10 -5 M

ADP 6.3 X 10-SM -- 1.4 >< 10-4M
HCO,~- 1.0 × 10 -3 M -- --
M g ++ -- 2 . 9 >< I O - ~ M b --
M n +÷ -- 9.4 >< 1 0 - 6 M b --
Co ++ -- 2.1 X10 -SM b --

Acetyl-CoA, -- 3.3 >< 10-~ M c --
Propionyl-CoA -- 1.1 >< 10-4 M c --
Crotonyl-CoA -- 1.35 X 10 -~ M c --
Phosphate 1.2 >< 10 -2 M -- --
Pyruvate 4.4 X 10 -~ M -- 4.6 >< 10 -3 M
a-Ketobutyrate 4.9 >< 10 -3 M -- 4.5 X 10 -3 M
Oxaloacetate 5.0 X 10 -5 M -- 2.0 X 10 -3 M
~-Methyloxaloacetate 8.0 X 10 -~ M -- 6.1 X 10-4 M
Obtained from plots of reciprocal initial rate and reciprocal substrate concentration.
b Obtained from plots of reciprocal initial rate and reciprocal free metal ion con-
centration after correction for metal ion bound to ATP.
Obtained as concentration of acyl-CoA required to give 50% maximal activation of
CO2 fixation by pyruvate carboxylase.
d Obtained from the effect of ATP, ADP, pyruvate, and oxaloacetate on the rate of
inactivation of pyruvate carboxylase by avidin or from titrations of the reduction
in enhancement of the bound manganese by pyruvate, oxaloacetate, a-ketobutyrate,
and B-methyl oxaloacetate.
" T h e Ka for acetyl-CoA may be as low as 4 X 10-6 M under optimal conditions.
The value is increased by unfavorable Mg++/ATP ratios, by high absolute con-
centrations of A T P and by unknown factors present in some samples of commer-
cial CoASH.
metal ion activator (after correction for binding by ATP or ADP)
exhibit simple Michaelis-Menten behavior. Pyruvate shows Michaelis-
Menten behavior at concentrations below 1 raM, but at higher concen-
t r a t i o n s a p p a r e n t a c t i v a t i o n is o b s e r v e d w h i c h b e c o m e s m o r e m a r k e d a t
low A T P c o n c e n t r a t i o n s . T h e a c y l - C o A s h o w s s i g m o i d b e h a v i o r . F o r t h e
nucleotide and a-keto acid substrates dissociation constants obtained by

the proton relaxation rate or avidin inactivation analysis methods are
included.lU, 2o
The equilibrium constant of the pyruvate carboxylase reaction at
pH 8.0 and a free Mg ~ concentration of 4.5 mM has been determined as
10.2. 21
Inhibitors
Three main classes of reversible inhibitors of pyruvate carboxylase
have been described: (1) nucleotides that are competitive inhibitors with
respect to ATP; TM (2) analogs of the activator acyl-CoA's which are
competitive inhibitors with respect to acetyl-CoA ;18 (3) pyruvate analogs
and many dicarboxylic acids and their derivatives which are specific
inhibitors of the transcarboxylation step of the pyruvate carboxylase
reaction [Eq. (2)] and inhibit the enzyme as a result of interaction with
the bound manganeseY° The inhibitor constants obtained for representa-
tives of each of these classes of inhibitors are summarized in Table IV.
In contrast to pyruvate carboxylase purified from yeast, 22 pyruvate
carboxylase from chicken liver is not inhibited by L-aspartate (0.2-20
raM) at either saturating or nonsaturating concentrations of acetyl-CoA.
Pyruvate carboxylase is inactivated irreversibly by incubation with
(1) avidin, due to binding of the biotin residues of the active site; (2)
sulfhydryl reagents such as p-chloromercuribenzoate and 5,5'-dithiobis (2-
nitrobenzoic acid) at concentrations less than 0.1 m M ; " and (3) denatur-
ing agents, e.g., guanidine salts, sodium dodecyl sulfate. Low concentra-
tions (0.4 M) of urea cause dissociation of the enzyme to a 6.75 S subunit
which resembles that induced by incubation at 2 ° . At higher concentra-
tions of urea (6-8M) precipitation of inactive protein is observed.
Guanidine chloride (2M) and sodium dodecyl sulfate (1%) cause
dissociation of the enzyme to smaller subunits (S2o,w = 2.7 S).1~
Tissues and Species Distribution
High levels of pyruvate carboxylase activity are found only in
kidney and liver, although some activity may be present in brain. The
enzyme is associated primarily with mitochondria in all vertebrate tissues
and species examined. Preliminary results suggest that the procedure
described for chicken liver may be applied with minor modifications to
the purification of pyruvate carboxylase from calf liver. 28 Pyruvate
~A. S. Mildvan, M. C. Scrutton, and M. F. Utter, J. Biol. Chem. 241, 3488 (1966).
H. G. Wood, J. J. Davis, and It. Lochmuller, J. Biol. Chem. 241, 5678 (1966).
u E. Palacian, G. de Torrontegui, and M. Losada, Biochem. Biophys. Res. Commun.
24, 644 (1966).
= J. C. Wallace, M. R. Olmsted, and M. F. Utter, unpublished observations, 1967.
T A B L E IV
SOME INHIBITORS OF PYRUVATE CARBOXYLASE
Inhibitor K~
1. Nucleotides ~
CTP 9.6 X 10-SM
UTP 4.5 X10 -4M
TTP 1.3 X 10 - a M
CDP 1.6 X10 -sM
5'-CMP 1.2 X~10-2 M
2. Acyl-CoA analogs b
Malonyl-CoA "8.3 X 10 -6 M
Methylmalonyl-CoA 1.0 X 10 -4"M
Acetyl-pantetheine 2 . 8 × 10-4 M
3. P y r u v a t e analogs and dicarboxylic acids*
Fluoropyruvate 1.7 × 10 -4 M
Phenylpymvate 4.8 × 10-* M
Oxalate 1.2 X 10-6M
Oxamate 1.6 × 10 -8 M
Malonate 2.2 × 10 -2 M
~Iesoxalate 2.1 × 10 -8 M
L-Malate 6.5 X 10 -~ M
Glyoxal 5.6 X 10 -* M
The initial rate of CO2 fixation was measured as a function of the concentration of
ATP.
b The initial rate of CO2 fixation was measured as a function of the concentration of
acetyl-CoA a t levels above 3 X 10 -5 M.
c The initial rate of COs fixation was measured as a function of the concentration of
p y m v a t e except for mesoxalate when the initial rate of oxaloacetate decarboxylation
as a function of oxaloacetate concentration was measured. Uncompetitive or non-
competitive inhibition was observed.
carboxylase has been purified through stage 2 from pigeon and rat liver
by procedures identical to those described here, and satisfactory specific
activities were obtained. 2' A purification method for pyruvate carboxylase
from sheep kidney cortex mitochondria has been described by Ling and
Keech? 5
2, B. R. Landau, M. C. Scrutton, and M. F. Utter, unpublished observations, 1965.

UA. M. Ling and D. B. Keech, Enzymolo¢ia 30, 367 (1966).
[39] Pyruvate C a r b o x y l a s e f r o m S a c c h a r o r n y c e s cerevisiae

By M. R. YOUNG,BERNADINETOLBERT,and M. F. UTTER
Mg ++
Pyruvate % ATP -b HCO3- ~ ~ oxaloacetate ~ ADP q- P,.
As indicated in a previous article [38], pyruvate carboxylase from
avian liver has been studied intensively and its properties well estab-
lished. Although the enzyme from yeast has not yet been examined
thoroughly, it appears to differ from the avian enzyme in several funda-
mental ways?-' Therefore, a partial purification of the yeast enzyme and
a description of some of its properties are included.
Assay Method
Principle. The most convenient method of measuring the activity of
this enzyme, as noted in the preceding article, is a spectrophotometric
assay in which oxaloacetate production is measured with malate dehy-
drogenase (EC 1.1.1.37). Other investigators have in addition used
isotopic assays based on the incorporation of 14C0~ into oxaloacetic acid.
Reagents. The reagents described below differ slightly from those
presented in the preceding article and are prepared more readily. They
have proved entirely satisfactory for the assay of enzymatic activity
during the purification procedure. All reagents are dissolved in twice-
distilled water.
Tris-S04, 0.5M, pH 7.8, prepared at 25 ° from reagent grade
Trizma Base (Sigma) adjusted to pH 7.8 with concentrated
H2SO,
Pyruvate, 0.1 M, prepared from the potassium salt (Sigma, Type
III) neutralized to pH 6.8 with 1 M KOH
Disodium ATP, 16.5 mM, (Sigma, crystalline from equine muscle)
neutralized to pH 7.0 with 1 M KOH
KHCO~, 0.2 M
MgS04, 0.134 M
D P N H , 3.2 mM, disodium salt (Sigma Grade III)
' M. Losada, J. L. Canovas, and M. Ruiz-Amil, Biochem. Z. 340, 60 (1964).
2j. Gailiusis, R. W. Rinne, and C. R. Benedict, Biochim. Biophys. Acta 9'2, 595
(1964).
:'J. J. Cazzulo and A. O. M. Stoppani, Biochim. Biophys. Acta I00, 276 (1965).
4M. R. Young, Bernadine Tolbert, and M. F. Utter, unpublished observation, 1966.
[39] PYRUVATE CARBOXYLASE FROM S . cerevisiae 251
Malate dehydrogenase (Boehringer, Mannheim, 10 mg/ml) diluted

in 0.1% bovine serum albumin to give a solution of 100 units/ml.
This reagent is prepared fresh every other day.
Acetyl-CoA, 1.7 raM, prepared from acetic anhydride and CoASH
(P-L Biochemicals Inc.) by a modification of the procedure of
Simon and Shemin 5 and assayed spectrophotometrically as de-
scribed by Ochoa 6
Avidin (purchased from Nutritional Biochemicals Corporation),
prepared as a suspension of 7.7 mg per milliliter of water
For convenience, the first three reagents are combined (5 ml of
pyruvate, 10 ml of Tris-S04, and 10 ml of ATP). Aliquots of this mixture
are stored at --10 ° for as long as 1 week.
Procedure. Table I lists the amount of each reagent used to prepare 1
TABLE I
SPECTROPHOTOMETRIC ASSAY OF PYRUVATE CARBOXYLASE
Micromoles per
milliliter of Volume per milliliter
Component assay solution of assay solution
Pyruvate 10 0.50 ml of the

combined mixture
Tris-SO~ 100 --
ATP 3.3 --
KHCO3 20 0.10
MgSO, 6.7 0.05
Malate dehydrogenase -- 0,05
DPNH 0.16 0~ 05
Acetyl-CoA 0. 083 0.05
Enzyme plus water -- 0.20
ml of assay solution. All assays are started by the addition of enzyme

to the temperature-equilibrated assay mixture in the cuvette, Although
acetyl-CoA is not required by pyruvate carboxylase from yeast, its addi-
tion to the assay increases the observed reaction rate by 50-150%. Assays
are performed routinely with and without acetyl-CoA at each stage in
the purification. All calculations of units and specific activities are based
on assays containing acetyl-CoA.
Prior to stage 3 in the purification procedure there is marked con-
tamination of the preparations with other DPNH-utilizing systems. The
most effective control is obtained with enzyme which has been pre-
incubated for 10-15 minutes with an equal volume of the avidin suspen-
~See Vol. III [137].
aS. Ochoa, Biochem. Prep. 5, 19 (1957).
sion described above. Under these conditions the rate obtained after
inactivation of the enzyme by avidin may be as much as 75% that of
untreated enzyme. Therefore only an approximate estimate of the pyru-
vate carboxylase content of crude preparations can be made.
Units. Units are expressed as mieromoles of D P N H oxidized per
minute at 25 ° and pH 7.8. Specific activities are expressed as units per
milligram of protein per minute. After stage 3, protein is routinely
measured spectrophotometrically/ Less pure preparations contain ex-
traneous 260 m~ absorbing material, and the biuret method 8 is used.
Pyruvate carboxylase has been purified from commercial bakers'
yeast, 1-s but the enzymatic content of this material is low and the final
specific activities obtained have been correspondingly low. We have
found that yeast cells cultured aerobically on the medium described below
have a greatly enhanced pyruvate earboxylase content when lactate is
used as the carbon source. Crude extracts prepared from cells grown in
this manner have specific activities up to 40-fold those of commercial
bakers' yeast2 Cells grown in the same medium, but with glucose in
place of lactate as the carbon source, contain approximately one-third
as much pyruvate carboxylase.
Growth o/Cells. 8accharomyces cerevisiae (Harden and Young strain)
is maintained on agar slants of the following composition:
Glucose 5.0% (w/v)

Difco Bacto-Peptone 0.5%
Difco yeast extract 1.0~
KHsPO4 0.0%
Agar 2.0%
The composition (per liter of solution) of the subculture and growth

medium is found below.
Subculture material is prepared from slants inoculated 24 hours before
use and incubated at 30 °. A separate slant is used to inoculate each 3-liter
Fernbach flask (5 in all) containing 650 ml of the medium described
above. The subcultures are grown aerobically with vigorous shaking on a
rotary flask agitator for 16 hours at 30% Approximately 80 ml of sub-
culture material is transferred aseptically to each of 32 flasks (also
1See Vol. I I ! [73]. The factors 1.55 × A~0 -- 0.76 × Am are used.
'See Vol. I I I [73].
' M. Ruiz-Amil, G. de Torrontegui, E. Palaeian, L. Catalina, and M. Losada, J. Biol.
Chem. 240, 3485 (1965).
[39] PYRUVATE CARBOXYLASEFROM S. cerevisiae 253
Bacto-Peptone 5.0 g
Yeast extract 25.0 g
Na lactate (60%) 5 ml
CaCl~ 0.214 g
MgSO, 0.122 g
(NH4)2S04 6.0 g
KHsPO4 2.0 g
Ergosterol 0.012 g (in 3 ml absolute ethanol)
Tween-80 2.64 ml
Wheat germ oil 0.25 ml
containing 650 ml) to give an inoculum of 1 g of cells per liter of medium.

The flasks are then agitated as described above for 16.5--17 hours at 30 °.
The yield of cells is approximately 11 g per liter to give a total of 230-
250 g (wet weight) of cells. This represents a convenient amount of
yeast to carry through the purification procedure described below.
Harvesting and Washing. The cells are harvested at room tempera-
ture by means of a Sharples Super Centrifuge and then suspended in
twice their weight of cold (4 ° ) glass-distilled water. The suspension is
centrifuged at 1000 g for 20 minutes, and the supernatant liquid dis-
carded. The cells are washed again and stored overnight at 4 ° for use
the next day.
Reagents and Materials. All reagents are dissolved in water distilled

once from a metal still and a second time from glass.
(NH,),SO, (purchased from Mann Research Laboratories Inc.,

Enzyme grade), twice recrystallized from 10 mM EDTA at
alkaline pH. Percent saturations are calculated at 25 ° using the
data of Green and Hughes 1°
Tris base: trimethylolaminomethane (Sigma, reagent grade Trizma
Base)
EDTA: disodium ethylenediaminetetraacetate (Sigma, ED2SS)
DTE: 2,3-dihydroxy-l,4-dithiobutane (Sigma, dithioerythritol)
KC1 (Baker analyzed reagent)
Protamine sulfate (purchased from Nutritional Biochemicals Cor-
poration), prepared at 25 ° as a 2% suspension in 0.05 M Tris
buffer (pH 7.2)
Sephadex G-25, Pharmacia Fine Chemicals, Uppsala, Sweden. The
Sephadex was swollen and the columns prepared according to
Porath and Flodin21 All void volumes were determined using
I, See Vol. I 110].

uj. Porath and P. Flodin, Natt~re 183, 1657 (1959).
Blue Dextran
Cellulose-phosphate (Carl Schleicher and Schuell Co., Keene, New
Hampshire), prepared according to Peterson and Sober 12
Sagarose 8 (purchased from Gallard-Schlesinger Chemical Manu-
facturing Corporation). The gel was suspended in buffer and
poured into the column as a thick slurry
Unless otherwise stated, all the following steps are carried out at 4 °
in 50 mM Tris-C1 (pH 7.2) containing 5 mM EDTA and 0.1 mM DTE.
Stage 1. Preparation of the Crude Extract. Cells of Saccharomyces
cerevisiae (250 g) suspended in 500 ml of 50 mM Tris-HC1 buffer, pH
7.8, containing 5 mM EDTA, 0.1 mM DTE, and 1 mM MgCI~ are added
to 500 g of glass beads (Superbrite, Type 130-washed in EDTA) in an
Eppenbach colloid mill operated at 9 ° and run continuously at maximum
speed for 25 minutes. The chamber of the mill is rinsed with 100 ml of
the same buffer and the combined rinse plus crude extract is centrifuged
at 10,000 g for 10 minutes. The cloudy supernatant liquid (pH 6.3) is
adjusted to pH 7.2 with 1 M Tris base and recentrifuged until clear.
Stage 2. Heat Denaturation. The crude extract from above (in 100
ml aliquots) is heated rapidly with swirling in a 300-ml Erlenmeyer flask
to 49 ° and transferred immediately to a 50 ° water bath for 2 minutes.
The solution is cooled to 5 ° in a --70 ° (dry ice-ethanol) cooling bath.
The precipitate is removed by centrifugation at 35,000 g for 40 minutes
and discarded.
Stage 3. Protamine Sulfate Treatment. A 2% suspension of protamine
sulfate in water is added (800 mg total per 250 g of cells) to the super-
natant fraction from stage 3 with the pH maintained at 7.2 with 1 M Tris
base. A voluminous precipitate forms which is removed by centrifuga-
tion at 10,000 g for 20 minutes.
Stage 4. Ammonium Sulfate Fractionation. The clear supernatant
fraction (510 ml) from stage 3 is taken to 45% saturation by the
addition of 141 g of solid ammonium sulfate. The precipitate, which
contains all the pyruvate carboxylase activity, is collected by centrif-
ugation at 10,000 g for 30 minutes and the supernatant liquid is dis-
carded. The precipitate is transferred to a 50 ml polycarbonate ccntrifuge
tube and extracted successively with a series of solutions of decreasing
ammonium sulfate concentration. All extracting solutions are prepared
at 4 ° by dissolving solid ammonium sulfate in the standard Tris buffer
and readjusting the pH to 7.2 with 1 M Tris Base. The solutions, used
in the order given, are 14 ml 45%, 8 ml 35%, 8 ml 30%, 4 ml 30%, 8 ml
25%, and 4 ml 25%. The precipitate is kept suspended with a magnetic
1:See Vol. V [1].
[39] PYRUVATE CARBOXYLASE FROM S . cerevisiae 255
stirrer for 10 minutes and the supernatant liquid is recovered by centrif-

ugation at 35,000 g for 10 minutes. The magnetic stirring bar is left in
the centrifuge tube during centrifugation to prevent transfer losses. The
supernatant liquid is decanted and the residual precipitate is suspended
in the next extracting solution, P y r u v a t e carboxylase of high specific ac-
tivity is found primarily in the two 30% extractions (see Table I I ) .
Occasionally the specific activity of either the 35% or the first 25%
extraction is high as well.
Stage 5. Sephadex G-~5. The extractions from stage 4 arc turbid and
are usually stored overnight at 4°; during this time further precipitation
occurs. The appropriate extractions are combined the following morning
TABLE II
PURIFICATION OF YEAST PYRUVATE CARBOXYLASE
Pyruvate Specific
Volume carboxylase Protein activity Recovery
Purification stage (ml) (total units) (mg) (units) (%)
1. Crude extract 560 780 11,435~ 0.07 100

2. Heat denaturation 520 810 7,722 a 0 11 104
3. Protamine sulfate 510 820 4,794 a 0.17 105
treatment
4. Ammonium sulfate
fractionation
45% 14.1 -- 120 -- --
35% 8.1 166 94 1.77 --
30% (I) 8.2 366 126 2.90 --
30% (II) 4.1 169 56 3.02 69
250/0 (I) 8.2 93 154 0.60 --
25% (II) 4.1 36 85 0.42 --
5. Sephadex G-25 11.0 510 150 3.40 65
6. Cellulose phosphate 1.2 300 49 6.12 39
7. Sagarose 8b 4.8 194 7.7 25.0 25
Determined according to the biuret method (see Vol. III [73]). Occasional prepara-
tions contain large amounts of 260 m~ absorbing material throughout the prepara-
tion, and the spectrophotometric method cannot be used.
bValues in this line have been calculated from another experiment.
and centrifuged at 35,000 g for 15 minutes. The clear supernatant liquid
(approximately 12 ml) is taken to 40% saturation with solid ammonium
sulfate. The resulting precipitate is collected by centrifugation at 35,000
g for 15 minutes, and the pellet is dissolved in 2 ml of standard buffer.
The concentrated enzyme is applied to a 1 X 25 cm column of Sephadex
G-25 equilibrated with the standard Tris buffer and then eluted with the
same buffer at a flow rate of 1 ml per minute. Fractions of 1.0-1.5 ml
are collected. P y r u v a t c carboxylase appears as soon as one void volume
has passed through the column and is eluted in a volume of 8-12 ml.
The active fractions are turbid and are pooled and centrifuged for 10
minutes at 35,000 g. Pyruvate carboxylase is recovered in the super-
natant fraction with very littJe loss of activity from stage 4 and with
a slight increase in specific activity (see Table II).
Stage 6. Cellulose Phosphate. The pooled fractions from stage 5. are
applied to a column (2 X 30 cm) of cellulose phosphate equilibrated with
the standard Tris buffer. Elution is carried out with the same buffer
at a flow rate of 0.5 ml per minute. Pyruvate carboxylase is not adsorbed
and appears in the effluent as soon as one volume (70 ml) of buffer has
passed through the column. Two milliliter fractions are collected and the
enzyme is recovered in approximately 22 ml. 0nly those fractions with
a specific activity greater than 5 are combined (14 ml) and carried
through the next step. The pooled fractions are taken to 50% saturation
with solid ammonium sulfate and centrifuged at 35,000 g for 15 minutes.
The precipitate is dissolved in a minimum volume of 50 mM Tris-C1
(pH 7.2) containing 5 mM EDTA, 0.1 mM DTE, 0.2 M KC1, and 1%
ammonium sulfate.
Stage 7. Sagarose 8. The concentrated enzyme from stage 6 is applied
to a 1.1 X 57 cm column of Sagarose 8 equilibrated with 50 mM Tris-Cl
buffer (pH 7.2) containing 5 mM EDTA, 0.1 mM DTE, and 0.2M
KCI. The enzyme is eluted with the same buffer using a flow rate of 4-5
ml per hour and is collected in 0.5-1.0 ml fractions. Pyruvate carboxylase
is retarded on this column and appears after the recovery of 55 ml of
buffer. Most of the activity is found in the 55-85 ml fraction, with the
bulk of the contaminating protein appearing in earlier fractions. The
fractions of highest specific activity are pooled, and the protein is
precipitated with solid ammonium sulfate. The precipitate is dissolved in
50 mM Tris-C1 buffer (ptt 7.2) containing 5 mM EDTA, 0.1 mM DTE,
and 1 M sucrose.
Purification Results, Stability and Storage. The results of a typical
purification are summarized in Table II and show a 350-fold increase in
specific activity over the crude extract. The specific activity obtained at
stage 7 is generally 25 but has been as high as 30. Procedures reported in
the literature show 130-fold 9 (specific activity 0.2) 25-fold/'~ and 80-
fold 1. purifications.
Enzyme purified through stage 4 can be kept for 2 weeks at 4 ° or
2 days at room temperature with 20% loss of activity. Enzyme purified
through stage 7 loses 30% of its activity in 15 days when stored in Tris
Is T. G. Cooper and C. R. Benedict, Biochem. Biophys. Res. Commun. 22, 285 (1966).
'* E. Palacian, O. de Torrontcgui, and M. Losada, Bioehem. Biophys. Res. Commun.
24, 644 (1966).
[39] PYRUVATE CARBOXYLASE FROM S. cerevisiae 257
buffer containing 1 M sucrose. After this initial loss of activity the en-
zyme appears to be stable for as long as 2 months.
Unlike pyruvate carboxylase from avian liver, the yeast enzyme
prepared as described above does not appear to be cold labile. Ruiz-Amil
et al2 state, however, that pyruvate carboxylase from commercial
bakers' yeast undergoes greater inactivation at 0 ° than at 22 °.
Physical Properties
Pyruvate carboxylase from yeast contains biotin as demonstrated by
the specific inhibition of the enzyme by avidin2 Enzyme purified through
stage 7 appears homogeneous in the ultracentrifuge. The sedimentation
coefficient' at 20 ° (not extrapolated to infinite dilution) of this material
is 15.6 (1 mg of protein per milliliter).
Inhibitors
Pyruvate carboxylase from yeast is inhibited irreversibly by incuba-
tion with avidin 2,9 and 0.1 mM p-chloromercuribenzoate. 2 The enzyme is
also inhibited by oxalate, ~ sodium ions, 9 and aspartate" (K~ = 1.9 X
10-4). Inhibition by aspartate is especially interesting since it appears to
be sigmoid with respect to aspartate concentration. It is noteworthy that
aspartate is completely inert with the avian enzyme.
Activators
Pyruvate carboxylase is stimulated by potassium ions 9 and reduced
glutathione. 2 In contrast to the enzyme from avian liver, which has an
absolute requirement for acetyl-CoA 15 and the enzyme from Pseudo-
monas, which is not activated by acetyl-CoA," pyruvate carboxylase
from yeast shows an appreciable rate in the absence of acyl-CoA com-
pounds and an increased rate in their presence. In addition, the speci-
ficity for acyl-CoA cofactors appears to be much broader for the yeast
enzyme than for avian pyruvate carboxylase. For example, the yeast
enzyme is activated by benzoyl-CoA,4 which is entirely inert with the
avian enzyme" and by methylmalonyl-CoA ' which is an inhibitor
(against acetyl-CoA) of the avian enzyme." Coenzyme A is about 75%
as effective as acetyl-CoA for the yeast enzyme ~-8 but is essentially in-
active with the liver enzyme." The most effective activator found to date
is palmityl-CoA. 4
Kinetic Properties
The optimum pH of this enzyme is 8.32 Ruiz-Amil et al? report
'~ D. B. Keec'h and M. F. Utter, J. Biol. Chem. 238, 2603 (1963).
16W. Seubert and U. Remberger, Biochem. Z. 334, 401 (1961).
"M. C. Scrutton and M. F. Utter, J. Biol. Chem. 242, 1723 (1967).
258 R E A C T I O N S L E A D I N G TO A N D F R O M T H E CYCLE [40]
apparent Michaelis constants for pyruvate (0.8 raM), HC03- (2.7 raM),
ATP (0.24 mM), and Mg+*~ (4.2 mM). Gailiusis et al. ~- have reported
that the yeast enzyme carries out a pyruvate-oxaloacetate exchange
reaction similar to that reported for pyruvate carboxylase from avian
liver. 18 This exchange reaction is inhibited by avidin and p-chloro-
mercuribenzoate and does not require acetyl-CoA, ATP, Mg ~, or reduced
glutathione. Cooper and Benedict12 report that the addition of acetyl-
CoA to this enzyme results in a lower apparent K~ value for bicar-
bonate. No significant change in the K,~ values for the other substrates
was detected.
i, M. C. Scrutton, D. B. Keech, and M. F. Utter, J. Biol. Chem. 240, 574 (1965).
[40] P y r u v a t e Carboxylase from Pseudomonas

By W. SEVBERT and H. WEICKER
Pyruvate q- CO2 A- ATP ~ oxaloacetate -k ADP A- P~ (1)
Assay Method
Principle. Pyruvate carboxylase is assayed by coupling the carboxyla-
tion of pyruvate [Eq. (1)] with the reduction of the oxaloacetate formed
to malate using NADH and malate dehydrogenase [Eq. (2)].~
Oxaloacetate -4- NADH q- H + ~ malate -b NAD + (2)
Pyruvate -4- CO2 -4- ATP A- NADH -4- H + --~
malate + ADP -4- P~ q- NAD+ (3)
Under the condition of the assay (see procedure), the equilibrium of
the overall reaction [Eq. (3)] is in favor of malate. The amount of
pyruvate earboxylated is equivalent to the amount of NADH oxidized.
With an excess of malate dehydrogenase (see procedure), the rate of
NADH-oxidation is proportional to the concentration of pyruvate ear-
boxylase. The oxidation of NADH is followed spectrophotometrically at
366 mg (Eppendorf photometer).
Reagents
Tris (hydroxymethyl) aminomethane-HC1 buffer, (Tris-HCl), 0.1 M,
pH 7.2
' W . Scubert and U. :Remberger, Biochem. Z. 334, 401 (1961).
258 R E A C T I O N S L E A D I N G TO A N D F R O M T H E CYCLE [40]
apparent Michaelis constants for pyruvate (0.8 raM), HC03- (2.7 raM),
ATP (0.24 mM), and Mg+*~ (4.2 mM). Gailiusis et al. ~- have reported
that the yeast enzyme carries out a pyruvate-oxaloacetate exchange
reaction similar to that reported for pyruvate carboxylase from avian
liver. 18 This exchange reaction is inhibited by avidin and p-chloro-
mercuribenzoate and does not require acetyl-CoA, ATP, Mg ~, or reduced
glutathione. Cooper and Benedict12 report that the addition of acetyl-
CoA to this enzyme results in a lower apparent K~ value for bicar-
bonate. No significant change in the K,~ values for the other substrates
was detected.
i, M. C. Scrutton, D. B. Keech, and M. F. Utter, J. Biol. Chem. 240, 574 (1965).
[40] P y r u v a t e Carboxylase from Pseudomonas

By W. SEVBERT and H. WEICKER
Pyruvate q- CO2 A- ATP ~ oxaloacetate -k ADP A- P~ (1)
Assay Method
Principle. Pyruvate carboxylase is assayed by coupling the carboxyla-
tion of pyruvate [Eq. (1)] with the reduction of the oxaloacetate formed
to malate using NADH and malate dehydrogenase [Eq. (2)].~
Oxaloacetate -4- NADH q- H + ~ malate -b NAD + (2)
Pyruvate -4- CO2 -4- ATP A- NADH -4- H + --~
malate + ADP -4- P~ q- NAD+ (3)
Under the condition of the assay (see procedure), the equilibrium of
the overall reaction [Eq. (3)] is in favor of malate. The amount of
pyruvate earboxylated is equivalent to the amount of NADH oxidized.
With an excess of malate dehydrogenase (see procedure), the rate of
NADH-oxidation is proportional to the concentration of pyruvate ear-
boxylase. The oxidation of NADH is followed spectrophotometrically at
366 mg (Eppendorf photometer).
Reagents
Tris (hydroxymethyl) aminomethane-HC1 buffer, (Tris-HCl), 0.1 M,
pH 7.2
' W . Scubert and U. :Remberger, Biochem. Z. 334, 401 (1961).
[40] PYRUVATE CARBOXYLASE FB.OM Pseudomo~as 259
Magnesium chloride, 0.1 3I

Sodium pyruvate, 0.1 M
Potassium bicarbonate, 0.1 M
NADH, 10 mM
ATP, 0.1 M, pH 7.2
Serum albumin, 50 mg/ml
Malate dehydrogenase, 0.5 mg sust)endcd in 1 ml of 2.8 M am-
monium sulfate solution; spccific activity 720 units/~mg
P~'ocedure. The reaction mixture (in 3 ml cuvettes, d---- 1 cm) con-
tains: buffer, 1.1 ml; magnesium chloride, 0.15 ml; potassium bicarbonate,
0.2 ml; pyruvate, 0.02 ml; NADH, 0.05 nil; ATP, 0.02 ml; serum albumin,
0.02 ml; and malate dehydrogenase, 0.005 ml; T = 30 °.
The reaction is initiated by the addition of pyruvate carboxylase. In
order to compensate for the activities of lactate dehydrogenase (Eq. 4)
present in crude extracts, the readings of the optical density arc taken
against a blank containing all components except ATP.
Pyruvate -t- NADH + H + ~- lactate + NAD + (4)
Under these conditions, the decrease of the optical density is linear
during the first 3-4 minutes. This decrease is used to calculate the enzyme
activity.
Units. One unit is defined as the amount of enzyme catalyzing the
oxidation of 1 micromole of N A D H per minute under the standard condi-
tions. Specific activity is expressed as units per milligram of protein. The
molecular extinction coefficient of N A D H at 366 mu is taken as 3.3 X 10'~
(cm2/mole).2 Protein is calculated from the 280/260 absorption. 3'~
Growth o] Cells. The organism is aerobically grown at 30 ° in a
medium containing the following components per liter: NH4-acetate,
4.4 g; Ko.HP04, 9.45 g; KH2P04, 2.72 g; NH~NOa, 1.0 g; MgS04.7 H~O,
0.2 g; CaC12.2 H20, 0.1 g; FeSO~.7 H_oO, 0.1 g; NH~-molybdate, 0.6 rag;
MnSO4.H20, 0.6 mg.
Stock cultures containing 0.05% citronellic acid instead of ammonium
acetate are stored at 2 ° on 2% agar slants. Cultures are transferred
monthly.
For large-scale production, the organism is cultured in 15 liter volumes
in 20 liter carboys. The medium is inoculated with a 500 ml inoculum
obtained by two successive subcultures in the liquid medium. The large-
scale cultures are aerated with sterile air; after 36 hours of growth, the
pH is adjusted to 7.2-7.5 every 12 hours by the addition of 5-10 ml of
glacial acetic acid. The cells are harvested by sedimentation in a Sharples
2 H. J. l=[ohorst, Biochem. Z. 328, 509 (1956).
a 0. Warburg and W. Christian, Biochem. Z. 310, 384 (1941).
' E. Layne, Vol. III, p. 447.
centrifuge after 3-4 days of growth. The cell pellet is washed twice with
0.1 M Tris-HC1 buffer, pH 7.2, and stored at --15 °. Average yield:
3.0-3.5 g, wet weight, per liter after centrifugation for 40 minutes at
40,000 g.
The purification of pyruvate carboxylase from Pseudomonas has been
described previously. 1 Improvements of the purification procedure de-
veloped more recently are included here. All operations are performed
at 0 °.
Step 1. Extraction. The cell pellet (100 g) is suspended in 200 ml of
0.1 M Tris-HCl buffer, pH 7.2, containing 10 mM glutathione and 1 mM
EDTA. In aliquots of 20-30 ml, the cells are disrupted by two subsequent
treatments with ultrasonic vibration for 1 minute (75 Watt/cm, ~ 20
KHz). In between, the solutions are cooled for 1 minute to avoid warm-
ing up above 5 ° to 7 ° . The suspension is centrifuged for 30 minutes at
37,000 g, and the residue is extracted with another 100 ml of the same
medium. Insoluble material is again separated by centrifugation at
37,000 g. Volume of the combined supernatants: 300-320 ml.
Step g. Heat Inactivation. The crude extract (in a l-liter Erlenmeyer
flask) is heated in a water bath at 90-95 ° (5 liters) to a temperature of
55 ° within 2-2~/~ minutes. After transfer to another water bath at 55 °,
the extract is kept for an additional 2 minutes at this temperature, and
is cooled subsequently to 5 ° within 2 minutes in a ice-salt mixture.
Denatured protein is separated by centrifugation at 37,000 g for 10
minutes. Volume of the filtrate: 240-260 ml.
Step 3. Protamine SulIate Precipitation. Under mechanical stirring,
20 ml of 2% protamine sulfate is added to the filtrate resulting from the
heat inactivation. After 10 minutes, the precipitate is separated by cen-
trifugation and discarded.
Step 4. Precipitation with Ammonium SuIIate. The filtrate of the
protamine sulfate precipitation is brought to 5 5 ~ saturation with solid
ammonium sulfate (32.6 g/100 ml). The salt is added slowly over a
period of 30 minutes with mechanical stirring. After the addition of
ammonium sulfate, the mixture is stirred for another hour. The precipi-
tate is isolated by centrifugation for 20 minutes at 37,000 g.
Step 5. Fractionation with Saturated Ammonium Sul]ate Solution.
The precipitate from the last step is dissolved in 0.1 M Tris-HC1 buffer
(10 mM glutathione and 1 mM EDTA), pH 7.2, in order to obtain a
final protein concentration of 10 mg/ml. For each 100 ml of the solution,
67 ml of ammonium sulfate solution (saturated at 2 °) is added slowly
over a period of 30 minutes under mechanical stirring. The precipitate is
separated by centrifugation (10 minutes at 37,000 g) after stirring for an
[40] PYRUVATE CARBOXYLASEFROM Pseudomonas 261
additional half an hour; it is discarded. Most of the pyruvate carboxylase

activity is precipitated by the addition of 11 ml of saturated ammonium
sulfate solution to each 100 ml of the supernatant under the same condi-
tions as described above. The precipitate is then sedimented and stored
overnight at --15 ° .
Step 6. Chromatography o7~ DEAE-Sephadex A 50. The precipitate is
taken up in 5-10 ml of 50 mM Tris-HC1 buffer, pH 7.2, and dcsalted by
passage through a Sephadex G-25 column (3 X 30 cm). Separation of
inactive proteins is achieved by subsequent chromatography on DEAE-
Sephadex A-50 (capacity: 3.5 ± meq/g; particle size: 40-120 tt; columr~
size: 2 X 35 cm) equilibrated with 50 mM Tris-HCl buffer, pH 7.2.
After the protein solution has been added to the DEAE-Sephadex
column two inactive protein peaks are eluted by washing the column with
200-300 ml (4-6 hours) of 50 mM Tris-HC1 buffer, pH 7.2, and 500-800
ml (overnight) of 0.167M Tris-HCl buffer of the same pH. A small
portion of pyruvate carboxylase is eluted by subsequent washing with
0.2 M Tris-HC1 buffer, pH 7.2 (0.02-0.03 enzyme units/ml). When the
specific activity of pyruvate carboxylase has reached a value of 1.0-1.2
(generally after 200-300 ml of 0.2M Tris-HC1 buffer), most of the
enzyme is eluted with 0.25 M Tris-HCl buffer, pH 7.2. Generally, 60%
of the enzymatic activity appears in the first two fractions (each 20-25
ml) of the 0.25 M Tris-HC1 eluate.
To concentrate pyruvate carboxylase, the combined fractions are
saturated with ammonium sulfate to about 90-95% and centrifuged after
half an hour for 30 minutes at 40#00 g. Generally, 30% of the activity
is lost by this procedure.
Properties
Stability. Pyruvate carboxylase from Pseudomonas is an unstable
enzyme. Dilute solutions of the enzyme lose activity within 15 hours at
0% The enzyme is usually kept at --15 ° in 0.1 M Tris-HC1 buffer (+10
mM glutathione), pH 7.2, saturated with 60% ammonium sulfate. Under
these conditions, the enzyme loses about 50% of its activity during onc
month.
Activators and Inhibitors. Pyruvate carboxylase contains biotin. The
identity of biotin with the prosthetic group of the carboxylase could be
proved by the specific inhibition of the enzyme by avidin2 In contrast
to the mammalian pyruvate carboxylase ~ and the enzyme from yeast, ~
pyruvate carboxylasc from P.~eudomona.~ i.~ not activated by acetyl-CoA.
'D. B. Keet.h a~ld M. F. Utter, J. Biol. Chem. 238, 2603 (1963).

"J. Gailiusis, R. W. Rinne, and C. R. Benedict, Bioctzim. Biopttys. Acta 92, 595
(1964).
The enzyme is activated by Mg**; this activation is saturated at 4.6
mM. The enzyme is inhibited by 0.1 m M Cialit [2-(ethylmercurimer-
eapto) benzoxazole-5-carbonic acid], 0.1 m M oxalate and 10 m M oxamide
(oxalic acid monoamide), p H 7.0.
Kinetic Properties. The apparent Michaelis constants for A T P and
bicarbonate are 0.11 m M and 2.2 mM, respectively.
PURIFICATION OF PYRUVATE CARBOXYLASE FROM Pseudomonas
Protein Enzyme Specific activity

Fraction (mg) units a (units/mg)
Crude extracO 9,900-11,250 380-430 0.037-0.042

Filtrate of heat 3,650-3,980 252-330 0. 068-0.09
inactivation
Filtrate of protamine 3,160-3,540 249-312 0.079-0.09
sulfate precipitation
Precipitate with solid 970-1,280 253-315 0.23-0.33
ammonium sulfate
Fractionation with saturated ammonium sulfate solution
Fraction from 260-308 120-194 0.4-0.74
40-46% saturation
(ratio 280/260 > 1.03)
Chromatography on DEAE-Sephadex A 50
Eluate with 0.25 M Tris-HC1 buffer pH 7.2
First fraction 6.4-7.4 46-57 6.2-8.8
Second fraction 11.0-16.5 25--60 2.3-3.6
One unit of enzyme is required to catalyze the oxidation of 1 micromole of NADH
per minute under standard conditions.
From 100 g of wet cells.
[41] Malate-Lactate Transhydrogenase from

Micrococcus lactilyticus
By S. H. G. ALLEN
COO- COO-
l I
CH~ CH3 CH~ CH3
r i t [
C=O +CHOH ~ CHOH+C-~O
I J [ I
COO- COO- COO- COO-
Oxaloacetate l,-Lactate L-Malate P y r u v a t e
Assay M e t h o d
Principle. The transhydrogenase is readily reversible, and thus
The enzyme is activated by Mg**; this activation is saturated at 4.6
mM. The enzyme is inhibited by 0.1 m M Cialit [2-(ethylmercurimer-
eapto) benzoxazole-5-carbonic acid], 0.1 m M oxalate and 10 m M oxamide
(oxalic acid monoamide), p H 7.0.
Kinetic Properties. The apparent Michaelis constants for A T P and
bicarbonate are 0.11 m M and 2.2 mM, respectively.
PURIFICATION OF PYRUVATE CARBOXYLASE FROM Pseudomonas
Protein Enzyme Specific activity

Fraction (mg) units a (units/mg)
Crude extracO 9,900-11,250 380-430 0.037-0.042

Filtrate of heat 3,650-3,980 252-330 0. 068-0.09
inactivation
Filtrate of protamine 3,160-3,540 249-312 0.079-0.09
sulfate precipitation
Precipitate with solid 970-1,280 253-315 0.23-0.33
ammonium sulfate
Fractionation with saturated ammonium sulfate solution
Fraction from 260-308 120-194 0.4-0.74
40-46% saturation
(ratio 280/260 > 1.03)
Chromatography on DEAE-Sephadex A 50
Eluate with 0.25 M Tris-HC1 buffer pH 7.2
First fraction 6.4-7.4 46-57 6.2-8.8
Second fraction 11.0-16.5 25--60 2.3-3.6
One unit of enzyme is required to catalyze the oxidation of 1 micromole of NADH
per minute under standard conditions.
From 100 g of wet cells.
[41] Malate-Lactate Transhydrogenase from

Micrococcus lactilyticus
By S. H. G. ALLEN
COO- COO-
l I
CH~ CH3 CH~ CH3
r i t [
C=O +CHOH ~ CHOH+C-~O
I J [ I
COO- COO- COO- COO-
Oxaloacetate l,-Lactate L-Malate P y r u v a t e
Assay M e t h o d
Principle. The transhydrogenase is readily reversible, and thus
[41] MALATE-LACTATE
TRANSHYDROGENASE 263
enzyme activity can be assayed in either direction2, ~ The specific activ-

ity of the enzyme is 50% higher with L-malate and pyruvate as sub-
strates, than it is with L-lactate and oxaloaeetate. Routinely, then be-
cause of the higher activity obtained and because of the relative
instability of oxaloacetate solutions, the enzyme is assayed with L-
malate and pyruvate as substrates. Two assay procedures were used.
The direct assay depends upon the increase in absorbance at 258 m~ duc
to oxaloacetate formation. A molar extinction coefficient for oxaloacetate
was determined experimentally to be E25s = 8.4 X 102 M -~ cm-'. Initial
rates with this assay are linear with time and enzyme concentration. The
reverse reaction, i.e., the disappearance of oxaloacetate, can also be
measured with this type of assay. The indirect assay, employing N A D H
oxidation, which was approximately 7 times more sensitive than the
direct assay, measured the formation of oxaloacetate from L-malate and
pyruvate by coupling the transhydrogenase with the malate dehydro-
genase.
transhydrogenase
L-Malate -t- pyruvate oxaloacetate + L-lactate
Oxaloacetate -t- N A D H + H + , - L-malate + NAD +
Net: P y r u v a t e + N A D H -4- H + ~ L-lactate + NAD +
Initial rates with this assay were also linear with time and enzyme
concentration. The reverse reaction, i.e., the appearance of pyruvate from
oxaloacetate and L-lactate, can also be measured by this type of assay
except that lactate dehydrogenase rather than malate dehydrogenase is
used. With all these assays one unit of enzyme is defined as that amount
catalyzing the oxidation of 1 micromole of either L-malate or L-lactate
per minute.
Direct Assay
Reage~ts
1. Tris-HC1 buffer, 0.5 M pH 7.8
2. Sodium pyruvate, 0.1 M (Sigma Chemical Co.)
3. Tris-L-malate, 0.2 M (Sigma Chemical Co.)
4. Distilled water
5. Malate-lactate transhydrogenase
Procedure. The reagents listed above are added to a 0.5 ml quartz
speetrophotometric cell (1 em light path) in the following order and
~M. I. Dolin, E. F. Phares, and M. V. Long, Biochem. Biophys. Res. Commun. 21,
303 (1965).
~'S. H. G, Allen, J. Biol. Chem. 241, 5266 (1966).
amounts: Tris-HCl buffer, 0.05 ml; sodium pyruvate, 0.10 ml; Tris-L-
malate, 0.05 ml; distilled water, 0.04 ml; malate-lactate transhydro-
genase, 0.01 ml of an appropriate dilution.
Usually larger but proportional volumes of reagents 1 through 3 are
combined to form a mixture that could be added as a single volume of
0.20 ml. All reactants except the transhydrogenase are kept at room tem-
perature, or in a bath at the same temperature as the spectrophotometric
cell chamber (usually 25 ° ) prior to adding the enzyme which initiates
the reaction. The cell contents are mixed well and the reaction at 258 m~
is measured. Since pyruvate itself absorbs some light at this wavelength a
cuvette with all the reagents except the enzyme can be used as a reagent
blank.
In measuring the rate in the reverse direction, 0.05 ml of 0.2M
all-lithium lactate (Sigma Chemical Co.) and 0.01 ml of 10 mM Tris-
oxaloacetate, pH 6.5, are substituted for reagents 2 and 3. All other condi-
tions are the same as described above. Oxaloacetic acid is adjusted to pH
6.5 with Tris base using bromcresol green indicator, and this solution is
made fresh daily.
Indirect Assay
Reagents
1. Tris-HC1 buffer, 0.5 M, pH 7.8
2. NADH, 4 mM (Sigma Chemical Co.)
3. Sodium pyruvate, 0.1 M (Sigma Chemical Co.)
4. Tris-L-malate, 0.2 M (Sigma Chemical Co.)
5. Malate dehydrogenase (Boehringer-Mannheim), dilution-0.01 ml
contains 0.1 unit (dilution from commercial prcparation made in
1% bovine serum albumin)
6. Distilled water
7. Malate-lactate transhydrogenase
Procedure. The reagents listed above are added to a 0.50 ml spectro-
photometric cell (1 cm light path) in the following order and amounts:
Tris-HC1 buffer, 0.05 ml; NADH, 0.01 ml; sodium pyruvate, 0.08 ml;
Tris-L-malate, 0.05 ml; malate dehydrogenase, 0.01 ml; distilled water,
0.03 ml; malate-lactate transhydrogenase, 0.01 ml of an appropriate
dilution.
Usually, larger but proportional volumes of reagents 1 through 5 are
combined to form a mixture that can be added as a single volume of
0.20 ml. All reactants except the transhydrogcnase are kept at room tern-
perature or in a bath at the same temperature as the spectrophotometric
cell chamber (usually 25 ° ) prior to adding the enzyme to initiate the
reaction, which is measured at 340 mt~.
[41] MALATE-LACTATE TRANSIIYDROGENASE 265
In measuring the rate in the reverse direction, 0.05 ml of 0.2M

all-lithium lactate (Sigma Chemical Co.), 0.01 ml of 10 mM Tris-oxalo-
acetate, pH 6.5 (Sigma Chemical Co.), and 0.01 ml of lactate dchydro-
genase (Boehringer-Mannheim) containing 0.1 unit are substituted for
reagents 3, 4, and 5. All other conditions are the same as described above.
Since oxaloacetate tends to decarboxylate at a slow rate, the rate of
pyruvate formation should be measured in a cuvette containing all the
reagents except transhydrogenase, at each level of oxaloacetate used. The
oxaloacetate is prepared as described above.
Micrococcus lactilyticus (perhaps more properly named Veillonella
gazogenes), was grown anaerobically at 30 ° for 2 or 3 days in 20 liter
bottles containing 15 liters of a medium consisting of 1% yeast extract,
1% tryptone, and 2% sodium lactate as described by Delwiche. 3 Cells
were harvested with a Delaval separator (model 100 LPS) at 4 °. Approxi-
mately 50 g of cells, wet weight, was obtained per 15 liters of medium.
In the preparation described here, 50 g of cells was used. Unless other-
wise noted, all subsequent steps were carried out at 0-4 °. A 30% wet
weight to volume suspension of cells was made in 0.1 M potassium-
phosphate buffer (K-P04), pH 7.0. The cells were ruptured by two con-
secutive treatments in a chilled French pressure cell (Aminco) with
20,000 psi pressure. Comparable results have also been obtained with the
use of sonieation or the colloid mill (Gifford-Wood Co.) with 200 tz glass
beads. The unbroken cells and cell debris were removed by centrifugation
for 20 minutes at 20,000 g. Approximately 90 ml of dark brown extract
was obtained which contained 3300 mg of protein (step 1, see the table),
as measured by the biuret reaction. The specific activity of the trans-
hydrogenase in the crude extract was generally close to 15 (range 10-20).
Step 2. The extract was desalted by passage through a Sephadex
G-25M (Pharmacia) column (5 X 50 cm), and the sample was then
diluted to 250 ml with cold distilled water. Approximately 60 ml of
packed moist DEAE-cellulose (Selectacel type 40, 0.97 meq/mg, Sch-
leicher and Schuell) which had been washed and equilibrated with 5 mM
K-PO, buffer, 7.0 was added. The mixture was stirred at 0 ° for 1 hour;
then a portion was centrifuged and the transhydrogenase activity was
determined ifi an 0.02 ml aliquot of the clear supernatant solution.
Usually all the transhydrogenase was adsorbed; if it was not, more
DEAE-cellulose was added and the process was repeated. When all the
enzyme was adsorbed, the mixture was filtered (Whatman No. 4) at 4 °
and the opalescent filtrate was discarded. The DEAE-cellulose was then
E. A. Delwiche, E. F. Phares, and S. F. Camon, J. Bacteriol. 71, 598 (1956).

suspended in 300 ml of 5 mM K-P04 buffer, 7.0, stirred for 20 minutes

at 0 ° and again filtered. The filtrate contained no transhydrogenase. The
DEAE-cellulose was next washed as above with 300 ml of 0.2 M K-P04
buffer, 7.0. This filtrate contained most of the transhydrogenase (35,200
units) with a specific activity of 38 (step 2, see the table). The remainder
of the adsorbed transhydrogenase could be removed from the DEAE-
cellulose by a second wash with 300 ml of 0.2 M K-P04 buffer. The
specific activity of the enzyme in this second fraction was 11 and was
not used in further purification. The first 0.2 M eluate was brought to
90% saturation by addition of solid ammonium sulfate (Mann, enzyme
grade), and the precipitate was centrifuged at 16,000 g for 45 minutes.
The precipitate could be stored indefinitely at --55 ° .
Step 3. The ammonium sulfate precipitate from step 2 was dissolved
in 50 mM Tris-HCl buffer, pH 7.5, and the resulting solution was passed
through a Sephadex G-25 column (4 X 35 cm). The desalted protein was
next adsorbed onto a DEAE-Sephadex A-50 (Pharmacia) column (4 X 20
cm) which had been equilibrated with 5 mM K-PO, buffer, pH 7.0. The
column was washed with 210 ml of 0.1 M K-PO, buffer, pH 7.0, and a
protein peak, as measured by 280/260 absorbance, which did not contain
transhydrogenase activity was removed from the column. The trans-
hydrogenase was then eluted with 0.2M K-PO, buffer, 7.0, and the
activity coincided with the protein peak. The fractions containing the
highest specific activity were pooled and resulted in a preparation con-
gaining 81 mg of protein and approximately 14,000 units of enzyme (step
3, see the table). Pooling of the fractions on both sides of this protein
peak resulted in the recovery of another 14,000 units of enzyme which
had a slightly lower specific activity. Analytical ultracentrifugation of a
sample of protein from the peak fractions showed the preparation to be
homogeneous as judged by schlieren photographs.
PURIFICATION OF MALATE-LACTATE TRANSHYDROGENASE
Total Specific Total

protein activity units Recovery
Step (mg) (units/rag) (~moles/min) (%)
1. Extract 3300 15 49,600 --

2. DEAE-cellulose, first 0.2 M 927 38 35,200 71
eluate
3. DEAE-Sephadex
chromatography
Fraction 93-98 81 171 13,900 28
Fractions 92, 99--108 122 117 14,274 29
Totals, 92-108 28,174 57
[41] MALATE-LACTATE
TRANSHYDROGENASE 267
Properties
Equilibrium. The reaction is readily reversible with a Keq of 1.8 +__0.4
favoring pyruvate and L-malate.
pH Optimum. The pH optimum is 7.5-8.5, and 50% of the maximum
activity can be noted at pH 6.3 and 9.5.
Stability. The enzyme ix relatively stable in the purified state since
when stored at --15 ° as all ammonium sulfate suspension at about 10-20
mg of protein per milliliter, there is about 40% loss in activity aftcr a
year.
Molecular Weight and Sedimentation Coefficient. These values as
determined on enzyme preparations of specific activity ~150 were as
follows: Molecular weight 99,000 ± 9000, as determined from sucrose
gradient studies assuming molecular weights of 68,000, 96,600, and
150,000 for bovine serum albumin, yeast hexokinase, and yeast alcohol
dehydrogenase, respectively. This value is in fair agreement with that of
115,000 reported for this enzyme by Dolin et al., 1 who used the short
column equilibrium method;4 The S~o,w was 4.6 S as determined both by
sucrose gradient and the analytical ultracentrifuge.
Km Values. The K,, values for pyruvate and L-malate were deter-
mined by the indirect assay. When the concentration of pyruvate was
varied from 0.32 mM to 16 mM an apparent Kpyr of 2.4 mM was ob-
tained. When the concentration of L-malate was varied from 0.68 mM to
16 mM an apparent K,n~ of 1.4 mM was obtained. The Km values for
oxaloacetate and L-lactate were also determined with the indirect assay.
When the concentration of oxaloacetate was varied from 2.6 p ~ / t o 0.51
mM, an apparent K o ~ of 50 ta~r was obtained. When the concentration
of L-iactate Klac was varied from 0.26 mM to 4 mM an apparent K~¢
of 1.9 mM was obtained.
Requirements. No metal ion or coenzyme requirement has been noted
for this transhydrogenase. Exogenous NADH or NADPH cannot be
coupled to the transhydrogenation. Furthermore, as reported previously,
no artificial hydrogen carriers or election donors have been found to
couple with the enzyme2 A rather large number of a-keto and a-hydroxy
acids other than oxaloacetate, pyruvate, L-malate, and L-lactate can
serve as substrates for the enzymes. D-Lactate is not a substrate, dl-a-
Hydroxybutyrate is as good a hydrogen donor as malate but dl-fl-hy-
droxybutyrate is only 1.4% as active. As chain length increases, the
activity with a-hydroxy acids decreases; for example a-hydroxycaproate
has only 0.2% of the activity noted with malate. Other components,
such as 2-methyl lactate and isocitrate, are not substrates for the enzyme.
' D. A. Yphantis, Biochemistry 3, 297 (1964).
61]. F. Phares and M. V. Long, Abstr. 180th Meeting Am. Chem. Soc. 1956, p. 62c.
Sulfhydryl groups on the enzyme are necessary for activity. Enzy-

matic activity is inhibited 4 6 ~ by 1 0 / ~ / and 71% by 0.2 mM p-hy-
droxymercuribenzoate (HMB). The inhibition by H M B could be com-
pletely reversed by addition of thiols to the enzyme treated with HMB.
Mechanism and Discussion
The malate-lactate transhydrogenase is thought to catalyze the first
step in the fermentation of lactate by M. lactilyticus. Thus far the
enzyme has not been found in mammalian tissues or in extracts of
Escherichia coli or Propionibacterium shermanii grown anaerobically on
lactate containing media. Its presence in other microorganisms or plants
has not been determined.
A reduced pyridine nucleotide which is probably N A D H has been
shown to be firmly linked to the enzyme?, ~ The pyridine nueleotide can-
not be removed from the enzyme by either charcoal absorption or by acid
ammonium sulfate precipitation. In fact the prosthetic group remains
tightly bound to the protein during the entire purification procedure.
Treatment by boiling the protein at pH 10 for 3 minutes does cleave the
pyridine nucleotide from the enzyme. Spectral analysis of the enzyme
reveals an absorbance band in the 340-350 m~ region. This absorbanee
can be increased to a maximum by addition of either of the hydrogen
donors, L-lactate or L-malate, or reduced by addition of either of the
hydrogen acceptors, oxaloacetate or pyruvate. Careful addition of hy-
drogen donor to the fully oxidized form indicates that 3 moles of donor
per mole of enzyme are needed to fully reduce the prosthetic group as
measured by absorbance at 345 m~. Dolin also reported the presence of
3 NAD prosthetic groups per molecule of enzyme as measured enzy-
matically. 1 Fluorescence spectra also show the expected shifts upon
addition of either the hydrogen donor or acceptor substrate. As isolated
the enzyme appears to contain approximately 40% of the prosthetic
group in the reduced form.
The transfer of hydrogen between substrates has been demonstrated
by the enzyme mediated transfer of tritium from laetate-2-3H to the
pyridine prosthetic group. ~ When lactate 2-8tt was added to the enzyme,
an increase in fluorescence of 5 5 ~ occurred. The protein was separated
from the excess of lactate-2-SH by gel filtration, and it was found that
the protein contained 83,750 cpm. After treatment of this protein with
1 micromole of oxaloacetate, the fluorescence of the protein decreased
9 3 ~ and the protein was again separated from small molecular weight
compounds by gel filtration. The pyridino protein isolated was still in
the oxidized form as indicated by fluorescence measurements, but con-
tained only 960 epm out of the 67,000 cpm placed on this second gel
~1] MALATE-LACTATE TRANSHYDROGENASE 269
column, whereas 54,000 cpm were recovered in a peak eluted subsequent

to the protein. Unlabeled malate and lactate were added to the pooled
fractions which contained this radioactive peak. Celite chromatography
revealed that all the radioactivity was present as malate. These results
indicate that a direct transfer of hydrogen occurs to the pyridine nucleo-
tide on the enzyme and that the transfer of reducing equivalents coincides
with the increase in both fluorescence and absorbance at 345 m#. The
reaction can be carried out in a two-step manner; i.e., both substrates
need not be present simultaneously for the transfer of hydrogen to occur.
When the radioactive lactate is separated from the enzyme by gel filtra-
tion, the transhydrogenase remains reduced and contains the tritium.
Oxidation of the pyridinoprotein with oxaloacetate as measured by
fluorescence liberates the tritium from the enzyme, yielding oxidized
enzyme and tritium-labeled malate. Since virtually all the radioactivity
is removed from the pyridine nucleotide, it can be concluded that the
transfer is stereospecific and that the same hydrogen is involved in both
steps of the reaction. Thus the mechanism appears to be a straight-
forward transfer of reducing equivalents between substrates mediated
by an enzyme-bound pyridine nucleotide. Kinetic studies, however, do
not conform to the typical "ping-pong" mechanism. Dolin 6 has pre-
sented evidence for a mechanism involving non-fluorescent intermediate
complexes. The exact mechanism is at present not clear.
*Dolin, M. I., Abstr. 7th Intern. Congr. Biochem., Tokyo, 1957, p. 779. (Abstr.
No. F-121).
270 REACTIONS
[42] P h o s p h o e n o l p y r u v a t e Carboxykinase from

:Pig L i v e r M i t o c h o n d r i a
[EC 4.1.1.32 GTP: oxaloacetate carboxy-lyase(transphosphorylating)]
By M. DANIELLANE, H. C. CHANG,and ROBERTS. MILLER
Mn++
Phosphoenolpyruvate + HCO~- -~ IDP (or GDP) . •
oxaloacetate + ITP (or GTP)
A s s a y Method
Principle. Mitochondria]phosphoeno]pyruvatecarboxykinasecatalyzes
the IDP- (or GDP-) dependent carboxylationof phosphoenolpyruvateto
form oxaloacetate and ITP (or GTP). '-s The enzyme is assayed' most
reliably in the presence of NADH and malate dehydrogenaseby measur-
ing the rate of incorporation of 14C-bicarbonate into malate (acid-stable
'~C-activity) or the rate of NADH oxidation spectrophotometrically.
Carboxykinase activity also can be determined in the reverse direction,
in which case the ITP-dependent decarboxylation of oxa]oacetate is
measured. In the latter case the rate of P-eno]pyruvate and IDP forma-
tion is followed spectrophotometrically (NADH oxidation) by coupling
to the pyruvate kinase- and lactate dehydrogenase-catalyzedreactions.
Reagents
Imidazole buffer (Cl-), 0.5 M, pH 6.6
Phosphoenolpyruvate (Nas+), 25 mM
I D P (Na÷), 25 mM
MnCl2, 20 mM
KH'4C08 1.0M (approximately 105 cpm per micromole; specific
activity must be accurately known), for the '~C-bicarbonate
fixation assay
KHC03, 1.0M, for the spectrophotometric assay
1H. C. Chang and M. D. Lane, J. Biol. Chem. 241, 2413 (1966).

~H. C. Chang, H. Maruyama, R. S. Miller, and M, D. Lane, J. Biol. Chem. 241,
2421 (1966).
, M. F. Utter and K. Kurahashi, J. Am. Chem. Soc. 75, 758 (1953).
' M. F. Utter and K. Kurahashi, J. Biol. Chem. 207, 787 (1954).
~M. F. Utter, K. Kurahashi, and I. A. Rose, J. Biol. Chem. 207, 803 (1954).
* M. F. Utter and K. Kurahashi, J. Biol. Chem. 207, 821 (1954).
'J. L. Graves, B. Vennesland, M. F. Utter, and R. J. Pennington, J. Biol. Chem.
223, 551 (1956).
SK. Kurahashi, R. J. Pennington, and M. F. Utter, J. Biol. Chem. 226, 1059 (1957).
[42] PHOSPHOPYRUVATE CARBOXYKINASE--PIG LIVER 271
NADH, 0.1 M, for the 14C-bicarbonate fixation assay

NADH, 2.5 raM, for the spectrophotometric assays
Malate dehydrogenase, suspension in 70% saturated (NH,)2S0,
containing 120 units/ml
Glutathione, 0.1 M
HC1, 2 M
Liquid scintillator, 0.25 g of 1,4-bis-2(5-phenyloxazolyl) benzene
(POPOP), 10 g of 2,5-diphenyloxazole (PPO), and 100 g of
recrystallized naphthalene per liter of dioxane
Tris buffer (C1-), 0.5 M, pH 7.5
ITP (Nan*), 0.1M
Oxaloacetate, 10 raM. Prepare fresh daily and bring to pH 7.5 with
NaOH at least 1 hour before use to ensure keto-enol equilibration
MgClz, 20 mM
Crystalline pyruvate kinase, suspension in 60% saturated
(NH~)~S04 containing 1 mg/ml
Crystalline lactate dehydrogenase, suspension in 60% saturated
(NH4)..S04 containing 1 mg/ml
Procedures for '~C-Bicarbonate Fixation Assay. The IDP- and Mn ÷*-

dependent carboxylation of P-enolpyruvate results in the formation of
ITP and oxaloacetate. The reaction velocity is followed, in the presence
of NADH and malate dehydrogenase, by determining the rate of incor-
poration of HI~CO~- into malate (acid-stable 1~C activity). The HI~C0~--
fixation carboxylation assay reaction mixture contains the following
components (in micromoles unless specified) : imidazole (Cl-) buffer, pH
6.6, 100; KH14C03 (approximately 105 cpm per micromole), 50; P-enol-
pyruvate, 1.25; IDP, 1.25; MnCl2, 1.0; GSH, 2.0; NADH, 2.5; malate
dehydrogenase, 5 units; and P-enolpyruvate carboxykinase, up to 0.004
unit in a total volume of 1.0 ml. The final pH is 7.0. The reaction is
initiated by addition of P-enolpyruvate; after a 15-minute incubation
at 30 °, it is terminated by addition of 1 ml of 2 N HC1. The acid-stable
14C activity in a 0.5-ml aliquot is taken to dryness in a scintillation
counting vial at 85 ° for 60 minutes in a forced-draft oven. After addition
of 1 mI of H~O and then 10 ml of liquid scintillator to the vial, acid-
stable 1~C activity (as 14C-malate) is determined using a liquid scintilla-
tion spectrometer. Initial velocity of bicarbonate fixation follows zero
order kine~ics for at least 20 minutes and is proportional to enzyme con-
centration up to a level of 0.004 unit of carboxykinase.
Procedure ]or Spectrophotometric Carboxylation Assay. The more
rapid spectrophotometric carboxylation assay can be employed with car-
boxykinase preparations that have been carried through step 4 (cellulose
phosphate chromatography) of the purification procedure. The reaction

mixture and conditions for this assay are modified from those described
for the H~4COa--fixation carboxylation assay (above) to include un-
labeled instead of ~4C-bicarbonate, and less NADH (0.15 micromole).
The initial velocity of NADH oxidation is followed for 2 minutes at 340
m~ (1 cm light path) after initiating the reaction with P-enolpyruvate.
The earboxylation reaction follows zero order kinetics with up to 0.055
unit of carboxykinase.
Procedure ]or Decarboxylation Assay. The ITP- and Mn+÷-dependent
decarboxylation of oxaloacetate catalyzed by the carboxykinase leads to
the formation of IDP and P-enolpyruvate. This reaction is coupled to
pyruvate kinase- and lactate dehydrogenase-catalyzed reactions and the
overall reaction rate is determined by following NADH oxidation
spectrophotometrically. This assay can be used only for carboxykinase
preparations carried beyond step 4 of the purification procedure because
preparations from earlier steps contain NADH-oxidizing activity. The
reaction mixture contains the following components (in micromoles un-
less specified) : Tris (C1-) buffer, pH 7.5, 100; oxalaeetate (neutralized),
0.5; ITP, 3.0; MnC12, 1.5; MgCl~, 1.0; GSH, 2.0; NADH, 0.15; pyruvate
kinase, 1.25 units; lactate dehydrogenase, 2.5 units; and carboxykinase,
up to 4 X 10-3 unit in a total volume of 1.0 ml. The final pH is 7.4.
The rate of nonenzymatic decarboxylation of oxaloacetate (to pyruvatc
and C02) is determined at 30 ° by following the rate of NADH oxidation
at 340 m~ for 2 minutes in the presence of all the components except
carboxykinase. Enzymatic decarboxylation is then initiated by the ad-
dition of earboxykinase, and the NADH oxidation rate at 30 ° is deter-
mined again. The rate of oxaloacetate deearboxylation in the presence of
carboxykinase is corrected for the nonenzymatic rate (determined in
the absence of carboxykinase). It is important that controls be included
in which I T P is omitted to make certain that the carboxykinase prepa-
ration is not significantly contaminated with malate dehydrogenase. The
rate of the enzymatic deearboxylation follows zero order kinetics for at
least 2 minutes and is proportional to enzyme concentration up to a
level of 4 X 10-s unit (carboxylation assay) of earboxykinase. Deear-
boxylation rate is expressed as micromoles of oxaloacetate decarbox-
ylated per minute.
Units. A unit of P-enolpyruvate carboxykinase is defined as that
amount of enzyme which catalyzes the carboxylation of 1.0 micromole of
P-enolpyruvate per minute under the conditions of the ~'C-bicarbonate
fixation or speetrophotometric carboxylation assay. Protein is determined
spectrophotometrically as described by Layne 9 and specific activity is
DE. Layne, Yol. III, p. 451.
[42] PHOSPHOPYRUVATE C A R B O X Y K I N A S E - - P I G LIVER 273
expressed in terms of units per milligram of protein (spectrophotometri-

eally determined).
Other Methods o] Assay. P-enolpyruvate carboxykinase catalyzes
an ITP- (or GTP-) and Mn++-dependent exchange between H14COs- and
oxaloacetate which is considerably faster than either the overall earbox-
ylation or decarboxylation reaction.I, -~ The H~4COa--oxaloacetate ex-
change can also be used as a sensitive assay for P-enolpyruvate carboxy-
kinase. 1
The purification procedure described is based on that of Chang and

Lane? All the operations are conducted at approximately 4 ° unless
otherwise specified. The results of each purification step are summarized
in the table.
Mitochondrial Acetone Powder. Fresh pig liver, 1 kg, is homogenized
in 2 liters of cold 0.25 M sucrose (containing 0.5 mM EDTA) for 1 min-
ute (three 20-second periods) at top speed in a Waring blendor (4-liter
capacity). One additional liter of sucrose solution is added, and the mix-
ture is rehomogenized for 20 seconds. The homogenate is centrifuged at
1500 g for 15 minutes; the supernatant suspension is filtered through 4
layers of cheesecloth and then recentrifuged at 60,000 g in a refrigerated
Sharpies centrifuge (flow rate, approximately 0.2 liter per minute). The
sedimented particles (principally mitochondria) are resuspended with
about 100 ml of 0.25 M sucrose (0.5 mM EDTA) and added slowly with
rapid stirring into 20 volumes of acetone at approximately --5 ° . The
precipitate is allowed to settle for approximately 5 minutes, then most of
the supernatant solution is decanted and the suspension is filtered under
vacuum with a Biichner filter. The precipitate is washed on the filter
with dry acetone followed by peroxide-free ether. After the last volume
of ether has been drawn through the filter, the precipitate is quickly
transferred to a vacuum desiccator and the last traces of ether are
removed in a vacuum. The acetone powder (yield, approximately 50 g per
kilogram of liver) is stable for at least 4 months when stored at --20 °.
While the procedure described deals with 1 kg of liver as starting quan-
tity, experience in our laboratory indicates that 20 kg of liver can be
processed in 1 day if an industrial size Sharples centrifuge (model 16) is
used at a flow rate of 0.5 liter per minute.
Extraction and Ammonium Sul]ate Fractionation. Acetone powder,
100 g, is extracted for 3 hours with 2 liters of 10 mM phosphate buffer
(pH 7.5) with slow stirring. The suspension is centrifuged at 30,000 g
for 10 minutes and the clear supernatant extract is retained. This solution
is brought to 45% saturation 1° with solid ammonium sulfate (0.277 g per

milliliter of extract) introduced slowly with magnetic stirring. After
standing for 30 minutes, the supernatant solution is recovered after
centrifugation. Sufficient solid ammonium sulfate (0.099 g per milliliter of
supernatant solution) is added to the supernatant solution to bring it to
60% saturation. The suspension is allowed to stand for 30 minutes,
centrifuged, the precipitate dissolved in 70 ml of 5 mM phosphate buffer,
pH 7.0 (containing 0.5 mM EDTA and 5 mM GSH), and then dialyzed
against 3 liters of the same buffer for 10 hours.
DEAE-Cellulose Chromatography. The dialyzed enzyme from the
previous step (about 7 g of protein) is applied to a DEAE-cellulose
column (4.5 X 36 em packed volume; Schleicher and Schuell Type 20)
equilibrated previously with 0.SM phosphate and then the dialysis
buffer. Elution is accomplished with 5 mM phosphate, pH 7.0 (containing
5 mM mereaptoethanol) ; the column effluent is monitored continuously
for ultraviolet-absorbing compounds (253 m~, LKB Uvicord absorptiom-
eter), and carboxykinase activity is located by the H14CO3--fixation
assay. P-enolpyruvate earboxykinase activity is eluted with the protein
breakthrough peak. The most active fractions containing about 90%
of the activity applied to the column are pooled, placed in dialysis bags,
and then dialyzed against an ammonium sulfate solution (0.5 raM
EDTA and 0.5 mM GSH), pH 6.5, of sufficient concentration to reach
60% saturation at equilibrium.
Cellulose Phosphate Chromatography. The protein precipitate from
the pooled DEAE-cellulose column fractions is recovered by centrif-
ugation, dissolved in about 30 ml of 5 mM phosphate buffer, pH 7.0
(0.5 mM EDTA and 0.5 m M GSH), and then dialyzed against 2 liters
of the same buffer for 12 hours. The dialyzed solution (about 1.8 g of
protein) is applied to a column (4.5 X 40 cm) of cellulose phosphate 11
which has been equilibrated with the dialysis buffer. Stepwise gradient
elution is accomplished by placing 800 ml of 5 mM phosphate, pH 7.0,
into the mixing chamber attached to the column and introducing the fol-
lowing phosphate buffers (K+), all pH 7.0, into a separatory funnel
~°AI1 of the "percentage of ammonium sulfate saturation" figures obtained with
solid ammonium sulfate refer to percentage of saturation at 25°. Enzyme grade
ammonium sulfate (low heavy metal ion content) from Mann Research Labora-
tories was used in all the ammonium sulfate enzyme fractionations.
i~Cellulose phosphate was obtained from Schleieher and Sehuell and was equilibrated
by successively washing with 0.1 N NaOH, water (until neutral), 0.1 N HCI, water
(until neutral), 50 mM potassium phosphate, pH 7.0, and finally 5 mM potassium
phosphate, pit 7.0. The equilibrated cellulose phosphate should not be stored longer
than 2 weeks. Deviations from this equilibration procedure often result in failure
of the column to retain the P-enolpyruvate carboxykinase.
[42] PHOSPHOPYRUVATE CARBOXYKINASE--PIG LIVER 275
attached to the mixing chamber: 160 ml of 5 mM, 800 ml of 0.2 M, and

800 ml of 0.4 M. The effluent is monitored continuously for protein, col-
lected fractionally, and fractions are assayed for carboxykinase activity
as described in the previous section. P-enolpyruvate carboxykinase is
eluted as a distinct protein peak after approximately 650 ml of eluate
have been collected. The "active" fractions are pooled (about 250 ml) and
precipitated with (NH~)2S04 by the dialysis technique described under
"DEAE-Cellulose Chromatography."
Hydroxylapatite Chromatography. The precipitated protein, which is
recovered from the preceding step by centrifugation, is dissolved in 4 ml
of 5 mM phosphate buffer, pH 7.0 (0.5 mM EDTA and 0.5 mM GSH),
and dialyzed against the same buffer for 10 hours. The dialyzed enzyme
solution (about 100 mg of protein) is applied to a column (2.0 X 12 cm)
of hydroxylapatite12 already equilibrated with the dialysis buffer (EDTA
omitted). Stepwise elution is carried out under pressure (2 psi from
nitrogen gas cylinder) with 40 ml of 5 mM, 150 ml of 50 mM, and 100-
200 ml of 0.1M phosphate buffer (K+), pH 7.0, containing 5 mM
mercaptoethanol. P-enolpyruvate carboxykinase activity and protein in
the eluted fractions (5 ml) are determined as described under "DEAE-
cellulose Chromatography." Elution of carboxykinase usually occurs dur-
ing the application of the 50 mM phosphate buffer to the column after
approximately 150 ml of eluate has been collected. Fractions containing
peak carboxykinase activity are pooled and precipitated at 60% am-
monium sulfate saturation (pH 6.5) by the dialysis technique described
earlier. The enzyme, stored under 60% saturated ammonium sulfate (pH
6.5, 0.5 mM EDTA and 0.5 mM GSH), loses activity at a relatively slow
rate (about 10% per month).
As indicated in the table, a 210-fold purification of P-enolpyruvate
carboxykinase from the initial liver mitochondrial acetone powder ex-
tract is achieved in excellent yield (48%) by the procedure outlined.
The degree of purification from the whole liver homogenate is probably
higher since preparation of the mitochondrial acetone powder extract
constitutes a purification. Carboxykinase assays conducted on the whoh,
homogenate have been found to be unreliable.
Properties
Specificity. P-enolpyruvate is the only substrate known to be car-
boxylated by P-enolpyruvate carboxykinase. IDP and GDP, as well as
ITP and GTP, are nearly equally active in the carboxylation and de-
carboxylation reactions, respectively.2 The corresponding uridine nucleo-
'~A. Tiselius, S. Hjert6n, and ~. Levin, Arch. Biochem. Biophys. 65, 132 (1956).
PURIFICATION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE
Specific activity
Total Decarboxyl-
activity Carboxyl- ation b
Total (carbox- ation (~moles/
protein" ylation) (units/mg min/mg Yield
Step (g) (units) protein °) protein ~ (%)
1. Acetone powder extract, 28.0 672 0.024 -- 100

2. 45--60% saturated (NH4)~SO~ 7.2 535 0.074 -- 80
fraction
3. DEAE-cellulose eluate 1.83 481 0. 263 -- 71
4. Cellulose phosphate chroma- 0.112 358 3.2 22 53
tographic fraction
5. Hydroxylapatite chrome~ 0.064 320 5.0 d 35 d 48
tographic fraction
• Protein determined spectrophotometrically as described by Layne (Vol. III, p. 451).

Decarboxylation rate determined with the decarboxylation assay.
c Extract of 100 g of mitochondrial acetone powder which can be obtained from 2 kg
of liver (wet weight).
Specific activity, expressed in terms of refractometrically determined protein, is
9.1 units per milligram of protein for carboxylation and 63.4 micromoles per minute
per milligram of protein for decarboxyl'ation.
tides exhibit slight activity, whereas, adenine and eytidine nucleotides

are inactive. 2 Mg ÷÷ can replace Mn ++ in the carboxylation reaction at
pH 7.5; however, the maximum velocity is only 30% that with Mn**.
pH Optima. Maximum initial velocities for the carboxylation, de-
carboxylation, and oxaloacetate-H14C03 - exchange reactions are attained
between pH 6.4 and 6.7. 2 While the acid slopes of the pH optimum
curves are nearly identical, the alkaline slopes of the earboxylation and
exchange reaction are far steeper than that of the decarboxylation
reaction.
Molecular Properties. Pig liver mitochondrial P-enolpyruvate car-
boxykinase has a sedimentation coefficient (S s0.
° w -----5.21 S) and a molec-
ular weight determined by sedimentation equilibrium of 73,300.1 The
partial specific volume of the enzyme calculated from its amino acid
composition is 0.736.1 The relation between absorbance at 280 m~ and
refractometrically determined protein concentration is given by the
equation, c----0.613 ~lom~ where c is protein concentration in mg/ml
and A is absorbance at 280 m/~ (1 cm light path). The absorbance ratio
A_-8o:A_o6ois 1.75. To convert protein determined by the method of Layne 9
[43] PHOSPHOPYRUVATECARBOXYLASE--PEANUT COTYLEDONS 277
to refractometrically determined protein the former should be multiplied

by a factor of 0.552. The carboxykinase contains 15 sulfhydryl groups
per molecule which account for its total half-cystine content) 3
Kinetic Properties and Inhibitors? The K~ values for HCQ-
P-enolpyruvate, IDP, GDP, and Mn ÷÷ (total) at pH 7.5 in the carbox-
ylation reaction are 25 mM, 0.12 raM, 33 #M, 20 #M, and 0.33 mM,
respectively. The K~ values for oxaloacctate, ITP, GTP, and Mn *÷
(total) at pH 7.5 in the decarboxylation reaction are 0.15 mM, 0.58
raM, 0.16 mM, and 0.43 mM, respectively. The carboxykinase is revers-
ibly inhibited by p-CMB. DL-Phospholactate is a competitive inhibitor
with respect to P-enolpyruvate and has a K~ of 0.9 mM. The insensitivity
of P-enolpyruvate carboxykinase action to avidin indicates that biotin
is not a prosthetic group for this enzyme.
,3 H. C. Chang and M. D. Lane, unpublished observations.
[43] P h o s p h o e n o l p y r u v a t e C a r b o x y l a s e f r o m
Peanut Cotyledons
[EC 4.1.1.31 Orthophosphate: oxaloacetate earboxy-lyase (phosphorylating))
By M. DANIEL LANE,H. MAaUVAMA,and R. L. EASTF2~AV
Mg++
Phosphoenolpyruvate + HC03- , oxaloacetate + P/
Assay Method
Principle. Phosphoenolpyruvate carboxylase catalyzes the irreversible
carboxylation of phosphoenolpyruvate to form oxaloacetate and ortho-
phosphate. 1 Carboxylase activity is determined conveniently in the
presence of NADH and malic dehydrogenase by following either the rate
of incorporation of H14CO3- into malate (acid-stable 14C-activity) or
the rate of NADH oxidation spectrophotometrically. This enzyme, which
is distributed widely in plant tissues '-* and microorganisms, 5-6 has not
been found in animal tissues.
1H. Maruyama, R. L. Easterday, H. C. Chang, and M. D. Lane, J. Biol. Chem. 241,

2405 (1966).
1R. S. Bandursld and C. M. Greiner, J. Biol. Chem. 204, 781 (1953).
a T. T. Tchen and B. Yennesland, J. Biol. Chem. 213, 533 (1955).
' H. Maruyama and M. D. Lane, Biochim. Biophys. Acta 65, 207 (1962).
*I. Suzuki and C. H. Werkman, Arch. Bioehem. Biophys. 761 103 (1958).
ej. L. Chnovas and H. L. Kornberg, Biochim. Biophys. Acta 96, 169 (1965).
[43] PHOSPHOPYRUVATECARBOXYLASE--PEANUT COTYLEDONS 277
to refractometrically determined protein the former should be multiplied

by a factor of 0.552. The carboxykinase contains 15 sulfhydryl groups
per molecule which account for its total half-cystine content) 3
Kinetic Properties and Inhibitors? The K~ values for HCQ-
P-enolpyruvate, IDP, GDP, and Mn ÷÷ (total) at pH 7.5 in the carbox-
ylation reaction are 25 mM, 0.12 raM, 33 #M, 20 #M, and 0.33 mM,
respectively. The K~ values for oxaloacctate, ITP, GTP, and Mn *÷
(total) at pH 7.5 in the decarboxylation reaction are 0.15 mM, 0.58
raM, 0.16 mM, and 0.43 mM, respectively. The carboxykinase is revers-
ibly inhibited by p-CMB. DL-Phospholactate is a competitive inhibitor
with respect to P-enolpyruvate and has a K~ of 0.9 mM. The insensitivity
of P-enolpyruvate carboxykinase action to avidin indicates that biotin
is not a prosthetic group for this enzyme.
,3 H. C. Chang and M. D. Lane, unpublished observations.
[43] P h o s p h o e n o l p y r u v a t e C a r b o x y l a s e f r o m
Peanut Cotyledons
[EC 4.1.1.31 Orthophosphate: oxaloacetate earboxy-lyase (phosphorylating))
By M. DANIEL LANE,H. MAaUVAMA,and R. L. EASTF2~AV
Mg++
Phosphoenolpyruvate + HC03- , oxaloacetate + P/
Assay Method
Principle. Phosphoenolpyruvate carboxylase catalyzes the irreversible
carboxylation of phosphoenolpyruvate to form oxaloacetate and ortho-
phosphate. 1 Carboxylase activity is determined conveniently in the
presence of NADH and malic dehydrogenase by following either the rate
of incorporation of H14CO3- into malate (acid-stable 14C-activity) or
the rate of NADH oxidation spectrophotometrically. This enzyme, which
is distributed widely in plant tissues '-* and microorganisms, 5-6 has not
been found in animal tissues.
1H. Maruyama, R. L. Easterday, H. C. Chang, and M. D. Lane, J. Biol. Chem. 241,

2405 (1966).
1R. S. Bandursld and C. M. Greiner, J. Biol. Chem. 204, 781 (1953).
a T. T. Tchen and B. Yennesland, J. Biol. Chem. 213, 533 (1955).
' H. Maruyama and M. D. Lane, Biochim. Biophys. Acta 65, 207 (1962).
*I. Suzuki and C. H. Werkman, Arch. Bioehem. Biophys. 761 103 (1958).
ej. L. Chnovas and H. L. Kornberg, Biochim. Biophys. Acta 96, 169 (1965).
Reagents
KH~4C08, 0.1M (approximately 105 cpm per micromolc; specific
activity must be accurately known), for the 14C-bicarbonate
fixation assay
KHCOa, 0.1 M, for the spectrophotometric assay
P-enolpyruvatc (Na~) 25 mM
MgC12, 0.1 M
GSH, 0.1 M
NADH, 0.1 M, for the ~4C-bicarbonate fixation assay
NADH, 2.5 mM, for the speetrophotometric assay
Malate dehydrogenase suspension in 70% saturated (NH4)~S0~
containing 120 units/ml
Liquid scintillator, 0.25 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene
(POPOP), 10 g of 2,5-diphenyloxazole (PP0), and 100 g of
recrystallized napthalene per liter of dioxane
Procedure ]or 14C-Bicarbonate Fixation Assay. The Mg÷÷-dependent

carboxylation of P-enolpyruvate results in the formation of oxaloacetate
and orthophosphate (see Reaction). The reaction rate in the presence of
NADH and malic dehydrogenase is determined by following the rate of
incorporation of H~'C0s into malate (acid-stable ~4C activity). The
H~4C03-fixation carboxylation assay reaction mixture contains the fol-
lowing components (in micromoles except as indicated): Tris (C1-)
buffer, pH 7.8, 80; KH14C03 (approximately 105 cpm per micromole),
10; P-enolpyruvate, 2.0; MgC12, 2.0; GSH, 5.0; NADH, 2.0; malate
dehydrogenase, 14 units; and P-enolpyruvate carboxylase, up to 0.004
unit in a total volume of 1.0 ml. The final pH is 7.9. After a 15-minute
incubation at 30 °, the reaction is terminated by addition of 1 ml of 2 N
HCI. A 0.5 ml aliquot is taken to dryness in a scintillation counting vial
at 85 ° for 60 minutes in a forced draft oven. After addition of 1 ml of
H20 to the vial, then 10 ml of liquid scintillator, acid-stable 14C
activity (as 14C-malate) is determined with a liquid scintillation spec-
trometer. Initial velocity follows zero order kinetics for 15 minutes and
is proportional to enzyme concentration up to a level of 0.004 unit of
P-enolpyruvate carboxylase.
Procedure ]or Spectrophotometric Assay. A spectrophotometrie car-
boxylation assay is used generally for P-enolpyruvate carboxylase prepa-
rations carried beyond step 3 (0-55% saturated (NH4)~S04 fraction;
see table) of the purification procedure. The reaction mixture and condi-
tions are modified from those described for the HI'COs-fixation carbox-
~3] PHOSPHOPYRUVATECARBOXYLASE--PEANUT COTYLEDONS 279
ylation assay (above) to include, unlabeled, instead of 14C-labeled,

bicarbonate and less NADH (0.15 ~mole). The rate of NADH oxidation
is followed at 340 m~ (1 cm light path; 30 °) for 2-3 minutes after
initiating the reaction with P-enolpyruvate. The carboxylation rate fol-
lows zero order kinetics for at least 2 minutes and is proportional to
enzyme concentration up to a level of 0.05 unit of carboxylase.
Units. A unit of P-enolpyruvate carboxylase is defined as that amount
of enzyme which catalyzes the carboxylation of 1.0 micromole of P-enol-
pyruvate per minute under the assay conditions described Protein is
determined spectrophotometrically as described by Layne,~ and specific
activity is expressed in terms of units per milligram protein.
The purification procedure described is based on that of Maruyama

et al. 1 All the procedures are carried out at 4 ° unless specified. The
results of the purification procedure are summarized in the table.
Initial Extract and Aging Treatment. Large seeded, Virginia-type
shelled peanuts 8 (Arachis hypogeae), 6 kg, are dusted with 8 g of Arasan
50-Red (E. I. du Pont de Nemours and Company, Inc.; active ingredient,
tetramethylthiuram disulfide) and placed between several layers of
paper toweling in Pyrex baking dishes covered with perforated aluminum
foil. The peanuts are watered with 0.2% sodium propionate in tap water
and are germinated for 4 days in the dark at 28-30 °. Additional sodium
propionate solution is added as needed. Cotyledons are removed, washed
several times with distilled water, and then homogenized in 2 volumes
(w/v) of 50 mM phosphate buffer, pH 7.0 (2 X 10-4M EDTA and
5 X 10-3 M 2-mercaptocthanot), for four periods of 30 seconds at top
speed in a Waring blendor. The homogenate is filtered through cheese-
cloth and centrifuged for 1 hour at 14,000 g; the supernatant solution is
decanted, and filtered again through cheesecloth. To the resulting
extract, referred to as "initial extract" are added sufficient neutralized
GSH to produce a concentration of 0.5 mM and a few drops of toluene.
This extract is aged for 25 hours at 30 ° in a water bath, stored overnight
at 4 °, and then centrifuged at 14,000 g for 50 minutes.
Ammonium Sulfate Fractionation. The clear yellow supernatant
solution from the previous step is brought to 55% saturation with solid
7E. Layne, Vol. III, p. 451.

*Peanut seed used in our laboratory was obtained from the Peanut Growers Co-
operative, Franklin, Virginia. Since the Virginia botanical type is dormant for a
variable period after harvest, it is important to specify at the time of purchase
that seeds are intended for immediate germination.
ammonium sulfate s by gradual addition of the salt (0.351 g/ml) with

gentle stirring. After the suspension has stood overnight, it is centrifuged
and the precipitate is dissolved in 300 ml of 20 mM phosphate buffer, pH
6.5, 10 mM 2-mercaptoethanol and 0.2 mM EDTA, and the enzyme
solution is dialyzed against 8 liters of the same buffer for 8 hours.
After dialysis, the enzyme solution is centrifuged to remove the volumi-
nous precipitate (globulins) formed during dialysis. The supernatant
solution (60-80 mg of protein per milliliter) is diluted with the dialysis
buffer to produce a protein concentration of 40 mg/ml. Saturated am-
monium sulfate, 1° pH 7.5 (0.471 mg per milliliter of enzyme solution),
is chilled to 4 ° and the enzyme solution is added to it with gentle
mixing. The resulting 32% saturated ammonium sulfate solution is al-
lowed to stand for 30 minutes and is then centrifuged at 15,000 g for 15
minutes. The supernatant solution is added to sufficient chilled saturated
ammonium sulfate, pH 7.5 (0.172 ml per milliliter of supernatant solu-
tion), to produce a 42% saturated ammonium sulfate solution. After
it has stood for 8-10 hours, the suspension is centrifuged as before; the
precipitate is dissolved in 225 ml of 20 mM phosphate buffer, pH 6.5
(10 mM 2-mercaptoethanol and 0.5 mM EDTA), and then dialyzed
against 8 liters of the same buffer for 8 hours.
Ecteola- and DEAE-Cellulose Chromatography. The dialyzed enzyme
solution from the previous step is applied to an Ecteola-cellulose column
(4.5 em diameter; 11.2 g of dry Ecteola-cellulose per gram of protein;
exchange capacity approximately 0.3 meq/g; Type 20 epichlorohydrin
triethanolamine cellulose, Carl Schleicher and Schuell Co.) equilibrated
previously with 20 mM phosphate buffer, pH 6.5 (10 mM 2-mercapto-
ethanol and 0.5 mM EDTA). The enzyme is eluted with the same buffer
and appears in the breakthrough peak. The column effluent is monitored
continuously at 253 n ~ (LKB Uvicord absorptiometer) and carboxylase
activity is located with the spectrophotometric assay method. The pooled
earboxylase-containing fractions (approximately 280 ml, 8 g of protein,
and 8 0 ~ of the carboxylase activity applied) are applied immediately to
a DEAE-cellulose column (4.5 X 45 em; exchange capacity 0.8 meq/g;
Type 20 diethylaminoethyl cellulose, Carl Sehleieher and Sehuell Co.)
equilibrated previously with 20 mM phosphate buffer, pH 6.5 (10 mM
2-mercaptoethanol and 0.5 mM EDTA). Stepwise, gradient elution is
accomplished by placing 950 ml of the same buffer in a mixing chamber
attached to the column and introducing the following phosphate buffers,
' A l l the "percentage of a m m o n i u m sulfate saturation" figures obtained with solid
a m m o n i u m sulfate refer to percentage of saturation at 25 ° .
*~A m m o n i u m sulfate is saturated at room temperature and neutralized with NH~OH
so t h a t when diluted 5-fold the p H is t h a t indicated.
[43] PHOSPI1OPYRUVATECARBOXYLASE--PEANUTCOTYLEDONS 281
all pH 6.5, into a separatory funnel attached to the mixing chamber: 2

liters of 0.2 M and 2 liters of 0.4 M containing 10 mM 2-mercaptoethanol
and 0.5 mM EDTA. The effluent is monitored continuously for protein
and collected fractionally; the fractions are assayed for P-enolpyruvatc
carboxylase activity. Enzyme activity appears in the cluate after about
2400 ml has been collected. The most "active" fractions (approximately
600 ml) containing 50 or 60% of the carboxylase activity applied to
the column are pooled, placed in dialysis bags, and then dialyzed against
an ammonium sulfate solution, pH 6.5 (0.5 mM EDTA and 10 mM
2-mercaptoethanol), of sufficient concentration to reach 60% saturation
at equilibrium.
Hydroxylapatite Chromatography. The precipitated protein (250-350
rag) recovered from the preceding step by centrifugation is dissolved in
15 ml of 20 mM phosphate buffer, pH 6.5 (10 mM 2-mercaptoethanol),
and applied to a hydroxylapatite 11 column (2 >( 15 cm) previously equili-
brated with the same buffer. Stepwise elution is carried out under pres-
sure (2-4 psi) with 10 ml of 20 raM, 80 ml of 0.1 M, 100 ml of 0.2 M,
100 ml of 0.3 M, and 100 ml of 0.5 M phosphate buffers, pH 6.5, con-
taining 10 mM 2-mercaptoethanol. P-enolpyruvate carboxylase activity
and protein concentration in the eluted fractions (3 ml) are determined
as described earlier. Elution of peak enzyme acitvity occurs during the
addition of the 0.3 M buffer. These fractions are pooled (70-80 ml) and
precipitated at 60% ammonium sulfate saturation (pH 6.5) in the
presence of 10 mM 2-mereaptoethanol and 0.5 mM EDTA by the dialysis
technique described earlier.
Sephadex G-200 Gel Filtration. The enzyme suspension (10-20 mg of
protein) from hydroxylapatite chromatography is centrifuged, the pre-
cipitate is redissolved in 1 ml of 20 mM phosphate buffer, pH 6.5 (con-
taining 0.5 mM EDTA, 0.5 mM GSH, and 0.1 mM P-enolpyruvate), and
the solution is applied to a Sephadex G-200 column (1.5)< 85 cm; Phar-
macia Fine Chemicals, Inc.) equilibrated previously with the same
buffer. Sephadex G-200 columns of this dimension have a flow rate of
about 5 ml per hour under a hydrostatic head of 20 cm of buffer. The
enzyme is eluted with the equilibrating buffer and appears in the
eluate after approximately 45 ml have been collected. Enzymatic activ-
ity and protein concentration in the eluted fractions are determined as
described previously. The Ve: Vo ratio (where Ve = elution volume and
Vo ~ void volume) was found to be 1.36. This value corresponds to a
molecular weight of 3 to 3.5 X l05 when related to a plot of log molec-
ular weight against Ve:Vo obtained from Sephadex G-200 elution data
~ A. Tiselius, S. Hjert~n, and O. Levin, Arch. Biochem. Biophys. 65, 132 (1956).
for a series of proteins of known molecular weight. Fractions containing
maximum carboxylase specific activity are pooled and precipitated at
6 0 ~ ammonium sulfate saturation, p H 6.5 (0.5 m M E D T A , 0.5 m M
GSH, and 0.1 m M P-enolpyruvate), by the dialysis technique described
earlier.
As indicated in the table, a 2800-fold purification of P-enolpyruvate
carboxylase from the initial extract is achieved in 5% yield with the
procedure outlined. The purity of carboxylase preparations having a
specific activity of 27-28 units per milligram of protein is approximately
8 0 ~ as determined by sedimentation velocity analysis. 1 When stored as
a suspension under 60% saturated ammonium sulfate (containing 0.5
m M E D T A and GSH) at 4 ° the enzyme is stable for at least 2 or 3
months. In dilute solution at low ionic strength" (5 m M Tris-HCl, pH
7.6), carboxylase activity is lost at about 10-15% per hour.
PURIFICATION OF PHOSPHOENOLPYRUVATE CARBOXYI~SE
Total Specific
activity~ activity Yield
Step Proteins (units) (units/mg) (%)
1. Initial extractc 598 6230 0.0104 100

2. Aged extract 207 6820 0.033 109
3. 0-55% saturated (NH4)~SO4fraction 45.3 4960 0.109 80
4. 32-42% saturated (NH4)~S04fraction 10.4 3720 0.357 60
5. Ecteola-cellulose eluate 7.00 3020 0.432 48
6. DEAE-cellulose chromatographic 0.286 1730 6.08 28
fraction
7. Hydroxylapatite chromatographic 0.020 305 15.2 4.9
fraction
8. Sephadex G-200 gel filtrate 0.010 292 28.1J 4.7
a Determined spectrophotometrically according to the method of Layne (Vol. III,
p. 451).
Determined with the spectrophotometric P-enolpyruvate carboxylase assay method
with exception of initial and aged extracts; H ~4COs- fixation assay was used for the
latter extracts. A unit is the amount of enzyme required to catalyze the carboxyla-
tion of 1.0 micromole of P-enolpyruvate per minute under the assay conditions.
c Initial extract was obtained from 6 kg of peanuts after germination and homogeni-
zation.
d Specific activity based on refractometrically determined protein is 49.6 units/mg
(see section "Molecular Properties" for details).
Properties
Kinetic Properties and Inhibitors. The p H optimum for P-enolpy-
ruvate carboxylase determined with the spectrophotometric carboxyla-
tion assay is approximately 7.9. The Km values determined for HC0a-,
[44] PHOSPHOPYRUVATECARBOXYLASE--S. typhimurium 283
P-enolpyruvate, and Mg *+ are 0.31 mM, 0.5-0.6 raM, and 0.3--0.4 raM,
respectively, at pH 7.9. The carboxylase is reversibly inhibited by
p-chloromercuribenzoate. 4 0 r t h o p h o s p h a t e and DL-phospholactate are
competitive inhibitors with respect to P-enolpyruvate and have K ' s of
5.5 mM and 0.11 mM, respectively. 12 The insensitivity of P-enolpyruvate
carboxylase action to avidin indicates that biotin is not a prosthetic
group for this enzyme. 4
Molecular Properties. P-enolpyruvate carboxylase from peanut coty-
ledons has a sedimentation coefficient (S,.o,~) of 13.9 S and an estimated
molecular weight (Sephadex G-200 gel filtration experiments) of 350,000.
To convert protein determined by the method of Layne 7 to refracto-
metrically determined protein, the former should be multiplied by a
factor of 0.567. The absorbance ratio (A2so:A26o) of the purified enzyme
is approximately 1.8.
1-.R. L. Easterday and M. D. Lane, unpublished observations, 1965.
[44] Phosphoenolpyruvate Carboxylase from

Salmonella typhirnurium, S t r a i n L T 2
[EC 4.1.1.31 Orthophosphate:oxaloacetate carboxy-lyase (phosphorylating) ]
By P. MAEBA and B. D. SANWAL
Assay Method
Principle. The activity of phosphoenolpyruvate carboxylase is assayed
routinely by a coupled system in which the oxaloacetate formed is reduced
to malate by N A D H + H ÷ in the presence of malate dehydrogenase. The
rate of oxidation of NADH is measured spectrophotometrically as the
decrease of absorbanee at 340 ml~. A summary of the reaction system is
given below:
P-enolpyruvate + H C O a - ~ oxaloacetate + t', (1)
Oxaloacetate ~- NADH -t- H + ~ malate + NAD + (2)
P-enolpyruvate + HCOa- -t- NADH -t- H + ~ malate + P~ + NAD + (3)

Reagents
NADH, 2 mM
MgCI~.6 H20, 0.6 M
NaHCOa, 0.6 M, prepared fresh
[44] PHOSPHOPYRUVATECARBOXYLASE~S. typhimurium 283
P-enolpyruvate, and Mg *+ are 0.31 mM, 0.5-0.6 mM, and 0.$-0.4 raM,
respectively, at pH 7.9. The carboxylase is reversibly inhibited by
p-chloromercuribenzoate. 4 Orthophosphate and DL-phospholactate are
competitive inhibitors with respect to P-enolpyruvate and have K~'s of
5.5 mM and 0.11 mM, respectively. 12 The insensitivity of P-enolpyruvate
carboxylase action to avidin indicates that biotin is not a prosthetic
group for this enzyme. 4
Molecular Properties. P-enolpyruvate carboxylase from peanut coty-
ledons has a sedimentation coefficient (S~.o,~) of 13.9 S and an estimated
molecular weight (Sephadex G-200 gel filtration experiments) of 350,000.
To convert protein determined by the method of Layne 7 to refracto-
metrically determined protein, the former should be multiplied by a
factor of 0.567. The absorbance ratio (A2so:A26o) of the purified enzyme
is approximately 1.8.
1: R. L. Easterday and M. D. Lane, unpublished observations, 1965.
[44] Phosphoenolpyruvate Carboxylase from

Salmonella typhirnurium, S t r a i n L T 2
[EC 4.1,1.31 Orthophosphate:oxaloacetatc carbox3"-lyase (phosphorylating)]
By P. MA~BA and B. D. SANWAL
Assay Method
Principle. The activity of phosphoenolpyruvate carboxylase is assayed
routinely by a coupled system in which the oxaloacetate formed is reduced
to malate by N A D H + H ÷ in the presence of malate dehydrogenase. The
rate of oxidation of NADH is measured spectrophotometrically as the
decrease of absorbance at 340 mt~. A summary of the reaction system is
given below :
P-enolpyruvate + H C O 3 - ~ oxaloaeetate + t', (1)
Oxaloacetate + NADH + H + ~ malate + NAD + (2)
P-enolpyruvate + HCO3- + NADH + H + ~ malate + P~ + NAD + (3)
Reagents
NADH, 2 mM
MgCI~.6 H20, 0.6 M
N a H C Q , 0.6 3I, prepared fresh
Sodium phosphoenolpyruvate, 0.1 M

Malate dehydrogenase, 0.15 mg/ml. An ammonium sulfate suspen-
sion of pig heart enzyme (obtained from Boehringer and Soehne)
is diluted to give this concentration.
Procedure. The assay mixture contains Tris-HC1 buffer, 2.0 ml;

NADH, 0.1 ml; MgCl~, 0.05 ml; NaHC08, 0.05 ml; phosphoenolpyru-
vote, 0.1 ml; and malate dehydrogenase, 0.1 ml; suitably diluted enzyme
preparation and distilled water to make a total volume of 3.0 ml. The
oxidation of NADH is measured in 1 cm light path silica cuvettes with
the use of a spectrophotometer at 24-25 °. The reaction is started by the
addition of the enzyme. NADH oxidizing activity is present in cruder
preparations; when this is so, a control cuvette lacking phosphoenol-
pyruvate is necessary. The absorbancy change due to the control is
subtracted from the readings obtained from the test cuvette.
Units. One unit of phosphoenolpyruvate carboxylase is defined as the
amount of enzyme causing an absorbancy change of 0.001 per minute
with the above assay system. Specific activity is expressed as units per
milligram of protein. Protein is de~ermined by the colorimetric method
of Lowry et al. 1
Media and Preparation o] Crude Extract. Salmonella typhimurium
strain LT 2 is grown in 20-liter earboys with forced aeration at 30 ° in
a glucose-salts medium containing K2HP04, 10.5 g; KH2P0,, 4.5 g;
(NH4)~S04, 1.0 g; MgS04, 0.05 g; and glucose, 40 g, per liter of distilled
waber. The cells are harvested in the late log phase with a Sharples cen-
trifuge. The cells are washed once with NaC1, 0.14 M, then suspended in
2 volumes (w/v) of ice cold Tris-ttC1 buffer, pH 8.0, containing MgC12.
The cell suspension is disrupted sonically in 100 ml batches with a
Raytheon 10 ke sonic oscillator fitted with a cooling jacket. Exposure
times of 45-60 minutes are required. The preparation is allowed to stand
overnight at 2 ° . All following steps are carried out at 0-4 ° , and all
buffers used contain 1 mM MgCI~. The extract is centrifuged at 27,000 g
for 20 minutes to remove cell debris and precipitated material. The
supernatant is subjected to further centrifugation in a Spinco type 30
rotor for 90 minutes at 30,000 rpm; the gelatinous sediment, which
contains most of the NADH oxidase activity, is discarded. The super-
natant constitutes the crude extract and is used for the purification of
the enzyme.
10. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193,
265 (1951).
[44] CXR~OXYLASV---S. typhimurium
PHOSPHOPYRUVATE 285
Step 1. Precipitation with Streptomycin and Protamine Sul]ate. One-

fifth volume of a 5% streptomycin sulfate solution, pH 7.0, is added
slowly with stirring to the crude extract, followed immediately by the
addition of 1/20 volume of 2% protamine sulfate, pH 6.5. The suspension
is stirred for 15 minutes and then centrifuged at 10,000 g for 30 minutes.
l'he precipitate is discarded, and the supernatant is fractionated with
ammonium sulfate.
Step ~. Ammonium SulIate Fractionation. The supernatant is made
0.1 mM with dithiothreitol, then solid ammonium sulfate is added slowly
with constant stirring to give 40% saturation. The mixture is stirred for
15 minutes and then centrifuged 20 minutes at 10,000 g. The bulky
precipitate is discarded, and the supernatant is brought to 55% satura-
tion by the slow addition of solid ammonium sulfate. The suspension is
stirred for 30 minutes and the precipitate that is collected after eentrifu-
gation is dissolved in 50 mM Tris-HC1 buffer, pH 8.0. The final volume
at this stage is one-tenth that of the original crude extract.
Step 3. Reprecipitation with Ammonium Sul]ate. Two volumes of 2%
protamine sulfate, pH 6.5, are added to the preparation from the previous
step and stirred for 1 hour. The mixture is made 0.1 mM with dithio-
threitol and is brought to 40% saturation with respect to ammonium
sulfate. The precipitate is stirred for 20 minutes, then collected by centrif-
ugation and dissolved in 10 mM Tris-HCl buffer, pH 8.0, to a volume
cqual to one-tenth of the original crude extract. A further centrifugation
at I5,000 g for 10 minutes is required to remove insoluble material which
contains little activity.
Step ~. Calcium Phosphate Gel Adsorption and Elution. With stirring,
a suspension of calcium phosphate gel (41 mg/ml) is added to the enzyme
preparation so that the gel to protein ratio is 2:1. The mixture is stirred
for 20 minutes then centrifuged to pack the gel; the yellow supernatant
is discarded.
The calcium phosphate gel is washed once with 0.2M ammonium
sulfate and twice with 0.4 M ammonium sulfate, both pH 8.0. After each
washing, the gel is packed by centrifugation and the yellowish super-
natant is discarded. The enzyme is eluted from the gel with 0.6M
ammonium sulfate, pH 8.0; a total of 5 elutions may be required to
remove most of the enzyme from the gel. Each wash and elution step
consists of suspending the gel in the appropriate solution using one-third
the original volume adsorbed and stirring with a magnetic stirrer for
10 minutes. The eluted fractions are pooled and brought to 55% saturation
with ammonium sulfate. The precipitated enzyme, after centrifugation,
is dissolved in 5 mM Tris-HCl buffer, pH 8.0.
Step 5. DEAE-Cellulose Chromatography. The enzyme preparatio~
from the preceding step is dialyzed 3 hours against frequent changes of

5 mM Tris-HC1 buffer, pH 8.0, before it is applied to a DEAE-cellulose
column (2.5 X 40 cm) equilibrated previously with 5 mM Tris-HC1
buffer, pH 8.0. The enzyme is eluted with a linear gradient of KCI. The
mixing chamber contains 900 ml of the equilibrating buffer and the
reservoir contains 0.75 M KC1 in 900 ml of equilibrating buffer. Fractions
of 6 ml are collected, and the tubes containing enzyme activity are pooled.
The enzyme is eluted off the column at 0.25 to 0.3 M KC1 and is con-
centrated by adding ammonium sulfate to 75~ saturation, adjusting to
pit 8.0 with 1.0 N NaOH, stirring for 1 hour, and centrifuging.
Step 6. Ammonium Sulfate Extraction. The ammonium sulfate pre-
cipitate from the preceding step is extracted with 5 ml quantities of
various ammonium sulfate solutions at pH 8.0. Two initial extractions
with 26 and 21.5~ (w/v) ammonium sulfate contain little activity, and
after eentrifugation the supernatant fluids are discarded. The pellet is
extracted repeatedly with 18% (w/v) ammonium sulfate until most of
the enzyme has been extracted. The supernatant fluids from the latter
extractions are pooled, brought to room temperature in 30 minutes, and
then allowed to stand in a small, open beaker at 2-4 °. A precipitate
begins to appear within 2 hours. After 2 days the precipitate is harvested
by centrifugation and resuspended in one-tenth the original volume of
ammonium sulfate employed for extraction. In this state the enzyme is
stable for at least one month.
The purification scheme given is a modification of the method of
Chnovas and Kornberg ~ and of Sanwal and Maeba2 The accompanying
table outlines the procedure.
Purity. The enzyme is almost homogeneous, showing only one major
and several minor bands on disc electrophoresis in polyacrylamide gel.
The following enzymes were either absent or undetectable in the prepara-
tion: phosphoenolpyruvate kinase, phosphoenolpyruvate carboxykinase,
ATP-linked pyruvate carboxylase, nucleoside diphosphokinase, nucleo-
side triphosphate-nucleoside monophosphate kinase, DPN-specifie malate
dehydrogenase, glutamate-aspartate transaminase, condensing enzyme.
TPN'-linked glutamate dehydrogenase, aldolase, "malic enzyme," and
lactate dehydrogenase.
pH Optimum. Phosphoenolpyruvate earboxylase tested in Tris-ttC1
buffer is maximally active at ptt 8.7-9.2. On the more alkaline side,
pH 9.5-10.5, the activity drops only gradually, but at pH 7.5 the enzyme
shows less than one-fourth of the activity obtained at pH 9.0.
2j. L. C~hmvas and H. L. Kornberg, Proc. Roy. Soc, B I ~ , 189 (1966).

~B. D. Sanwal and P. Maeba, J. Biol. Chem. 241, 4557 (1966).
[44] PROSPHOPYRUVATECARBOXVLAsE--S. t y p h i m u r i u m 287
PURIFICATION OF PHOSPttOENOLPYRUVATE CARBOXYLASE FROM ~. typhimurium ~
Total Purifi-
Volume protein Total Specific Yield cation
Step and fraction (ml) (rag) units activity~ (%) (fold)
Crude extract 290 10,005 1,218,000 122c 100 1.0

1. Supernatant from 315 8,127 1,205,000 148 99 1.2
streptomycin and
protamine sulfate
precipitation
2. Ammonium sulfate 30 1,350 1,083,000 802 89 6.6
0.4-0.55 saturation
3. Reprecipitation with 30 459 900,000 1,961 74 16.1
protamine sulfate
and 0.4 saturation
with ammonium
sulfate
4. Calcium phosphate 6 81 684,000 8,444 56 69.4
gel elution
5. DEAE 150 15 250,000 16,666 20.5 136.9
chromatography
6. Ammonium sulfate 3 5.6 87,200 17,200 8.0 141.3
extraction
a From 120 g of cells, wet weight.
b Units per milligram of protein.
c This value can be considered only approximately because the presence of a strong
NADH-oxidizing activity interferes with the assay procedure.
Properties
The protein has a molecular weight of 183,000 ± 8000 as determined
by zone centrifugation in sucrose density gradients according to the
procedure of M a r t i n and Ames. 4
E n z y m e activity is greatly enhanced in the presence of nonpolar
solvents, e.g., ethanol, dioxane. This effect has been interpreted to mean
t h a t the protein has hydrophobic regions accessible to these reagents and
t h a t weakening of the hydrophobic interactions leads to activation. Areas
on the enzyme bearing a preponderance of negatively charged groups
have also been implicated in the protein structure. These areas bind poly-
lysine and other polycations resulting in changes in kinetic parameters
and in heat stability2
K i n e t i c constaT~ts. At p H 9.0 in T r i s - H C l buffer using a Mg +* con-
centration of 10 m M , the Km value for phosphoenolpyruvate has been
evaluated as 11.2 mM, and for bicarbonate as 1.13 m M . In the presence
'R. G. Martin and B. N. Ames, J. Biol. Chem. 236, 1372 (1961).
*B. D. Sanwal, P. Maeba, and R. A. Cook, J. Biol. Chem. 241, 1372 (1966).
288 REACTIONS L E A D I N G TO A N D F R O M T H E CYCLE [45]
of 15 mM phosphoenolpyruvate and 20 mM bicarbonate, the K~ for

Mg ~* is 1.8 mM. With saturating concentrations of activators, e.g., acetyl-
CoA, the K,, value for phosphoenolpyruvate changes to 1 mM.
Activation and Inhibition. Phosphoeno]pyruvate carboxylasc is an
important regulatory enzyme and is subject to inhibition by aspartate 6
and activation by acetyl-CoA, 2 fructose-l,6-diphosphate, T CDP, CTP,
and GTP. 5 The physiological role of the enzyme and the role that these
allosteric effectors play in end-product inhibition, anaplerotic sequences,
and compensatory feedback effects have been described by Kornberg 8 and
Sanwal et al?,~,,
The sites for the effectors are distinct from each other and thab of
polylysine. 5 Although the enzyme cannot be desensitized by treatment
with heat or mercurials, dioxane (10%) can bring about desensitization
to the inhibitory effect of aspartate and the activating effect of fructose
diphosphate. 5
The K~ value for aspartate evaluated with bicarbonate at 25 mM and
phosphoenolpyruvate at 20 mM is 0.8 mM. The Kaetivatlon values evalu-
ated at 1.66 mM phosphoenolpyruvate are 0.5 mM for acetyl-CoA, 4.0
mM for CDP, 1.2 mM for CTP, and 5.0 mM for GTP.
Phosphoenolpyruvate carboxylase from Escherichia coli K12 has been
purified by this method. The properties of this enzyme are the same as
that from Salmonella.
P. Maeba and B. D. Sanwal, Biochem. Biophys. Res. Commun. 21, 503 (1965).
7B. D. Sanwal and P. Maeba, Biochem. Biophys. Res. Comraun. 22, 194 (1966).
' H. L. Kornberg, Essays Biochem. 2, 1 (1966).
[45] Phosphoenolpyruvate C a r b o x y l a s e f r o m E s c h e r i c h i a coil

[EC 4.1.1.31 Orthophosphate:oxaloaeetat~ earboxy-lyase (phosphorylating)]
By J. L. CXNOVASand H. L. KORNBmG
acetyl-CoA
Phosphoenolpyruvate T C02 . , oxaloacetate q- P~
Assay Method
Principle. The formation of oxaloacetate from the carboxylation of
phosphoenolpyruvate (PEP) is measured by coupling its reduction to
malate with concomitant oxidation of reduced nicotinamide adenine
dinucleotide [NADH2]. This is measured as the rate of change in ex-
tinction at 340 m~.
288 REACTIONS L E A D I N G TO A N D F R O M T H E CYCLE [45]
of 15 mM phosphoenolpyruvate and 20 mM bicarbonate, the K~ for

Mg ~* is 1.8 mM. With saturating concentrations of activators, e.g., acetyl-
CoA, the K,, value for phosphoenolpyruvate changes to 1 mM.
Activation and Inhibition. Phosphoeno]pyruvate carboxylasc is an
important regulatory enzyme and is subject to inhibition by aspartate 6
and activation by acetyl-CoA, 2 fructose-l,6-diphosphate, T CDP, CTP,
and GTP. 5 The physiological role of the enzyme and the role that these
allosteric effectors play in end-product inhibition, anaplerotic sequences,
and compensatory feedback effects have been described by Kornberg 8 and
Sanwal et al?,~,,
The sites for the effectors are distinct from each other and thab of
polylysine. 5 Although the enzyme cannot be desensitized by treatment
with heat or mercurials, dioxane (10%) can bring about desensitization
to the inhibitory effect of aspartate and the activating effect of fructose
diphosphate. 5
The K~ value for aspartate evaluated with bicarbonate at 25 mM and
phosphoenolpyruvate at 20 mM is 0.8 mM. The Kaetivatlon values evalu-
ated at 1.66 mM phosphoenolpyruvate are 0.5 mM for acetyl-CoA, 4.0
mM for CDP, 1.2 mM for CTP, and 5.0 mM for GTP.
Phosphoenolpyruvate carboxylase from Escherichia coli K12 has been
purified by this method. The properties of this enzyme are the same as
that from Salmonella.
P. Maeba and B. D. Sanwal, Biochem. Biophys. Res. Commun. 21, 503 (1965).
7B. D. Sanwal and P. Maeba, Biochem. Biophys. Res. Comraun. 22, 194 (1966).
' H. L. Kornberg, Essays Biochem. 2, 1 (1966).
[45] Phosphoenolpyruvate C a r b o x y l a s e f r o m E s c h e r i c h i a coil

[EC 4.1.1.31 Orthophosphate:oxaloaeetat~ earboxy-lyase (phosphorylating)]
By J. L. CXNOVASand H. L. KORNBmG
acetyl-CoA
Phosphoenolpyruvate T C02 . , oxaloacetate q- P~
Assay Method
Principle. The formation of oxaloacetate from the carboxylation of
phosphoenolpyruvate (PEP) is measured by coupling its reduction to
malate with concomitant oxidation of reduced nicotinamide adenine
dinucleotide [NADH2]. This is measured as the rate of change in ex-
tinction at 340 m~.
[45] PHOSPHOPYRUVATE
CARBOXYLASE--E. coli 289
Reagents
MgCI2, 50
NADH2, 10 mM
KHC03, 0.1 M, freshly prepared
Acetyl eoenzyme A, 1 10 mM
PEP, potassium salt, 50 mM
Crystalline malate dehydrogenase
Enzyme
Procedure. Place in a silica cuvette (1 cm light path, approximately
1.5 ml volume) 100 micromoles of Tris-HC1 buffer, pH 8.5, 0.1 micromole
of NADH2, 5 micromoles of MgCI~, 10 micromoles of KHC03, 0.5 micro-
mole of acetyl-CoA, approximately 2 IU of crystalline malate dehydro-
genase, enzyme and water to 0.9 ml; a blank cuvette receives the same
materials, but with NADH2 omitted. Any changes in extinction at 340
n~, which may be caused by NADH2-oxidase in the enzyme preparation,
are recorded for 1-2 minutes (NADH2-oxidase activity is likely to cause
difficulty only when crude extracts are used, and most of this interfering
enzyme can be removed from such extracts either by adding ammonium
sulfate to 40% saturation and centrifuging, or by centrifuging the ex-
tracts at 100,000 g for 30 minutes). If no change in extinction is observed,
or if such extinction changes are small and constant, 0.1 ml of the P E P
solution is added, and the linear rate of decrease in extinction at 340 m~
is recorded thereafter.
the oxidation of 1 micromole of NADH~ per minute under the assay
conditions stated, and hence catalyzes AE = 6.22 units per minute. Spe-
cific activities are expressed as units of enzyme per milligram of protein.
Protein is determined by the method of Lowry et al. 2
This method has been used 3 for the purification of the enzyme from
E. coli strain W, grown aerobically at 30 ° on a medium containing 50
mM glycerol as carbon source. Cells are harvested toward the end of the
logarithmic growth phase, at densities of 0.6--0.75 mg dry weight, per
milliliter.
Step 1. Harvested cells are suspended in a final concentration of 30
IE. R. Stadtman, Vol. I I I [137].

s O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, 3. Biol. Chem. 193,
265 (1951).
J. L. C/movas and H. L. Kornberg, Proc. Roy. 8oc. B165, 189 (1966).
mg dry weight per milliliter in buffer, pH 8.0, containing 5 mM Tris-

HC1 and 1 mM MgCl~ and are disrupted in batches of 15 ml by exposure
for 5 minutes to an MSE 60W ultrasonic oscillator. The suspensions are
cooled in iced water during sonication. The resultant extracts are com-
bined and centrifuged at 20,000 g for 10 minutes at 2°; the precipitated
material is discarded. All subsequent operations are performed at 2 ° .
Step ~. Add to the supernatant solution, with constant stirring, 2%
(w/v) protamine sulfate dissolved in 2 mM sodium acetate buffer, pH
5.0; it suffices to add 1 mg of protamine sulfate for each 10 mg of
protein in the supernatant solution. Let the mixture stand for half an
hour and then centrifuge it at 20,000 g for 10 minutes; discard the pre-
cipitated material.
Step 3. Add ammonium sulfate to the supernatant solution, with
constant stirring, to 40% of saturation. After it has stood for 15 minutes,
centrifuge the material at 20,000 g for 10 minutes and discard the pre-
cipitate. Again add ammonium sulfate to the supernatant solution to
bring the concentration to 50% of saturation, let the mixture stand for
15 minutes, then centrifuge it. Dissolve the precipitate in a small quan-
tity (10-15 ml) of buffer, pH 8.0, containing 5 mM Tris-HC1 and 1 mM
MgClz; dialyze the sample overnight against 2 liters of the same buffer.
Remove and discard any material precipitated during dialysis.
Step ~,. To the dialyzed solution, add alumina Cr-gel (approximately
3.5 rag, dry weight, of alumina per 10 mg of protein). Stir the suspension
for 5 minutes, remove the precipitate by centrifugation, and discard.
Step 5. Wash DEAE-cellulose (Whatman DE-50) with 0.5 N NaOH,
and then with water. Suspend the washed material in buffer containing
5 mM Tris-HC1 and 1 mM MgC12; readjust the pH of the suspension to
8.0 by the addition of HC1. Wash the cellulose several times with the
buffer; remove by decantation particles which do not sediment. Pour
the slurry into a chromatographic column (2 cm X 30 cm), closed with
glass wool at its lower end, and equilibrate it with 2-3 liters of the same
buffer. Apply the material obtained after treatment with Cr-gel to the
top of the column, then elute it with an increasing gradient of KC1. This
gradient may be obtained conveniently by allowing 500 ml of a solution
containing 1 mM MgCl2, 5 mM Tris-HC1, pH 8.0, and 750 mM KC1 to
flow, with constant stirring, into 500 ml of buffer w.ithout KC1. The
resultant mixture is permitted to flow through the column at 50-60
ml/hour. Fractions, each containing 10 ml, are collected automatically.
The enzyme appears in those fractions eluted at concentrations of
chloride4 between 0.13 and 0.19M with maximal activity at 0.15M.
~Estimated by the procedure of P. W. West and N. Coll, J. Am. Water Works
Assoc. 49, 1485 (1957).
[45] CARBOXYLASE--E. coli
PHOSPHOPYRUVATE 291
Those fractions containing the enzyme at specific activities greater than

1.25 are combined, and the enzyme is precipitated by addition of solid
ammonium sulfate to a final concentration of 75~o of saturation.
Step 6. The precipitate is extracted with decreasing concentrations of
ammonium sulfate dissolved in 1 m M MgCl~-5 m M Tris-HC1 buffer,
pH 8.0. For each extraction, the suspension is stirred slowly for 10
minutes and is then centrifuged. [Reproducible results were obtained
when the extracting solutions used were 10 ml of 30% (w/v) armnonium
sulfate in the buffer, 5 ml of 26.5% (w/v) ammonium sulfate in the
buffer, and 5 ml of 21.5% (w/v) ammonium sulfate in the buffer; these
solutions were used in the order given.] Most of the P E P carboxylase
activity is extracted by the final solution used. The procedure is sum-
marized in the table.
PURIFICATION OF P E P CARBOXYLASE FROM E. coli, STRAIS W
Total Total PEP Specific

protein carboxylase activity
Volume content content (units/rag Recovery
Step and fraction (ml) (rag) (units)" protein) (%)
1. Crude sonic extract 235 3219 188 0.058
2. Supernatant solution 240 2448 192 0.078 102
after protamine sulfate
treatment
3. Material precipitated 15 401 136 0.34 72
by ammonium sulfate
(40-50% saturation)
4. Supernatant solution 23 267 127 0.47 67
after C~-gel treatment
5. Selected fractions from 40 33 72 2.18 38
DEAE-cellulose column
6. Material extracted by 4.3 6 58 9.68 31
21.5% (w/v) ammonium
sulfate
"One unit catalyzes the oxidation of 1 micromole of NADH2 per minute under the
assay conditions.
Properties
Stability. The 160-fold purified enzyme obtained in step 6 is rela-
tively stable when stored at 3 °, losing less than 2 0 ~ of its activity in 2
weeks; however, it loses nearly half of its activity when stored for this
length of time at room temperature.
Specificity and pH Optimum. The enzyme appears to be wholly
specific for phosphoenolpyruvate, and catalyzes its carboxylation over a
wide range of pH, with maximal activity at pH 8.5.
Activators and Inhibitors. The enzymatic reaction requires the pres-

ence of divalent metal ions (Mg ~ ~ Mn ~+ ~ Co ÷t) : the Km for Mg *÷ is
0.98 raM. Although the enzymatic formation of oxaloacetate from phos-
phoenolpyruvate and carbon dioxide is detectable when acetyl-CoA is
omitted, its rate is stimulated more than 30-fold by acetyl-CoA; other
acyl-CoA derivatives are less effective in promoting this increased activ-
ity (aeetyl-~ propionyl-~> butyryl-~ acrylyl-~ crotonyl-CoA). The
K~ for acetyl-CoA is 0.14 mM. The effect of acetyl-CoA is catalytic, and
increases the apparent affinity of the enzyme for PEP from K~----5.5
mM to 0.64 raM. It has been reported 5 that the PEP carboxylase of
Salmonella typhimurium, which is activated by acetyl-CoA, may also be
stimulated by fruc~se-l,6-diphosphate; in the presence of acetyl-CoA,
82 ~ L-aspartate inhibits the activity of this enzyme6 by about 50~b.
' B. D. Sanwal and P. Maeba, Biochem. Biophys. Res. Commun. 22, 194 (1966).
'P. Maeba and B. D. Sanwal, Bioehem. Biophys. Res. Commun. 21, 505 (1965).
[40] P h o s p h o e n o l p y r u v a t e Carboxylase f r o m
Pseudomonas AM1
[EC 4.1.1.31 Orthophosphate:oxaloaeet~tecarboxy-lyase(phosphorylating)]
By J. R. Q u A ~
Phosphoenolpyruvate -P C02 --~ oxaloacetate -~ P~
This enzyme was first discovered in spinach by Bandurski and
Greiner~ and has since been found in several species of bacteria. The
purification from Pse~omonas AM1 and the properties which are de-
scribed here have been published previously. 2
A s s a y Methods
The enzyme may be assayed by two methods: (1) measurement of
the amount of isotope fixed into nonvolatile products from NaHI~COa;
(2) measurement of the rate of oxaloacetate formation by coupling its
reduction to malate with excess malate dehydrogenase at the expense of
added D P N H ; the rate of dehydrogenation of NADH is measured
speetrophotometrieally.
Method (2) is a more rapid and convenient assay than method (1),
but, unless special precautions are taken involving the use of anaerobic
R. S. Bandurski and C. M. Greiner, J. Biol. Chem. 204, 781 (1953).
~P. J. Large, D. Peel, and J. R. Quayle, Biochem. J. 8,5, 243 (1962).
Activators and Inhibitors. The enzymatic reaction requires the pres-

ence of divalent metal ions (Mg ~ ~ Mn ~+ ~ Co ÷t) : the Km for Mg *÷ is
0.98 raM. Although the enzymatic formation of oxaloacetate from phos-
phoenolpyruvate and carbon dioxide is detectable when acetyl-CoA is
omitted, its rate is stimulated more than 30-fold by acetyl-CoA; other
acyl-CoA derivatives are less effective in promoting this increased activ-
ity (aeetyl-~ propionyl-~> butyryl-~ acrylyl-~ crotonyl-CoA). The
K~ for acetyl-CoA is 0.14 mM. The effect of acetyl-CoA is catalytic, and
increases the apparent affinity of the enzyme for PEP from K~----5.5
mM to 0.64 raM. It has been reported 5 that the PEP carboxylase of
Salmonella typhimurium, which is activated by acetyl-CoA, may also be
stimulated by fruc~se-l,6-diphosphate; in the presence of acetyl-CoA,
82 ~ L-aspartate inhibits the activity of this enzyme6 by about 50~b.
' B. D. Sanwal and P. Maeba, Biochem. Biophys. Res. Commun. 22, 194 (1966).
'P. Maeba and B. D. Sanwal, Bioehem. Biophys. Res. Commun. 21, 505 (1965).
[40] P h o s p h o e n o l p y r u v a t e Carboxylase f r o m
Pseudomonas AM1
[EC 4.1.1.31 Orthophosphate:oxaloaeet~tecarboxy-lyase(phosphorylating)]
By J. R. Q u A ~
Phosphoenolpyruvate -P C02 --~ oxaloacetate -~ P~
This enzyme was first discovered in spinach by Bandurski and
Greiner~ and has since been found in several species of bacteria. The
purification from Pse~omonas AM1 and the properties which are de-
scribed here have been published previously. 2
A s s a y Methods
The enzyme may be assayed by two methods: (1) measurement of
the amount of isotope fixed into nonvolatile products from NaHI~COa;
(2) measurement of the rate of oxaloacetate formation by coupling its
reduction to malate with excess malate dehydrogenase at the expense of
added D P N H ; the rate of dehydrogenation of NADH is measured
speetrophotometrieally.
Method (2) is a more rapid and convenient assay than method (1),
but, unless special precautions are taken involving the use of anaerobic
R. S. Bandurski and C. M. Greiner, J. Biol. Chem. 204, 781 (1953).
~P. J. Large, D. Peel, and J. R. Quayle, Biochem. J. 8,5, 243 (1962).
[45] PHOSPHOPYRUVATE CARBOXYLASE--Pseudomonas A M 1 293
cuvettes, D P N H oxidase activity precludes its use in crude extracts. In

practice, therefore, method (1) is used for assay of crude extracts and
in enzyme purification, method (2) for work with purified enzyme.
Radioactive Assay
Principle. The amount of isotope fixed into nonvolatile products
(mainly malate and fumarate) from NaH14C03 in the presence of
phosphoenolpyruvate and D P N H is measured by radioassay on metal
planchets. The assay depends on the reduction of the primary product,
oxaloacetate, to malate, catalyzed by malate dehydrogenase. In the
early stages of purification, malate dehydrogenase is usually present in
excess in the enzyme fractions. After step 3 it is necessary to add
excess malate dehydrogenase to the assay mixture.
Reagents
MgC12, 10 mM
Glutathione, 10 mM
NaH14C03 (containing 20/LC of 14C/ml), 0.2 M
Sodium phosphoenolpyruvate, 25 mM
DPNH, 30 mM
Malate dehydrogenase, obtained from C. F. Boehringer and Soehne,
Mannheim, Germany, must be freed from ammonium sulfate
before use, as ammonium ions inhibit the phosphoenolpyruvate
earboxylase. The stock suspension of the commercial malate
dehydrogenase is diluted 10-fold with 0.01 M Tris-HC1 buffer,
pH 7.5, and is dialyzed for 2 hours against 1.5 liters of the same
buffer at 0 °.
Procedure. A sample of enzyme extract is incubated at 30 ° with
reaction mixture which contains Tris-HC1 buffer, 0.5 ml; MgCl~, 0.1 ml;
glutathione, 0.2 ml; NaH14CO~, 0.1 ml; phosphoenolpyruvate, 0.1 ml;
DPNH, 0.1 ml, and where appropriate, 375 units (according to the
assay of 0choa 3) of malate dehydrogenase. Water is added to a total
volume of 2 ml. After 30 minutes the reaction is stopped by the addition
of 3 ml of boiling 95% (v/v) ethanol; 0.1 nil samples are applied to
metal planchets (1 inch diameter) and dried in a stream of warm air.
The planche~ are then irrigated with 0.1 ml of 90% w/v formic acid
and dried. The nonvolatile radioactive fixation products are assayed
with a gas-flow counter at about 15% efficiency.
Units. One unit of enzyme is the amount of enzyme required to fix
1 millimicromole of 1~C0~ into nonvolatile products in 30 minutes. Spe-

cific activity is expressed as units of enzyme per milligram of protein.
Principle. The rate of oxaloacetate formation is measured by coupling
its reduction to malate with DPNH in the presence of excess malate
dehydrogenase; the rate of the resulting dehydrogenation of DPNH is
measured spectrophotometrically at 340 m~.
Reagents
Tris-HC1 buffer, 0.2 M pH 8.5
MgCl2, 10 mM
NaHCO~, 0.2 M
Sodium phosphoenolpyruvate, 25 mM
DPNH, 20 mM
Malate dehydrogenase, freed from ammonium sulfate as described
in the previous assay
Procedure. The complete reaction mixture, contained in 1.5 ml quartz
cuvettes (light path, 1 cm), consists of Tris-HC1 buffer, 0.2 ml;
MgC12, 0.2 ml; NaHC0a, 0.1 ml; sodium phosphoenolpyruvate, 0.04 ml;
DPNH, 0.01 ml; 113 units of dialyzed malate dehydrogenase; enzyme
extract and water to 1 ml. The blank cell lacks sodium phosphoenol-
pyruvate. The reaction is started by the addition of sodium phosphoenol-
pyruvate and is followed by measurement of the decrease in optical
density at 340 m/~ consequent on dehydrogenation of DPNH. The tem-
perature of incubation is 22 ° .
Growth Conditions
Cultures of Pseudomonas AM1 may be obtained from both the
National Collection of Industrial Bacteria, Torry Research Station,
Aberdeen, Scotland (Culture No. 9133) and the American Type Culture
Collection, Rockville, Maryland (Culture No. 14718). It may be main-
tained on slopes containing 0.1 M methylamine hydrochloride, mineral
salts, and 1.5% agar. The mineral salts mixture is that of Jayasuriya,4
which has the following composition (rag/100 ml): KH2P04, 136;
Na2HP04, 213; (NH4)2SO~, 50; MgS04.7 H20, 20; CaCl.o.2 H.~0, 1;
FeS0,.7 H20, 0.5; MnS04.5 H_oO, 0.25; NaMoO~.2 H~0, 0.25. The
organism is subcultured every month onto fresh slopes, grown at 30°~ and
stored at 2 ° .
The organism is grown under forced aeration at 30 ° in 10 liter
' G . C. N. Jayasuriya, J. Gen. Microbiol. 12, 419 (1955).
[46] PHOSPHOPYRUVATECARBOXYLASE--Pseudomonas AM1 295
batches of liquid medium of similar composition to that of the slopes,

except that 0.5% (v/v) methanol replaces methylamine as carbon source
and agar is omitted. Yields of approximately 10 g of cell paste per 10
liters may be obtained, and this is stored at --15 ° .
Step I. Preparation oJ Cell-Free Extract. Cell-free extracts may be
prepared either by sonication or crushing in a Hughes press. The prepa-
ration described below utilizes the latter method. Frozen, methanol-
grown bacteria (21 g, wet weight) are crushed in a Hughes prese at --25 °.
The crushed cells are extracted with 10O ml of ice-cold 0.04 M Tris-HC1
buffer, pH 7.5, containing 10 mM mercaptoethanol and a few crystals
each of deoxyribonuclease and ribonuelease (Koch-Light Laboratories
Ltd., Colnbrook, Bucks, England). The resulting extract is centrifuged at
25,000 g for 10 minutes at 2°; the pellet is discarded. All subsequent
operations are performed at 2 ° .
Step 2. Treatment with Protamine Sul]ate. Protamine sulfate is added
to the extract in the proportion of one part to 10 parts of bacterial
protein (w/w). The resulting precipitate is removed by centrifugation
and discarded.
Step 3. Ammonium Sul]ate Precipitation and Dialysis. Solid am-
monium sulfate is added to the supernatant solution to 40% of satura-
tion. The precipitated protein is centrifuged down and discarded. Further
ammonium sulfate is added to bring the solution to 50% of saturation.
The resulting precipitate is collected by centrifugation and dissolved in
10 ml of 50 mM Tris-HC1, pH 7.5. This solution is then dialyzed over-
night against 1.5 liters of the same buffer.
Step 4. Ion-Exchange Chromatography. Diethylaminoethylcellulose
(DEAE-cellulose, Whatman DE50) (7 g) is slurried in 5 mM Tris-HCt
buffer, pH 7.5, and poured into a chromatographic colunm (2.5 cm X 15
cm). The column is washed with 1 liter of the same buffer and the en-
zyme extract is applied to the top of the column. The column is then
eluted with an increasing gradient of potassium chloride in 5 mM Tris-
HC1 buffer, pH 7.5. This is formed by connecting together the bottom~
of two 500 ml polythene bottles, the first containing 500 ml of 1 M
KC1, the other 500 ml of 5 mM Tris-HC1 buffer, pH 7.5. The second
bottle is stirred mechanically, and the overflow is fed on to the top of
the column. The levels of the solution in both bottles drops at the
same rate throughout, and a linear gradient of increasing chloride con-
centration is delivered in the outflow. Column fractions (4 ml) are
collected at a flow rate of 40 ml/hour. Under these conditions the car-
boxylating enzyme is mainly eluted in 6 fr'tctions around fraction
number 60, at a chloride concentration of 0.14--0.19M. The combined

fractions are stored at 2 ° .
PURIFICATION OF 1)nosPIIOPYRUVATE CARBOXYLASE FROM Pse~Momonas AM1
Specific
activity
Volume Activity Protein (units/mg Yield
Step (ml) (units"/ml)(mg/ml) protein) (%)
1. Cell-freeextract 99 2,479 11.4 217 100

2. Fraction treated with protamine 128 1,318 5.9 224 68.9
sulfate
3. 40--50% Ammoniumsulfate 11.4 13,800 7.9 1750 64.3
precipitate, after dialysis
4. Selected combinedfractions 24.5 3,750 0.62 6050 37.6
after chromatographyon
DEAE-cellulose
a One unit is the anmunt required to fix 1 millimicromoleof CO2 in 30 minutes.
Properties
Specificity. None of the following compounds serves as substrata when
tested at 1 mM concentration in the spectrophotometrie assay system:
sodium 3-phosphoglycerate, sodium pyruvate, sodium pyruvate plus ATP,
L-a-glycerophosphate, lithium hydroxypyruvate, sodium DL-glycerate, DL-
serine, and potassium DL-lactate. The enzyme preparation is free of
lactate dehydrogenase and glycerate dehydrogenase activities, but con-
tains a small amount of malate dehydrogenase activity.
Activators and Inhibitors. The activity of the enzyme is absolutely
dependent on the presence of bivalent metal ions, Mg ÷÷ ions being the
most effective activator; of other bivalent cations tested, only Mn**
ions show appreciable activity. The enzyme is competitively inhibited by
phosphate ions and noncompetitively inhibited by ammonium ions. The
presence of GDP (1 mM) is without effect on the activity of the car-
boxylase, and the presence of ADP (1 mM) causes a slight inhibition.
Stability. The purified enzymc is stable in 5 mM Tris-hydrochloride
buffer, pH 7.5, for at least a month at 2 °, but is completely inactivated
after storage at --15 ° for 14 days. The activity is completely destroyed
in 2 minutes at 50 °.
pH Optimum. The enzyme shows a sharp pH optimum at 8.5 in Tris-
HC1 buffer.
Kinetic Properties. The K,, values for phosphoenolpyruvate and Mg ÷÷,
measured at 22 ° and pH 8.5 in Tris-HC1 buffer, are 0.33 mM and 0.196
raM, respectively.
[47] PIIOSPHOENOLPYRUVATE
CAI:tBOXYTRANSPHORYLASE 297
[47] Phosphoenolpyruvate Carboxytransphosphorylase

from Propionibacterium shermanii 1
[EC 4.1.1.38 Pyrophosphate:oxaloacetate carboxy-lyase (phosphorylating)]
By HARLANDG. ~VooD, JUDITH J. DAVIS, and JAMES -~,I. WILLARD
Mg--
P-enolpyruvate + C02 + 1), , ~ oxaloacetate + 1'I', (1)
Mg++
P-enolpyruvate + P~ , pyruvate + PP~ (2)
Assay Methods
Principle. Carboxytransphosphorylase catalyzes Eqs. 1 and 2. ~,2 It is
best assayed spectrophotometrically at 340 m~ by determining formation
of oxaloacetate from P-enolpyruvate, COs, and P~ by coupling reaction
(1) with malate dehydrogenase. 1~ The oxidation of D P N H in the absence
of P-enolpyruvate is used as a control and this change in optical density
is subtracted from that observed with the complete system. When the
correction for the control is large, the assay is done stepwise. The
carboxytransphosphorylase reaction is allowed to proceed for 4 minutes
in the absence of D P N H and malate dehydrogenase and is stopped by
addition of trichloroacetie acid. The oxaloacetate is then determined in
the deproteinized neutralized solution with malate dehydrogenase. The
D P N H oxidase activity is usually so low after step 3 that the control
is no longer necessary.
Reagents ]or the Assay.
Phosphocnolpyruvate (Sigma)
fl-DPNH (Sigma)
MgC12, reagent grade
KH2FO,, reagent grade
K~HPO,, reagent grade
K H C Q , reagent grade
CoCl2, reagent grade
fl-Mercaptoethanol, reagent grade
Malate dehydrogenase from Calbiochem or prepared from propioni-
1This work was assisted by grant AT-(30-1)-1320 from the Atomic Energy Commis-
siOll.
"H. Lochmiiller, H. G. Wood, and J. J. Davis, J. Biol. Chem. 241, 5678 (1966).
*J. J. Davis and H. G. Wood, Federation Proc. 25, 278 (1966); and manuscript in
preparation.
298 R ~ . A C T I O NLEADING
bacteria 3 is diluted in 50 mM phosphate buffer, pH 6.8, to give

60 units of enzyme per milliliter. This solution is dialyzed against
100 volumes of 50 mM phosphate buffer for 3 hours to remove
sulfate ions, which inhibits carboxytransphosphorylase.
Enzyme. Carboxytransphosphorylase is diluted in 50 mM phos-
phate buffer, pH 6.8, containing 1 mM fl-mercaptoethanol. Thiol
compounds stimulate the rate of the reaction 4 or 5 times, la, 2
There is about a 10% loss of activity at 0 ° in this solution in
24 hours, 20% in 4 days. Sulfate must be removed from the
carboxytransphosphorylase preparation before it is assayed un-
less the dilution is sufficient to bring the sulfate below the
inhibitory level. Sulfate, 5 mM, inhibits the reaction ~ 1 5 ~ , 20
mM N50%, and 100 m M ,~80%. Phosphate, above 20 raM,
likewise is inhibitory; 100 m M inhibits about 50~. All buffers
so far tested except bicarbonate inhibit the reaction. The sulfate
or excess phosphate may be removed from the carboxytransphos-
phorylase by gel filtration on Sephadex G-50 or by dialysis
against 50 mM phosphate buffer, pH 6.8. The dialysis tubing
should be boiled twice for 15 minutes in 0.1 mM EDTA; washing
with EDTA is not sufficient. The concentrated enzyme (6-30
mg/ml) is held in 50 mM phosphate buffer, pH 6.8, at 0 ° or
frozen and is quite stable. Mercaptoethanol should not be added
during storage since over long periods of time the enzyme is
not stable in its presence.
Spectrophotometric Assay. The assay mixture contains in micromoles
per milliliter: P-enolpyruvate, 2.0; KHCO3, 30; potassium phosphate
buffer, pH 6.8, 10; MgC12, 12; CoCI2, 0.1; DPNH, 0.125; and in units
per milliliter, malate dehydrogenase, 2.0. A mix (SA mix) is prepared
containing 1.25 times the required strength of the following reagents:
20 mM P-enolpyruvate, 1.0 ml; 0.3M KHCOs, 1.0 ml; 0.1 M potassium
phosphate buffer (pH 6.8), 1.0 ml; 0.1 M MgC12, 1.2 ml; 2.5 mM DPNH,
0.5 ml; and H20, 3.3 ml, to make a total of 8.0 ml.
Before the mixture is used, CO~. is bubbled through it for 15 minutes
at 25 ° to saturate it with C02 and to bring the pH to about 6.5. When not
being used, the solution is stored at 0 °. It is stable for several days. If
the pH becomes 7 or more C02 should be bubbled through the mixture
again. All solutions except those of the malate dehydrogenase and
carboxytransphosphorylase are kept at 25°; the latter are stored in ice.
The assay is carried out in cuvettes with 10 mm light path and 2 mm
'S. H. G. Allen, R. W. Kellermeyer, R. L. Stjernholm, and H. G. Wood, J. Bacteriol.

87, 171 (1964).
[47] PHOSPHOENOLPYRUVATECARBOXYTRANSPHOSPHORYLASE 299
width in a volume of 0.3 ml containing the following: the SA mix (as

above, saturated with C02), 0.24 ml; CoC12 (3 m M ) , 0.01 ml; malate
dehydrogenase (60 units/ml), 0.01 ml; carboxytransphosphorylase in 50
m M phosphate buffer (pH 6.8) and 1 m M mercaptoethanol, 0.01-0.04
ml; and H20 to a final volume of 0.3 ml.
The reaction is started by addition of the earboxytransphosphorylase
and is conducted at 25 ° . The reaction rate often increases during the
first 2 minutes and then becomes constant and is linear with a carboxy-
transphosphorylase concentration below 0.02 unit per milliliter. Thus
far no method has been found to eliminate this initial lag in the presence
of Co +*. If Co +* is omitted, the rate is linear from the beginning but
usually is not as rapid as with Co +*.
Units. Units are expressed in micromoles of oxaloacetate produced per
minute at 25 °, and specific activities are expressed in units per milligram
of protein. Protein is measured spectrophotometrically 4 in purified prepa-
rations and by the biuret procedure 5 in crude extracts. The value obtained
by the biuret procedure on the purified enzyme and with serum albumin
as the standard is 6% higher than that obtained by the speetrophoto-
metric method. 1~
Purification of Carboxytransphosphorylase
Purification obtained in the different steps is summarized in Table I.
Reagents and Equipment ]or Purification of the Enzyme

Ammonium sulfate (special enzyme grade, Mann-Research Labora-
tories), used without further purification
TABLE I
PURIFICATION OF P-ENOLPYRUVATECARBOXYTRANSPHOSPHORYLASEa
Specific activity Recovery

Step and fraction (units/rag protein) (%)
1. Crude extract ~0.1 100

2. Batch elution, DEAE 0.15-0.25 ~90
3. Cellulose phosphate column 0.2-0.35 ~75
4. Ammonium sulfate 35-55% 0.3-0.4 ~-~70
5. Cellulose phosphate column 4.5-18 ~-~40
6. DEAE-cellulose column 8-21 ~26
7. Crystallization 23 ~'-15
Approximately 300 g of cells are obtained from i00 liters of medium; the cells
contain about 1500 units of enzyme, yielding about 10 mg of crystalline enzyme.
See Vol. I][I [73]. The factors 1.45 X A~o-0.74 X A~eo are used.
~J. Wetley and J. Lambeth, Biochim. Biophys. Acta 40, 364 (1960).
Cellulose phosphate (reagent grade, capacity 0.8 mect/g, Brown

Company) is washed successively with 0.1 N NaOH, 0.1 N HC1,
distilled water, and finally with 30 mM phosphate buffer, pH 6.5.
The fine particles are removed by repeated centrifugation at 1000
rpm for 5 minutes in an International Centrifuge, Model PR2.
DEAE-cellulose (type 40, capactiy 0.9 meq/g, Brown Company),
washed as described above for cellulose phosphate
Phosphate buffers: 30 mM (pH 6.5), 50 mM (pH 6.8), 80 mM
(pH 6.8), 0.15M (pH 6.8), and 0.225M (pH 6.8). Mercapto-
ethanol is added to the buffers to obtain a concentration of 1 mM
just before use for column chromatography.
Glass columns (4.5 X 40 and 2 X 40 cm) fitted with coarse sin-
tered-glass disks
Fraction collector and test tubes
Bottles, 1 liter and 2 liter
Dialysis tubing, 2 cm, boiled 2 times for 15 minutes in 10-4 EDTA
Magnetic stirrer
Barnstead Purity Meter (Still and Sterilizer Company, Boston,
Massachusetts). The conductivity of 1.0, 2.0, and 3.0M
(NH4) 2S0~ at a 1:50,000 dilution in distilled water is determined
to establish a linear plot of conductivity against concentration.
The salt concentration of an unknown is determined using a
1:50,000 dilution, if it contains greater than 0.4M {NH4)~S04
or a 1:5000 dilution if the concentration is less than 0.4M.
In the latter case the concentration of (NH4)~SO, as read from
the standard plot is divided by 10.
Sorvall refrigerated centrifuge, RC-2
Source o] Enzyme. Thus far carboxytransphosphorylase has been

reported only in Propionibacterium shermanii. The bacteria can be
grown on glucose, glycerol, or lactate, but the best yield of enzyme is
from cells grown on glycerol for 17-50 days. TM The conditions for growth
are described in this volume [36].
Step 1. Preparation o] the Crude Extract. The procedure is the same
as described in this volume [36]. Approximately 1500 units of car-
boxytransphosphorylase of a specific activity of 0.1 is obtained from 100
liters of culture.
Step 2. Batch Elution .from DEAF,. The procedure is the same as
described in this volume [36].
Step 3. Chromatography on Cellulose Phosphate. The procedure is the
same as described in this volume [36]. Carboxytransphosphorylase
[47] PHOSPHOENOLPYRUVATE
CARBOXYTRANSPHOSPHORYLASE 301
(and also malate dehydrogenase, ~ acetyl kinase, 3 CoA transferase, 3

phosphotransacetylase, 3 and methylmalonyl-CoA mutase 8) are in the ini-
tim effluent and in the eluate with 50 mM phosphate buffer. The protein
is precipitated by addition of solid (NH4)2S0~ to give 90~'o saturation at
0 ° (62 g per 100 ml).
Step ~. FractioT~ation with Ammo~ium Sul]ate. The protein from step
3 may be kept for at least 6 months at --20 ° as a precipitate or as a
suspension. For further fractionation it is dissolved in 50 mM phosphate
buffer, pH 6.8, to a protein concentration of about 20 mgflml. The
concentration of (NH~)2S0, in the solution is determined using Nessler's
reagent or by measuring the conductivity of a 1:50,000 dilution using a
Barnstead purity meter. The concentration of (NH4)2S0, in the enzyme
solution is brought to 35% saturation by addition of solid (NH4)2SO,.
After stirring 20 minutes, the precipitate is removed by centrifugation at
16,000 g for 20 minutes and discarded. The carboxytransphosphorylase is
then precipitated by addition of I2.1 g of (NH4)2SO, per I00 ml of
solution to obtain 55% saturation. Phosphotransacetylase also is pre-
cipitated, but the other enzymes mentioned above remain in solution.
They can be precipitated at 75 and 90% saturation2
Step 5. Chromatography on Cellulose Phosphate. All chromatography
is done at 4 °. Carboxytransphosphorylase is adsorbed by cellulose phos-
phate equilibrated with 30 mM potassium phosphate buffer, pH 6.5
(footnote 1) and is eluted by 80 mM potassium phosphate, pH 6.8. This
step gives a 10- to 50-fold purification.
A 4.5 X 32 cm column of cellulose phosphate is prepared in successive
layers of about 2-3 cm each. About one-tenth of the required cellulose
phosphate suspended in 30 mM potassium phosphate buffer is added to
the column and is allowed to settle. The buffer is then drained off to the
surface of the cellulose phosphate before addition of the next layer.
Very slight pressure if any (1 psi) is used to pack the column because
the flow rate is reduced greatly under pressure. The column is washed at
4 ° with at least 300 ml of 30 mM potassium phosphate buffer, pH 6.5
containing 1 mM mercaptoethanol. The protein from step 4 (5-8 g) is
suspended in 50-100 ml of 50 mM phosphate buffer, pH 6.8, and dialyzed
in 2 cm tubing for 6-8 hours against 2 liters of 30 mM phosphate buffer
(pH 6.8, 1 mM mercaptoethanol) in a 2-liter Erlenmeyer flask at 4 °
using a magnetic stirrer and with changes of buffer at 2 and 4 hours.
The conductivity is determined with a Barnstead purity meter using a
1:5000 dilution. The enzyme solution is diluted to give a salt concentra-
' R. W. Kellermeyer, S. H. G. Allen, R. Stjernholm, and H. G. Wood, J. Biol. Chem.
239, 2562 (1964). See also this volume [35].
l ,," I I . 0 - . * - - - - - - - 4 I---~"-14.8"-',,---~ ~"98-'~ , " 4.6 ~'
2.0 I0
16, . . _ 8
'E
c 12 6 ~
~_
&£ S
d.
o3
Q4 2
0 I I I I I I I r ..J
6 70 74 78 82 86 90 94 98 102
Fractions
FIG. 1. Cellulose phosphate column. Protein from step 4 (7.65 g, 3150 units)
which had been dialyzed was placed on a 4.5 X 32 cm column and was washed 13
hours (overnight) with 1200 ml of 30 mM phosphate buffer (pH 6.5, 1 mM mercapto-
ethanol) at a rate of ~1.5 ml per minute. Most of the protein passed directly
through the column and then the concentration gradually fell to 0.16 mg of protein
per milliliter. During the next 8 hours 685 ml of 80 mM phosphate buffer (pH 6.8,
1 mM mercaptoethanol) was collected in ~20 ml fractions until fraction 66 and
thereafter in ~10 ml fractions. The highest specific activity was 1O at fraction 84
and it fell thereafter, but the units per milliliter increased until fraction 96. The
values shown at the top of the figure between the arrows are specific activities of
of pooled fractions following concentration of the protein by precipitation with
80% saturated (NH4)2S04.
There is considerable variability in step 5. Often the column does not retain all
the carboxytransphosphorylase. In the experiment of Fig. 1 the initial effluent and
the eluate with 30 m M phosphate contained 1035 units of carboxytransphosphorylase ;
1666 units were recovered in the eluate with 80 mM phosphate, of which 940 were
in protein with a specific activity greater than 9.8. The total recovery was 2695 units
or 86% of the initial 3150 units.
t i o n e q u i v a l e n t of 30 m M (NH4)~SO4 if t h e c o n d u c t i v i t y i n d i c a t e s t h e
c o n c e n t r a t i o n is g r e a t e r t h a n t h i s value. T h e r e u s u a l l y is a n a p p a r e n t
loss of 5 0 % of t h e a c t i v i t y d u r i n g t h e d i a l y s i s , b u t t h e e n z y m e a p p e a r s
to be r e a c t i v a t e d on p a s s a g e t h r o u g h t h e cellulose p h o s p h a t e since t h e
t o t a l r e c o v e r y of u n i t s f r e q u e n t l y is 8 0 - 9 0 % . T h e s a l t also can be r e -
m o v e d b y gel f i l t r a t i o n u s i n g S e p h a d e x G-50, a n d t h i s m a y p r o v e to be
t h e m e t h o d of choice.
T h e p r o t e i n s o l u t i o n is a p p l i e d to t h e column, which t h e n is w a s h e d
with 30 m M p h o s p h a t e buffer, p H 6.5, c o n t a i n i n g 1 m M m e r c a p t o e t h a n o l ,
[47] PHOSPIIOENOLPYRUVATECARBOXYTRANSPtIOSPHORYLASE 303
until the concentration of the protein in the eluate decreases to 0.1-0.16

mg/ml. The carboxytransphosphorylasc is then eluted (Fig. 1) with
80 mM phosphate buffer. Selected fractions are combined and the protein
is precipitated by addition of 52.6 g of (NH,)2S04 per 100 ml to obtain
a concentration of 80% saturation. The precipitate is dissoh'ed in a sinai1
volume of 50 mM phosphate buffer, pH 6.8, to give 6-30 mg of protein
per milliliter. The enzyme is stable for at least 6 months in this solution
when stored at 0 °. Frequently the specific activities of the combined and
concentrated fractions are higher than that found in tile fractions col-
lected from the column (Fig. 1). The explanation is not apparent at pres-
ent. If cobalt is omitted from the assay, the values may be 35% lower
both in the samples prior to and after precipitation.
The results shown in Fig. 1 are typical of several experiments. The
cellulose phosphate column may be regenerated following use by washing
with 500 ml of 0.5M KCI, 500 ml of 0.5M phosphate, pH 6.8, con-
taining 0.1 mM EDTA and finally 500 ml of 30 mM phosphate, pH 6.5.
Step 6. Chromatography on DEAE. Carboxytransphosphorylase is
adsorbed by DEAE-cellulose equilibrated previously at 4 ° with 80 mM
phosphate buffer, pH 6.8, containing 1 mM mercaptoethanol and is
eluted with 0.15 M phosphate buffer, pH 6.8, containing 0.2 M KCl. The
column (2 X 30 cm) is packed in layers using about 5 psi pressure.
The carboxytransphosphorylase obtained from several cellulose phos-
phate columns (step 5) is usually used in step 6 (0.2-0.7 g of protein).
The salt concentration is reduced by dialysis against 50 mM phosphate
buffer, pH 6.8, or by gel filtration using Sephadex G-50 before the
protein is placed on the column. The colunm is then washed with 80 mM
phosphate, pH 6.8, containing 1 mM mercaptoethanol until the protein
concentration in the eluate falls to about 0.05 mg/ml. The carboxytrans-
phosphorylase is then eluted using a gradient obtained with 0.15M
phosphate buffer and 0.15M phosphate buffer containing 0.4M KC1,
each at pH 6.8 containing 1 mM mercaptoethanol (Fig. 2). The protein
in the fractions containing carboxytransphosphorylase is precipitated
with (NHJ~SO~ at 80% saturation (52.6 g per 100 ml) and is dissolved
in 50 mM phosphate buffer, pH 6.8, to give 6-30 mg of protein per milli-
liter. The concentrated protein frequently has a lower specific activity
than the material obtained directly from the column, but it is stable for
at least 6 months at 0 ° or frozen.
The results shown in Fig. 2 with a DEAE-ccllulose column are quite
typical. The details of recoveries are included in the legend.
Step 7. Crystallization. The carboxytransphosphorylase of step 6 and
that of step 5 with high specific activity (,-15) may be crystallized
readily. The (NHJ2SO, concentration of the protein solution (12-30 mg
1.7 .,~ (7.o)04.3)

~, (2.9) (10.6) F-> 162 ~ ~ 8.0
L5 t I. ~' 30
ii
I ~ 1" I t 'x
i "t /~ ~,~
1.3 ! ~ ?~ Z6
I.I
,I ~
~. / ~'~ ' " Z2
. , c
.E !
~. 0.7
=T .... i MO Protein/ml
SpAc 14 o
<~
o.s . ~ ;,i x . ~o
Units/ml I ~il
0.3 6
t \.
j \ '~.
0.1 ", "-~ 2
40 44 48 52 56 60 64
Froclion$
FIG. 2. Dt~,AE-cellulose column. Protein from step 6 in 42 ml of 50 mM phos-
phate buffer (0.7 g, specific activity 5.7, equivalent to 4000 units) containing 1.12
(NHD.,SO~ was dialyzed for 2 hours against 400 volumes of 50 mM phosphate buffer
(pI-I 6.8, 1 mM mercaptoethanol). The conductivity then was equivalent to 0.15 M
(Ntt,)=SO, and the solution was diluted with an equal volume of water to 114 ml.
There was no loss of activity. The protein was placed on a 2 X 30 cm column and
was washed 13 hours (overnight) with 80 mM phosphate (pit 6.8, 1 mM mercapto-
ethanol) at a rate of approximately 36 ml per hour (18 ml per fraction). The
maximum protein concentration was in fraction 7 (0.45 mg/ml) and gradually
decreased to ~0.03 mg/ml in fraction 26, There was no carboxytransphosphorylase
in these fractions. A gradient elution was then started using 800 ml of 0.15M
phosphate buffer (pl=I 0.8, 1 mM mercaptoethanol) in the mixing chamber connected
to the column. The volume in the mixing chamber was kept constant by the
addition of 0.4 M KCI in 0.15 M phosphate buffer (ptI 0.8, 1 mM mercaptoethanol).
The protein concentration began to increase at fraction 31 and reached 0.63 mg/ml
at fraction 40. There was a slight decrease followed by a peak of 1.7 mg/ml in
fraction 45. The carboxytransphosphorylase appeared in the second peak with the
maximum protein (1.44 mg/ml) at fraction 54 and a specific activity of 21. The
values shown in parentheses are the specific a('tivities of the individual fractions
after the protein was precipitated with (NH,)=SO,; those between the arrows are
for the protein from pooled fractions. 2678 units were recovered in the concentrated
protein fractions equivalent to 67% of the units added to the column; 2270 units
[47] PIIOSPHOENOLPYRUVATE CARBOXYTRANSPHOSPIIORYLASE 305
of protein per milliliter in 50 mM phosphate, pH 6.8) is determined and

then brought to 40% saturation by addition of solid (NH4).~S04. The
solution is mixed immediately to dissolve the (NH,)2SQ and it soon
becomes slightly turbid. After 24 hours in an ice bath, there usually is
evidence of schlieren on agitation. When viewed with a phase contrast
microscope using oil immersion at 970-fold magnification, small, very
slender needle-shaped crystals are observable. By 72 hours the schlieren
are readily observable and all or most of the material is in the form of
minute needles. Crystals were prepared from protein obtained from a
cellulose phosphate column, step 5 (specific activity = 18, 12.4 mg/ml,
2.9 ml, 630 units). The crystals were harvested by centrifugation and
taken up in 50 mM phosphate, pH 6.8; 19.5 mg was obtained with a
specific activity of 23 (450 units). The supernatant solution contained
15.6 nag and had a specific activity of 7.7 (120 units). The recovery thus
was 90%, 71% as crystals.
Properties of Carboxytransphosphorylase
Sedimentation Properties. Four times crystallized carboxytransphos-
phorylase gave a single homogeneous peak in the ultracentrifuge with a
sedimentation constant of S '322o,w-----16.7 S with 1.32 mg of protein per
milliliter. ~ Treatment with 6 M urea for 12 hours at 0 ° and then dialysis
against 50 mM phosphate buffer (pH 6.8, 10 mM mercaptoethanol)
gave a peak with a sedimentation constant S~w = 7 S, but the protein
had no activity.
The crystals from the first crystallization were found to have a major
peak with a sedimentation constant of 16.3 S, but there was some slower
moving material present with a sedimentation constant of 8.7 S. The
supernatant solution remaining after removal of the carboxytransphos-
phorylase crystals contains 8.7 S protein as the major component. By
separation of the 8.7 S protein from the 16.3 S protein by sedimentation
in a partition cell, the 8.7 S protein was found to have a specific activity
of 7.0. Therefore, both the 16.3 S protein and 8.7 S protein are active
(unpublished data).
Molecular Weight. The molecular weight has been estimated by the
Archibald method to be 430,000 ± 30,000 for the 16 S protein assuming
a specific volume of 0.75. TM
Electrophoretic Pattern. Four times crystallized earboxytransphos-
phorylase on electrophoresis at pH 6.8 migrated as a single boundary
with only slight evidence of inhomogeneity. The eleetrophoretie mobility
were in protein with a specific activity greater than 15. It is noted that the specific'
activity was less in the concentrated protein than in the fractions obtained directly
from the column.
(microns) was 8.7 X 10-~ cm 2 per second per volt ascending and 10.3 X
10-5 em 2 descending.1~
Reactions Catalyzed by Carboxytransphosphorylase. The enzyme
catalyzes not only the reversible reaction to oxaloacetate (Reaction 1),
but also in the absence of C Q the conversion of P-enolpyruvate to
pyruvate and pyrophosphate (Reaction 2). The latter is irreversible
experimentally and is called the pyruvate reaction. It is inhibited by
C02. Reaction 1 in the back direction (oxaloacetate to P-enolpyruvate)
may be assayed by coupling with pyruvate kinase, hexokinase, and
glueose-6-phosphate dehydrogenase?~' The pyruvate reaction may be
assayed by linking it with lactate dehydrogenaseY Reaction 1 in the
forward direction is about 7 times faster than the back reaction and the
pyruvate reaction under optimal conditions, i.e., no thiols, is about one-
fourth as fast as the forward reaction to oxaloacetate. ~
Substrate Specificity. Thus far only arsenate has been found to re-
place orthophosphate in the reaction with P-enolpyruvate. 7 PP, or nu-
cleotides such as ADP, GDP, IDP, and UDP will not substitute for P~.
Inorganic triphosphate will not serve as a donor in the reverse reaction. 1~
Metal Requirements. Carboxytransphosphorylase has two types of
metal requirements, these have been designated type I and type II2
Type I metals are freely dissociable and have Km values of about 1 raM.
Type II are tightly bound, and thus far this metal has not been identified.
With Mg ÷÷ present to meet the Type I requirements, Co +* frequently
stimulates the oxaloacetate reaction. Ba ÷÷, Ca ++, Fe ÷÷, Pb% Sr *÷ and Hg ÷÷
are ineffective as Type I metals. Ca +÷ is an inhibitor of Type I function.
Stability. It has been observed (Figs. 1 and 2) that the protein, when
assayed directly from the columns, may have a different specific activity
than it does after (NH4)~SO~ precipitation. It is probable that loss of
activity occurs when the protein dissociates from 16 S to an 8.7 S protein.
For example fraction 54 of Fig. 2 had a specific activity of 21 and the
activity decreased to 14.3 after precipitation with (NH4)2SO,. When
the concentrated protein was sedimented, the major portion of the pro-
tein had a sedimentation constant of 8.7 S and the minor peak was 16 S.
At the time the protein had a specific activity of 21, it probably was
mostly 16 S comparable to the crystalline protein with a specific activity
of 23.
Carboxytransphosphorylase is quite stable to acid pH; there is little
loss of activity in the crude extract at pH 4.5 (0.25 M acetate buffer) for
5 hours at 25 °, but inactivation occurs at pH 4.0 even at 0 °. At pH 8.2
(0.2 M NaHCO~) 60% inactivation occurs at 25 ° in 1/2 hour and at 0 °
P. M. L. Siu and H. G. Wood, d. Biol. Chem. 237, 3044 (1962).

[47] PHOSPHOENOLPYRUVATE CARBOXYTRANSPHOSPHORYLASE 307
in 1/2 hour 6% of the activity is lost. Above pH 8.2 there is a very rapid
loss of activity.
Carboxytransphosphorylase is most stable when stored in the absence
of thiols. The ammonium sulfate precipitates when taken up as a
concentrated protein (6-30 mg/ml) in 50 mM phosphate, pH 6.8, are
stable for at least 6 months either at --22 ° or unfrozen at 0 °. Dilute
solutions of the enzyme as used in the assays (with thiols for the
oxaloacetate reaction) lose approximately 10% of their activity in 24
hours at 0 °. Concentrated solutions of the enzyme (5-30 mg/ml) are
quite stable after Sephadex filtration or dialysis against 50 mM phos-
phate buffer if the dialysis tubing has been boiled in 0.1 mM EDTA
before use.
pH Optimum. The forward reaction has a broad pH optimum l with
little change in rate between pH 6.0 and 8.0. The optimmn of the back
reaction is between pH 6.8 and 7.6, and of the pyruvate reaction between
6.3 and 7.1.2
Inhibitors. Carboxytransphosphorylase is strongly inhibited by
EDTA and other metal chelators. When EDTA is present in the assay
at 0.1 mM inhibition is practically complete with 10 mM Mg ÷÷ present
but no Co ++ and partial inhibition occurs at 1 #M. The inhibition is
reversed by dilution of the EDTA to about 0.1 vM or by addition of
cobalt in excess of the EDTA. Thiol is required for the inhibition.
Carboxytransphosphorylase is inhibited by P-hydroxymercuribenzo-
ate. 7 When incubated at 25 ° for 10 minutes in the presence 10 tdl//HMB
there was 50% inhibition. Ethylmaleimide and iodoacetate at 0.1 mM
do not inhibit strongly. The mercurial may act as a metal rather than
a thiol inhibitor.
Thiols stimulate the oxaloacetate reaction but inhibit the pyruvate
reaction; this may be because of formation of metal complexes by the
thiol.
So far all buffers tested inhibit the carboxytransphosphorylase re-
action (phosphate, Tris-HC1, glycylglycine, imidazole, and inaleate).
Glycerol 2-phosphate is the least inhibitory, 20% at 40 mM and pH 7.4.
Malate and PPi strongly inhibit the forward reaction; 2 mM PP~
giving 80% inhibition and 10 mM malate 50% inhibition. Sulfate at
20 mM inhibits 40%, and the inhibition is noncompetitive with phos-
phate or other components of the forward reaction. 1'~The back reaction is
inhibited by bicarbonate, P~ and PP, (10 mM bicarbonate, 40%; 10 mM
Pi, 25%, and 2 mM PPi, 80%).
The reaction is not inhibited by avidin since it is not a biotin
enzyme.
Substrate Affinity Constants. The Km values for the substrates in the
TABLE II
K,~VALUES OF SUBSTRATES AND METALS OF CARBOXYTRANSPHOSPHORYLASE IN THE
FORWARD AND BACK 0XALOACETATE REACTION AND IN THE FYRUVATE I~EACTION
Forward Back Pyruvate

reaction reaction reaction
Substrate and metals (mM) (mM) rmM)
P-enolpyruvate° 0.53 -- 0.054

Orthophosphate ~ 1.17 -- 1.03
Mg++ (as type I) a 1.25 1.3 0.63
Mn ++ (as type I)~ 0.50 b 0.04
Co++ (as type I) ~ 0.50 ~ 0.23
NaHCOa a 4.0 - - - -
Oxaloacetate ° -- 0.47 --
Inorganic pyrophosphate~ -- 0.22 --
a H. Lochmfiller, H. G. Wood, and J. J. Davis, J. Biol. Chem. 241, 5678 (1966).
The K,~ for Co++ and Mn ++ was not determinedin the back reaction because the
linking enzymes became limiting with these metals.
forward and back reactions and for the p y r u v a t e reaction are shown in
T a b l e I I . T h e y were determined under conditions described for the assay.
Equilibrium Constants. s The equilibrium constant, K, on,c, is expressed
as follows:
[°xal°acetate2-][HPPa-] = (9 =t= 2) X 107

[P-enolpyruvate3-][HP~-][HCO~-][H +]
The constant when expressed in terms of total concentration of each
reactant (i.e., the sum of all ionic species and metal complexes) is p H
and metal ion dependent and has been designated K~na,. s
[oxaloacetateT][PPT]
K'~al = [P-enolpyruvateT][PT][CO,T]
The K'an~, values have been estimated to be 6 at p H 6, 25 at p H 7, and
40 a t p H 8 when the free M g *+ concentration is 0.5 m M and K ÷ is 0.1 M
(no Co+*). With the free M g ÷÷ concentration at 5 raM, the values become
60 at p H 6, 500 a t p H 7, and 900 at p H 8.
M e c h a n i s m ol the Reactions. Carboxytransphosphorylase catalyzes a
PP~ dependent exchange of ~C02 into oxaloaeetate. Only type I metals
are required for this exchange (unpublished d a t a ) . This fact can be
demonstrated with E D T A , which inhibits the function of the enzyme
bound T y p e I I metal but does not inhibit the exchange. The enzyme
bound metal (no E D T A ) as well as M g ÷÷ is required, however, for the
exchange of P~ into PP~ and P-eno]pyruvate or for the complete reaction.
SH. G. Wood, J. J. Davis, and H. Lochmiiller, J. Biol. Chem. 241, 5692 (1966).
SYNTHETASE 309
It is postulated (unpublished data) that the pyrophosphate and the

oxaloacetate are linked to the protein by Mg~+; a reversible decarboxyla-
tion to pyrophosphoenolpyruvate would then account for the exchange
reaction with CO~. The cleavage of the pyrophosphate bond probably
requires coordination with the type II metal. Thus both type I and type
II metals would be required for the overall reaction and the exchange of
P~ into pyrophosphate and P-enolpyruvate.
[48] P h o s p h o e n o l p y r u v a t e S y n t h e t a s e
By R. A. CooPER and H. L. KORNBERG
Mg++
ATP ~ pyruvate ,_-__' PEP -t- AMP -t- PO~3- -{- 3 H +
Studies with isotopically labeled compounds suggest that PEP syn-
thesis proceeds in the following manner: 1
ATP -}- enzyme ~ enzyme ~ P04 ~- AMP -{- PO4a- q- 3 H + (1)
Enzyme N PO~ -b pyruvate ~ PEP ~- enzyme (2)
Reaction (1) is not completely understood; the transfer of a pyrophos-
phoryl group is indicated by the finding2,u that the fl-phosphate of ATP
is incorporated into PEP and the v-phosphate gives rise to POJ-.
Assay Methods
Method 1
Principle. PEP synthetase activity is measured as the rate of the
ATP-dependent removal of pyruvate. The enzymatic formation of PEP
and the ATP-dependent removal of pyruvate are shown 2 to be equivalent
even when crude bacterial extracts are used. However, since the formed
PEP undergoes further transformations in crude extracts, the initial rates
must be measured.
Reagents
Tris-HCl, 0.5 M, pH 8.4
MgC12, 0.1 M
ATP, 0.1 M, pH 6.8
Sodium pyruvate, 5 mM
2R. A. Cooper and H. L. Kornberg, Biochim. Biophys. Acta 141, 211 (1967).
' R. A. Cooper and H. L. Kornberg, Proc. Roy. ~%c. B168, 263 (1967).
" K. Berman, N. Itada and M. Cohn, Biochim. Biophys. Acta 141, 214 (1967).
SYNTHETASE 309
It is postulated (unpublished data) that the pyrophosphate and the

oxaloacetate are linked to the protein by Mg~+; a reversible decarboxyla-
tion to pyrophosphoenolpyruvate would then account for the exchange
reaction with CO~. The cleavage of the pyrophosphate bond probably
requires coordination with the type II metal. Thus both type I and type
II metals would be required for the overall reaction and the exchange of
P~ into pyrophosphate and P-enolpyruvate.
[48] P h o s p h o e n o l p y r u v a t e S y n t h e t a s e
By R. A. CooPER and H. L. KORNBERG
Mg++
ATP ~ pyruvate ,_-__' PEP -t- AMP -t- PO~3- -{- 3 H +
Studies with isotopically labeled compounds suggest that PEP syn-
thesis proceeds in the following manner: 1
ATP -}- enzyme ~ enzyme ~ P04 ~- AMP -{- PO4a- q- 3 H + (1)
Enzyme N PO~ -b pyruvate ~ PEP ~- enzyme (2)
Reaction (1) is not completely understood; the transfer of a pyrophos-
phoryl group is indicated by the finding2,u that the fl-phosphate of ATP
is incorporated into PEP and the v-phosphate gives rise to POJ-.
Assay Methods
Method 1
Principle. PEP synthetase activity is measured as the rate of the
ATP-dependent removal of pyruvate. The enzymatic formation of PEP
and the ATP-dependent removal of pyruvate are shown 2 to be equivalent
even when crude bacterial extracts are used. However, since the formed
PEP undergoes further transformations in crude extracts, the initial rates
must be measured.
Reagents
Tris-HCl, 0.5 M, pH 8.4
MgC12, 0.1 M
ATP, 0.1 M, pH 6.8
Sodium pyruvate, 5 mM
2R. A. Cooper and H. L. Kornberg, Biochim. Biophys. Acta 141, 211 (1967).
' R. A. Cooper and H. L. Kornberg, Proc. Roy. ~%c. B168, 263 (1967).
" K. Berman, N. Itada and M. Cohn, Biochim. Biophys. Acta 141, 214 (1967).
2,4-Dinitrophenylhydrazine (DNPH), 0.1%, dissoh'ed ill 2 N HCI :~

NaOH, 10%
Ammonium molybdate, 2.5%, dissolved in 5 N H2S04
Reducing reagent 4
Procedure. The following volumes of the rcageats are pipcttcd into a

16 mm X 125 mm test tube: Tris-HC1 buffer, pH 8.4, 0.10 ml; MgCl..,
0.05 ml; ATP, 0.05 ml; sodium pyruvate, 0,15 ml; and enzyme solution
plus water to bring the volume to 0.5 ml. ATP is omitted from the
control tube. The tubes are equilibrated at 30 ° and the reaction is started
by the addition of the enzyme solution in an amount sufficient to catalyze
the removal of about 0.1 micromole of pyruvate in 5 minutes. Samples
(0.1 ml) are removed immediately, after 5 minutes, and after 10 minutes
of incubation and put into test tubes containing 0.9 ml of water plus 0.33
ml of 0.1% 2,4-DNPH solution. After 10 minutes at 30 °, 1.67 ml of 10%
NaOH is added, and the extinction at 445 m~ is measured after a further
10 minutes. Under these conditions 1 micromole of pyruvate has an
extinction of 6 absorbance units.
Method 2
Principle. PEP synthetase activity can also be measured as the pyru-

vate-dependent formation of inorganic phosphate from ATP. This
assay is of little use with crude extracts, since the ATPase activity of
crude extracts results in phosphate release in the absence of pyruvate.
Procedure. The following volumes of reagents are pipetted into a
16 mm X 125 mm test tube: Tris-HCl, pH 8.4, 0.5 ml; MgC12, 0.25 ml;
ATP, 0.25 ml; sodium pyruvate, 0.75 ml; water and enzyme solution to
bring the total volume to 2.5 ml. Sodium pyruvatc is omitted from the
control tube. The tubes are equilibrated at 30 ° , and the reaction is
started by the addition of enzyme solution. Samples (0.5 ml) are re-
moved immediately and after 5 and 10 minutes of incubation and run
into test tubes containing 0.5 ml of 10% trichloroacetic acid. The
precipitated protein is removed by centrifugation, and a sample (0.8 ml)
is transferred to a second test tube containing 1.9 ml of water. Am-
monium molybdate solution (0.3 ml) plus reducing reagent (0.03 ml)
are added; the extinction at 660 m/~ is measured after a further 20
minutes.4 In this system, 1 micromole of phosphate has an extinction of
1.24 absorbance units.
~T. E. Frledemann and G. E. Haugen, J. Biol. Chem. 147, 415 (1943).
C. H. Fiske and Y. SubbaRow, J. Biol. Chem. {)6, 375 (1925).
SYNTHETASE 311
Method 3
Prb~c~ple. The P E P synthetasc reaction is reversible, and the phos-
phatc-dependent formation of pyruvate from P E P and AMP can be
measured by coupling it to the lactate dehydrogenase reaction. This
assay cannot be used with crude extracts because of their high NADH-
oxidase activity. It can, however, be used satisfactorily after step 1 of
the purification procedure.
Reagents
Na-K phosphate buffer, 0.5 M, pH 6.8
MgCl.,, 0.1 M
AMP, 0.1 M, pH 6.8
PEP, 0.1 M, pH 6.8
NADH2, 5 mM
Crystalline lactate dehydrogenase (LDH), 100 #g/ml
Procedure. The following reagents listed are pipetted into a silica
cuvette (1.5 ml volume, 10 mm light path): phosphate buffer, 0.2 ml;
MgCl.~, 0.05 ml; NADH~, 0.025 ml; L D H solution, 0.02 ml; AMP
solution, 0.01 ml; P E P solution, 0.01 ml; water and enzyme solution to
1 ml. The reaction is started by the addition of enzyme solution sufficient
to catalyze a change in extinction at 340 mt~ of up to 0.1 absorbance unit
per minute; the AE34om~is measured for about 5 minutes.
Methods 1-3
Units. One unit of P E P synthetase activity is defined as that amount
of enzyme catalyzing the formation of 1 micromole of P E P per minute
at 30 ° . Specific activity is expressed as units per milligram of protein.
P E P synthetase has been purified extensively from lactate-grown
Escherichia coli strain B. 1 In the purification procedure described here,
the final product, of specific activity 9.7, is at least 90% pure, judged
by acrylamide gel electrophoresis. Protein was determined with the
Folin-Ciocalteu reagent?
Growth o] Cells. E. coli strain B was grown at 30 ° in 15-liter batches of
medium containing: Na.~HP04.12 H~O, 193.3 g; KH2PO~, 28.6 g; NH4C1,
40 g; MgS04-7 H.~O, 1.2 g; C'tCl:-6 H~O, 0.6 g; FeS04.7 H~O, 0.06 g;
MnCl:.4 H~O, 0.06 g; sodium lactate solution (70%), 100 ml. The
medium was prepared immediatt, ly before use with freshly distilled water
265 (1951).
and inoculated with 700 ml of an overnight culture grown at 30 ° in the

same medium. The 15 liter culture was grown with vigorous aeration,
and the cells were harvested in a Sharples centrifuge when growth had
reached about 1.2 mg dry weight of cells per milliliter. The cells were
washed with 5 mM Tris-HCl-1 mM MgCl~ buffer, pH 7.4, and were
resuspended in the same buffer at a cell density of approximately 40 mg
dry weight of cells per milliliter.
Step I. Preparation o] Crude Extract. The cell suspension, in 40 ml
portions, was cooled in melting ice and sonicated for 5 minutes with a
Branson Soniprobe, type l130A, operating at 4-5 amp. The suspension
was then centrifuged at 22,000 g for 20 minutes at 15 °. The supernatant
solution was removed and stored; the pellet was resuspended in 5 mM
Tris-HCl-1 mM MgC12 buffer, pH 7.4, and resonicated. The two super-
natant solutions were combined. All subsequent steps were carried out
at 18-20%
Step 2. Prot¢mine Sul[ate Treatment. The crude extract was adjusted
to pH 5.5 by the addition of 1 N acetic acid, stirred for 10 minutes and
the precipitate removed by centrifugation. The extract was then frac-
tionated by the addition of protamine sulfate solution (40 mg/ml, dis-
solved in 50 mM Tris-maleate buffer pH 5.5), so that 1 mg of protamine
sulfate was added for each 20 mg of bacterial protein. The mixture was
stirred for 15 minutes then centrifuged at 22,000 g for 10 minutes at 18%
The precipitate was suspended in 0.25 M N a - K phosphate buffer, pH 6.8.
The fractionation was repeated several times, the same amount of
protamine sulfate being used each time; the P E P synthetase precipitates
in the brownish third or fourth fraction. This fraction was dialyzed against
2 X 200 ml portions of 5 mM Tris-HCl-1 mM MgC12-5 mM EDTA buffer
(TME buffer) at pH 6.8 for 14 hours and was then centrifuged at
140,000 g for 30 minutes.
Step 3. DEAE-Cellulose Chromatography. The enzyme solution from
step 2 was made 0.1 M with respect to KC1 and was applied to a 23
cm X 2 cm column of DEAE-cellulose, which had been previously equi-
librated with T M E buffer, pH 6.8. The column was eluted with 0.2 M
KCI in T M E buffer pH 6.8 at a rate of 80 ml/hour and fractions each of
10 ml were collected. P E P synthetase activity is not eluted from the
column by this treatment; the enzyme was eluted by increasing the KC1
concentration to 0.4 M. This elution sequence must be conducted rapidly,
as appreciable enzyme activity is lost if the enzyme remains on the
column for a long time. It is therefore advisable to carry out this
sequence in less than 6 hours.
Step 4. Ammonium Sul]ate Fractionation. Pooled material from step
3 was fractionated with solid ammonium sulfate; almost all the enzyme
$YNTHETASE 313
was precipitated between 40% and 50% saturation. The precipitate was
dissolved in a small volume of TME buffer, pH 6.8
Step 5. Sephadex G-200 Fractionation. The sample from step 4 was
centrifuged at 25,000 g for 10 minutes to remove any insoluble material.
The supernatant solution was mixed with a little Blue Dextran solution,
and applied to a 66 cm X 2 cm column of Sephadex G-200 equilibrated
with TME buffer pH 6.8. The column was developed with TME buffer
at a flow rate of 30 ml/hr and fractions each of 3 ml were collected.
The enzyme was eluted just after the Blue Dextran, and fractions with
specific activities greater than 8.7 were pooled.
This procedure gives a final product about 90% pure (as judged by
electrophoresis on polyacrylamide gels) and in high yield. The table
gives a summary of the purification.
SUMMARY OF PURIFICATION
Volume Protein Specific Yield

Step (ml) (mg) Uaits~ activityb (%)
1. Crude extract 458 7460 1245 0.17 100
2. Protamine sulfate 19.5 1170 1150 1.0 92
treatment
3. DEAE-cellulose, 30 366 1053 2.8 84
pooled fractions
4. Ammoniumsulfate 3.7 153 860 5.6 69
fractionation
5. SephadexG-200, 30 54 521 9.7 42
pooled fractions
° One unit is amount of enzymecatalyzingthe formationof 1 micromoleof PEP per
minute at 30°.
b Units per milligramprotein.
Properties
Stability. The enzyme is cold labile, and crude extracts lose half their
PEP-synthetase activity in about 4 hours if stored in ice. Similarly,
enzymatic activity is rapidly lost at pH values above 7. The enzyme is
remarkably stable at room temperature in Tris-HC1 buffers ranging
from pH 5.8 to 6.8, and, even in the absence of bacteriostatie agents, the
purified enzyme retains about 70% of full activity when stored for 2
months. At pH 6.8, half the enzymatic activity is lost in 1 hour at 50 °.
Specificity. CTP, GTP, or UTP at 10 mM concentration cannot
replace ATP in the reaction with pyruvate, nor will phenylpyruvate,
hydroxypyruvate, a-oxobutyrate, or glyoxylate at 1.5 mM concentration
replace pyruvate in the reaction with ATP.
Inhibitors. The enzyme requires M g " and is inhibited by EDTA in
excess of the metal ion concentration. NaF (20 mM) completely inhibits
the enzyme, p-hydroxymercuribenzoate (0.1 raM) inhibits 95%. AMP
(10 raM) inhibits the enzyme 33%, and P E P (2 mM) inhibits it 30%
in the standard assay. In the reverse reaction, ATP (1 mM) inhibits the
enzyme 50% in the standard assay.
p H Optimum and Kinetic Properties. 2 The pH optimum for P E P
synthesis is pH 8.4 (Tris-HCl buffer); for the reversal it is pH 6.8
(Na-K phosphate buffer). The K,, for pyruvate is 0.28 mM, and for ATP
0.19 mM, at pH 8.4. In the reverse reaction the K,n for P E P is 37 ~M;
for AMP 0.11 mM; and for P043- 38 raM.
[ 4 9 ] T h e M e t a b o l i s m of I t a c o n a t e a n d
M e s a c o n a t e in M a m m a l i a n L i v e r
By HENRY A. LARDY
Introduction
The natural occurrence of itaconate, mesaconate, and both dextro-
rotatory and levorotatory eitramalate in a variety of plant materials
prompted investigations of the metabolism of these organic acids in in-
tact animals I and in liver mitochondriaY Guinea pig liver mitochondria
in a medium containing 5 mM MgCI2, 17 mM phosphate buffer, pH 7.3,
and 100 mM KCI oxidize itaconate completely to C02 at rates compara-
ble to the oxidation of succinate or fl-hydroxybutyrate; rat liver mito-
chondria oxidize itaconate about one-fourth as rapidly and mitochondria
from rat brain or kidney, or pigeon liver did not catalyze the oxidation
of itaeonate. 2 The rate of itaconate oxidation by rat liver mitochondria
is enhanced by the addition of small amounts of a cosubstrate such as
fl-hydroxybutyrate or a-ketoglutarate. Guinea pig liver mitochondria
oxidize mesaconate one-eighth as rapidly as itaconate and methyl suc-
cinate one-sixth as rapidly; d/-citramalate is not oxidized at a detectable
rate. 2 Citraconate is not detectably oxidized in intact guinea pig liver
mitochondria, but extracts of these mitochondria do convert this acid
slowly to the same products that are derived from itaconate (as described
below).
' R. Emmerich, Z. Physiol. Chem. 261, 61 (1939).

"~J. Adler, S. F. Wang, and H. A. Lardy, J. Biol. Chem. 229, 865 (1957).
excess of the metal ion concentration. NaF (20 mM) completely inhibits
the enzyme, p-hydroxymercuribenzoate (0.1 raM) inhibits 95%. AMP
(10 raM) inhibits the enzyme 33%, and P E P (2 mM) inhibits it 30%
in the standard assay. In the reverse reaction, ATP (1 mM) inhibits the
enzyme 50% in the standard assay.
p H Optimum and Kinetic Properties. 2 The pH optimum for P E P
synthesis is pH 8.4 (Tris-HCl buffer); for the reversal it is pH 6.8
(Na-K phosphate buffer). The K,, for pyruvate is 0.28 mM, and for ATP
0.19 mM, at pH 8.4. In the reverse reaction the K,n for P E P is 37 ~M;
for AMP 0.11 mM; and for P043- 38 raM.
[ 4 9 ] T h e M e t a b o l i s m of I t a c o n a t e a n d
M e s a c o n a t e in M a m m a l i a n L i v e r
By HENRY A. LARDY
Introduction
The natural occurrence of itaconate, mesaconate, and both dextro-
rotatory and levorotatory eitramalate in a variety of plant materials
prompted investigations of the metabolism of these organic acids in in-
tact animals I and in liver mitochondriaY Guinea pig liver mitochondria
in a medium containing 5 mM MgCI2, 17 mM phosphate buffer, pH 7.3,
and 100 mM KCI oxidize itaconate completely to C02 at rates compara-
ble to the oxidation of succinate or fl-hydroxybutyrate; rat liver mito-
chondria oxidize itaconate about one-fourth as rapidly and mitochondria
from rat brain or kidney, or pigeon liver did not catalyze the oxidation
of itaeonate. 2 The rate of itaconate oxidation by rat liver mitochondria
is enhanced by the addition of small amounts of a cosubstrate such as
fl-hydroxybutyrate or a-ketoglutarate. Guinea pig liver mitochondria
oxidize mesaconate one-eighth as rapidly as itaconate and methyl suc-
cinate one-sixth as rapidly; d/-citramalate is not oxidized at a detectable
rate. 2 Citraconate is not detectably oxidized in intact guinea pig liver
mitochondria, but extracts of these mitochondria do convert this acid
slowly to the same products that are derived from itaconate (as described
below).
' R. Emmerich, Z. Physiol. Chem. 261, 61 (1939).

"~J. Adler, S. F. Wang, and H. A. Lardy, J. Biol. Chem. 229, 865 (1957).
[49] METABOLISM
OF ITACONATE AND MESACONATE 315
Itaconate M e t a b o l i s m by Soluble E n z y m e S y s t e m s
A soluble protein fraction from mitochondria is capable of degrading
itaconate when suitably fortified. The fraction is prepared 3 by extracting
0.6 g of acetone dried guinea pig liver mitochondria with 10 ml of water
or dilute, neutral buffer for 30 minutes at 0% The mixture is then centri-
fuged at 0 ° for 30 minutes at 80,000 g to obtain a clear, yellow extract.
This extract (0.5 ml) when fortified with 6 mM ATP, 7 mM MgCI~,
0.15 mM DPN, 1 mM CoA, and 25 mM phosphate, pH 7.3, in a final
volume of 2 ml catalyzes the conversion of itaconate via itaconyl-CoA to
a mixture of mesaconate, acetate, pyruvate, lactate, malate, citrate,
~-ketoglutarate, glutamate, and C02. The compounds containing more
than 3 carbon atoms probably arise by C02 fixation to pyruvate and
reduction of the oxaloacetate formed to malate or condensation with
acetyl-CoA to yield citrate. The pathway of itaconate degradation
elucidated by fractionating the mitochondrial extract and by the use of
known, purified enzymes is shown in Fig. 1.
Itaconate and mesaconate are converted by succinic thiokinase to
their corresponding coenzyme A thioesters. 2 The enzyme methyl gluta-
conase converts these two compounds to citramalyl coenzyme A to yield
an equilibrium mixture of 13% itaconyl-CoA, 58% citramalyl-CoA, and
29% mesaconyl-CoA at 20 ° and pH 7.4. 4 The stereochemical configura-
tion of the citramalate has not been determined.
Citramalyl-CoA is cleaved by an enzyme in the mitochondrial extract
to form acetyl-CoA and pyruvate; Mg ++ does not appear to be required
for this cleavage. A somewhat analogous reaction is the cleavage of
fl-hydroxy fl-methyI glutaryl-CoA to form acetyl-CoA and acetoacetyl-
CoA. However, the enzyme that catalyzes the latter reaction has a Mg *÷
requirement and it does not cleave citramalyl-CoA. It remains an open
question whether the cleavage of citramalyl-CoA to pyruvate and acetyl-
CoA is accomplished by a specific enzyme or by a nonspecific enzyme
whose chief function is to catalyze a chemically related reaction. The
latter possibility seems more likely.
The enzyme fraction that catalyzes cleavage of citramalyl-CoA did
not incorporate pyruvate-14C into itaconate or mesaconate when incu-
bated with labeled pyruvate and acetyl-CoA; the cIeavage reaction
therefore appears to be irreversible under the conditions of the experi-
ment. Losada et al. 5 have reported the synthesis of citramalate from
pyruvate and acetyl-CoA by cell-free preparations from C h r o m a t i u m .
3G. Drysdale "rod H. A. Lardy, J. Biol. Chem. 202, 119 (1953).

4S. F. Wang, J. Adler, and H. A. Lardy, d. Biol. Chem. 236, 26 (1961).
M. Losada, A. V. Trebst, S. Ogata, and D. I. Arnon, Nalure 186, 753 (1960).
316 REACTIONS LEADING TO AND FROM THE CYCLE [4g]
o-o
!
o "~
+
lIi J ~i ---'-""
o
b'~ '0 0 "~
Na ,~
•-, ~o ,o~'~ o ~ooo

u ~ oo o,~
r~
u-o-o-o o-o ~
II ~ II o~ + m II ~
i~'. ~ ~ T
4-
0
4- 4-
=I' ®
'8 ='8
~-~f-~-~'
u
[49] METABOLISM
OF ITACONATE AND MESACONATE 317
Metabolism of Itaconyl-CoA by Bacteria

In addition to the pathway for degrading free mesaconate described by
Barker 6 as occurring in Clostridium, several microorganisms catalyze
transformations of itaconate, mesaconate, citramalate, or methyl suc-
cinate by the pathway described above for mammalian liver. Cooper and
Kornberg ',8 and Nagai ~,1° found several Pseudomonas species to catalyze
the sequence of reactions described in Fig. 1. In addition to possessing
coo- cog
I l
C (H)CH3 ClOO" Ci(OH)CH3
HCH --~- HC--C--CHa~ HCH
J I I
coo- coo- cog
Methyl s u c c i n a t e Mesaconate Citramalate
C
Ii-O
~O
O" Succinyl-CoA
CH3
l>yruvate
C
I
OO-
C (O H) C H a
+ ~ I + Succinate
HCH
O~C--SCoA
O--C--SCoA
Acetyl-CoA Citramalyl-CoA
FIO. 2. Pathway of methyl suceinate metabolism.
the succinate activating enzymes, Pseudomonas species were found to

contain a transferase that converted itaconate and citramalate to their
respective coenzyme A thioesters by reacting with succinyl-CoA. The
activation of itaconate and citramalate proceeded equally well in cells
grown on glucose or itaconate, but the itaconyl-CoA hydratase and the
citramalyl-CoA lyase were inducible enzymes. Itaconate-grown Pseudo-
e H. A. Barker, this volume [51, 52].
' R. A. Cooper and H. L. Komberg, Biochim. Biophys. Acla 62, 438 (1962).
"R. A. Cool,or and It. L. Kornberg, Biochem. J. 91, 82 (1964).
~J. Nagai, J. Biochem. 53, 181 (1963).
loj. Nagai, J. Biochem. 54, 34 (1963).
monas species also oxidize methyl succinate (both D and L isomers) to

mesaeonate, which is converted to pyruvate and acetyl-CoA via citra-
malate and citramalyl-CoA 11 (Fig. 2). This contrasts with the mamma-
lian system described by Gawron et al. TM in which only levorotatory
methyl succinate is dehydrogenated; its immediate product is mesa-
conate.
cog
I
COO- mesaconase HO--C--CH3
HC--C--CH 3 HCH
c oo- oo-
Mesaconate S(-g)-Citramalate
coo-
l
-OOC COO- citraconase H3C--C--OH
I I ~" J
HC--C~CH 3 HCH
1
COG
Citraconate
R(-)-Citramalate
FIG. 3. Conversion of mesaconate to dcxtrorotatory citramalate.
Subramanian and Rao 13-1s have intensively investigated the metab-

olism of branched C~-dicarboxylic acids by strains of P s e u d o m o n a s
isolated from garden soil by the enrichment-culture technique. The
organisms contain a mesaconase that converts mesaconate to dextro-
rotatory citramalate (Fig. 3). If grown on citraconate, the organism
contains mesaconase and a new enzyme, citraconase, that hydrates citra-
conatc to ]evorotatory citramalate. The configurations of the enanti-
11H. Katsuki, J. Nagai, A. Wada, I. Fukuma, and S. Tanaka, J. Biochem. Tokyo 53,
328 (1963).
1--O. Gawron, A. J. Glaid, III, T. P. Fondy, and M. M. Bechtold, J. Am. Chem. Soc.
84, 3877 (1962).
1, M. R. Raghavendra Rao, S. S. Subramanian, H. A. Rahatekar, and S. V. Paranjpe,
Biochem. Biophys. Res. Commun. 12, 78 (1963).
~S. S. Subramanian and M. R. Raghavendra Rao, Indian J. Biochem. 3, 19 (1966).
~ S. S. Subramanian, Thesis, National Chemical Laboratory, Poona, 1967.
~ S. S. Subramanian and M. R. R. Rao, J. Biol. Chem. 243~ 2367 (1968).
[50] GLUTAMATE MUTASE 310
morphic forms of citramalate have been established by G. Settimy, H.

Weber, and D. Arigoni (unpublished data cited by Stadler et aI27).
The pathway of citramalate metabolism was not established by
Subramanian," hut the evidence indicated that ~-ketoglutarate might
be formed by a new pathway.
'7 P. A. Stadler, A. J. Frey, and A. Hofmaan, Helv. Chim. Acla 46, 2300 (1963).
[50] Glutamate Mutase (Methylaspartate Mutase)

[EC 5.4.99.1 L-threo-Methylaspartate carboxy-aminomethylmutaseI
By H. A. BARKER
COO-
+1 COO-
H,~NCH +l
] H3NCH
CH2 ~ [
1 H --C--CH3
CH~ 1
I coo-
COO-
L-Glutamate threo-~-Methyl-
L-asp~rtate
General
Assay Method
Principle. Glutamate mutase consists of two separable proteins called
component E and component S. 1 Neither component alone shows mutase
activity, but together they catalyze the reversible conversion of L-glu-
tamate to fl-methylaspartate. In the presence of an excess of fl-methyl-
aspartase (L-threo-3-methylaspartate ammonia-lyase, 4,3.1.2), fl-methyI-
aspartate is deaminated to mesaconate (2-methyl fumarate) which can
be estimated by its absorbance at 240 or 250 m~. 2 Under suitable condi-
tions the rate of absorbanee increase is proportional to the concentration
of the rate-limiting mutase component.
The assay of each glutamate mutase component requires a partially
purified preparation of the other component. Consequently it is necessary
to partially purify one component without the use of an activity assay.
1F. Suzuki and H. A. Barker, J. Biol. Chem. 241, 878 (1966).
:H. A. Barker, R. I). Smyth, 1{. M. Wilson, and H. Weissbach, J. Biol. Chem. 234,
320 (1959).
morphic forms of citramalate have been established by G. Settimy, H.

Weber, and D. Arigoni (unpublished data cited by Stadler et aI27).
The pathway of citramalate metabolism was not established by
Subramanian," hut the evidence indicated that ~-ketoglutarate might
be formed by a new pathway.
'7 P. A. Stadler, A. J. Frey, and A. Hofmaan, Helv. Chim. Acla 46, 2300 (1963).
[50] Glutamate Mutase (Methylaspartate Mutase)

[EC 5.4.99.1 L-threo-Methylaspartate carboxy-aminomethylmutaseI
By H. A. BARKER
COO-
+1 COO-
H,~NCH +l
] H3NCH
CH2 ~ [
1 H --C--CH3
CH~ 1
I coo-
COO-
L-Glutamate threo-~-Methyl-
L-asp~rtate
General
Assay Method
Principle. Glutamate mutase consists of two separable proteins called
component E and component S. 1 Neither component alone shows mutase
activity, but together they catalyze the reversible conversion of L-glu-
tamate to fl-methylaspartate. In the presence of an excess of fl-methyl-
aspartase (L-threo-3-methylaspartate ammonia-lyase, 4,3.1.2), fl-methyI-
aspartate is deaminated to mesaconate (2-methyl fumarate) which can
be estimated by its absorbance at 240 or 250 m~. 2 Under suitable condi-
tions the rate of absorbanee increase is proportional to the concentration
of the rate-limiting mutase component.
The assay of each glutamate mutase component requires a partially
purified preparation of the other component. Consequently it is necessary
to partially purify one component without the use of an activity assay.
1F. Suzuki and H. A. Barker, J. Biol. Chem. 241, 878 (1966).
:H. A. Barker, R. I). Smyth, 1{. M. Wilson, and H. Weissbach, J. Biol. Chem. 234,
320 (1959).
320 Rv.ACTIONS
L~.ADma TO AND FROM THe. CYCL~ [50]
It is best to start with the purification of component S and carry this

through step 4 or preferably step 5. This can be done by relying entirely
on measurements of absorbance at 280 m~ to locate the component S-
containing fractions. At both stages of purification, the component S
preparations are free of component E. The partially purified component
S can then be used for activity assays required in the purification of
component E.
Reagents
2-Mercaptoethanol, 0.1 M and 20 mM. Prepare fresh daily
Glutamate-buffer-salt solution. The reagent is made by mixing
10 ml of 1 M Tris-HC1, pH 8.3, 2 ml of 1 M KC1, 2 ml of 0.1 M
MgCI2, and 2 ml of 1 M monosodium-L-glutamate
Co-deoxyadenosyl benzimidazolylcobamide,8 16 td~/. About 0.3 mg
of the crystalline compound is dissolved in 2 ml of water and
then diluted to give an absorbanee of 0.122 at 519 m#. The
solution must be protected from light
fl-Methylaspartase, 2 40 units per ml of 0.05% bovine serum
albumin. The,enzyme should have a specific activity of at least
80 units per milligram of protein
Mutase component E solution (for component S assay), 2 units per
milliliter of 10 mM potassium phosphate buffer, pH 7.6. The
preparation should have a specific activity of at least 0.5 unit
per milligram of protein and should be virtually free of com-
ponent S activity
Mutase component E solution (for component E assay). The
preparation is suitably diluted with 10 mM phosphate buffer pit
7.6 to a concentration of 0.1-2.5 units per milliliter
Mutase component S solution (for component E assay), 3 units/ml.
The preparation should be free of component E activity and
preferably should have a specific activity of at least 10 units/rag
since less highly purified preparations often contain an inhibitor.
The preparation is reduced before the assay by mixing 3 units of
component S (in 0.6 ml water) with 0.2 ml of freshly prepared
0.1 M 2-mercaptoethanol and 0.2 ml 0.1 M potassium phosphate
buffer pH 8.0, and incubating the solution in a 10 X 75 mm test
tube at 0 ° for 40-120 minutes
Mutase component S solution (for component S assay). To a
sample containing 0.1-5 units in 0.3 ml is added 0.1 ml of freshly
prepared 0.1 M mercaptoethanol and 0.1 ml of 0.1 M potassium
~H. A. Barker, R. D. Smyth, H. Welssbach, J. I. Toohey, J. N. Ladd, and B. E.
Volcani, J. Biol. Chem. 235, 480 (1960).
[50] GLUTAM/kTZ MUT.CSE 321
phosphate buffer, p H 8.0, and the solution is incubated in a

10 X 75 mm test tube at 0 ° for 40-120 minutes
Component E
Assay M e t h o d I, ~
Procedure. The assay is carried out in a silica cuvette, I cm light
path, suitable for holding 1 ml of reaction mixture. I t is convenient to
use a recording spectrophotometer, especially for assaying relatively
crude preparations which, because of their high absorbance at 240 m~,
allow the use of only small samples. The assay is done at 24-25 ° .
The reaction mixture contains glutamate-buffer-salt solution, 0.08 ml;
fl-methylaspartase, 0.05 ml; cobamide coenzyme, 0.05 ml; mutase com-
ponent S solution (for component E assay), 0.05 ml; a suitable amount
of mutase component E solution (for component E assay) in a total
volume of 1.0 ml. The reaction can be started by addition of either
coenzyme or component S. The solution is mixed and the absorbance is
read at 240 m~ continuously for 3 minutes or intermittently at 30
second intervals. The maximal rate of absorbanee increase, which is
reached within 45 seconds after mixing the solution and remains virtually
constant for I minute or more, is used as a measure of mutase activity.
In assaying partially purified component E preparations, the absorbance
is read against water. With crude preparations, which m a y give an initial
absorbance as high as 1.5 at a protein concentration giving a convenient
activity level, the reference cuvette should contain a sufficient concentra-
tion of a UV absorbing compound to give a differential absorbance of
less than 0.3. The initial absolute absorbance of the reaction mixture
should never exceed 2.0.
Units. A unit of component E activity causes an increase of 3.85
absorbance units per minute at 240 m~, corresponding to the formation
of 1 micromole of mesaeonate per minute under the conditions of the
assay. The unit so defined is dependent upon the use of a component S
preparation that does not cause significant inhibition at higher levels.
Specific activity is defined as units per milligram of protein determined
b y the method of Lowry et al? with bovine serum albumin as a standard.
Purification Procedure I, 8
All operations are carried out at 0-4 ° unless otherwise indicated.
Step I. Preparation o] the Extract. Frozen moist cells of Clostridium
R. L. Switzer and H. A. Barker, J. Biol. Chem. 242, 2658 (1967).
' O. H. Lowry, N. J. Rosebrough, A. L. Farr, and 1R. J. Randall, J. Biol. Chem. 193,
265 (1951).
'B. Baltimore and H. A. Barker, unpublished data.
tetanomorphum H1, grown in a glutamate-yeast extract medium and

harvested as previously described, 3 are used as a source of enzyme. To
100 g of frozen cclls are added 200 ml of 10 mM potassium phosphatc
buffer, pH 7.6, and the cells are thawed and homogenized. The suspen-
sion, in 50 ml batches to each of which is added 2 g of fine (FFF)
corundum powder, is sonicated for 10 minutes in a Raytheon 10 kc
sonicator. The suspension is then centrifuged for 30 minutes at 13,000 g,
and the sediment is discarded. The volume of the clear supernatant
solution is 280 ml. The extract should be shielded from light during this
and the following step.
Step 2. Charcoal Treatment. To the 280 ml of extract, add 34 g (2.3 g
per gram of protein) of moist, and washed charcoal (Nuchar C), stir
the suspension gently for 10 minutes, and then centrifuge at 13,000 g
for 30 minutes. Discard the precipitate. To remove suspended charcoal,
filter the extract with gentle suction through Whatman No. 1 filter paper.
Step 3. Protamine Precipitation. The protein content of the extract is
determined and the solution is diluted with water to a protein concentra-
tion of 15 mg/ml (total volume, 700 ml). To this solution add dropwise
157 ml of 1% protamine sulfate (sa]minc, Lilly) solution, in 10 mM
potassium phosphate, pH 7.0, equivalent to 150 mg protamine sulfate
per gram of protein, with mechanical stirring over a period of 20 minutes.
After stirring for an additional 10 minutes, the suspension is. centrifuged
for 20 minutes at 13,000 g, and the precipitate is discarded. The solution
is usually frozen rapidly and stored at this point.
Step ~. Fractionation on DEAE~Cellulose. The enzyme solution (830
ml) from the previous step is dialyzed with stirring in 1 ~ inch wide
cellophane tubing against 10 volumes of 3 mM potassium phosphate
buffer pH 6.8 for 3-4 hours. The conductivity of the dialyzed solution
should be equal to or less than that of 10 mM potassium phosphate, pH
6.8. Centrifuge the dialyzed solution, if turbid, for 20 minutes at 13,000 g
and discard the precipitate.
Prepare a 5.0 cm (diameter) X 35 cm column of DEAE-cellulose,
equilibrated with 10 mM phosphate buffer, pH 6.8. Apply the dialyzed
solution to the column at a rate of about 16 ml per minute and collect
fractions of 250 ml each. The enzyme should remain at the top of the
column associated with a narrow pink band. The column is eluted succes-
sively with the following potassium phosphate buffers, pH 6.8, at a rate
of 22 ml per minute: 4 liters 20 mM and 5 liters of 50 raM. After remov-
ing a considerable amount of inactive material with the 20 mM buffer,
component E elutes rapidly at first and then with considerable tailing
with the 50 mM buffer. About 70% of the activity can be collected in
about 3.5 liters of solution (fractions 21-36).
Step 5. Ultrafiltration. The protein in the pooled fractions from the

previous step (3500 ml; 0.08 mg of protein per milliliter) is concentrated
to about 7% of its initial volume by ultrafiltration through a 3 inch
diameter Diaflo membrane type UM-1 in a model 400 Amicon ultra-
filtration cell/ At a pressure of 90 psi the concentration requires 6-7
hours.
Step 6. Ammonium Sul]ate Precipitation, One-tenth volume (25 ml)
of 1 M potassium phosphate buffer, pH 7.8, and 5 ml of 50 mM EDTA,
pH 7.4, are added to the enzyme solution (250 ml; about 1.1 mg of
protein per milliliter) and are followed by the slow addition of 472 mg of
powdered ammonium sulfate per milliliter (0.70 saturation). After stir-
ring for another 30 minutes, the protein is collected by centrifugation for
20 minutes at 13,000 g and dissolved in 10 mM phosphate buffer, pH 6.8
(final volume, 10 ml). The solution, which is pale orange in color, can be
stored at --15 ° or used directly for the following step.
Step 7. Sephadex Chromatography. Prepare a 2.5 cm (diameter) X 90
cm column of Sephadex G-150, previously equilibrated with 50 mM
potassium phosphate buffer, pH 7.0, containing 5 mM mercaptoethanol.
Add 20 mM mercaptoethanol to the enzyme solution from the previous
step (9.5 ml; 16.5 mg of protein per milliliter) about 30 minutes before
it is run into the column. Develop the column with the same phosphate-
mercaptoethanol solution, using a flow rate of about 15 ml per hour.
Collect fractions having a volume of 3 ml. Component E elutes mainly
in fractions 57-75 with a total volume of about 57 ml. It is contaminated
by fl-methylaspartasc, which elutes somewhat more slowly. The active
fractions are combined and dialyzed in 7/s inch cellophane tubing for 6
hours against 100 volumes of distilled water with one change of the
external solution. The dialyzed enzyme solution is essentially free of
mercaptoethanol and can be stored at --15 °.
Step 8. Brushite Chromatography. Prepare a 3.7 cm (diameter) X 8.0
cm column of brushite 8 previously equilibrated with 5 mM potassium
phosphate buffer, pH 6.8. The conductivity of the enzyme solution should
not be higher than that of the buffer. The enzyme can be diluted moder-
ately with distilled water if necessary to decrease its conductivity, but
the final volume should not be much greater than that of the column.
This step should be completed in 3-4 hours.
The enzyme solution is run into the column and the protein is eluted
with a linear gradient using 600 ml (7.5 column volumes) of 5 mM
potassium phosphate buffer, pH 6.8, in the mixing chamber and 600 ml of
0.3 M phosphate buffer, pH 6.8, in the reservoir. Fractions of 15 ml each
~Amicon Corporation, 280 Binney Street, Cambridge, Massachusetts.
s W. T. Jenkins, Biochem. Prep. 9, 83 (1962).
are collected at the rate of 3 ml per minute. Component S and B-methyl-

aspartase elute first (fractions 5-25) and component E elutes in fractions
27-49. The active fractions are pooled.
Step 9. Concentration by Ultrafiltration. The enzyme solution (325-
400 ml; 0.1 mg of protein per milliliter) is concentrated to 25 ml (1.2 mg
of protein per milliliter) by ultrafiltration using the Amicon apparatus
described in step 5. This takes about 45 minutes.
Step 10. Ammonium Sul]ate Precipitation. The solution from step 9
is made 0.1 M with phosphate buffer, p H 7.8, and 1 m M with E D T A and
then is brought to 0.70 saturation by slow addition of powdered am-
TABLE I
PURIFICATION OF COMPONENT E OF GLUTAMATE MUTASE a
Total Specific
Volume activity Protein activity Yield
Step (ml) (units) a (mg) (units/rag) (~)
1. Extraction 280 400 14,800 0.027 100

2. Charcoal treatment 244 346 10,500 0. 033 87
3. Protamine 830 332 3,200 0.10 83
precipitation
4. DEAE-cellulose 3,500 280 285 0.99 70
chromatography
5. Ultrafiltration 250 230 175 1.3 57
6. (NH~)2SO4 9.5 221 157 1.4 55
precipitation
7. Sephadex column 58 141 61 2.3 35
8. Brushite column 400 124 39 3.2 31
9. Ultrafiltration 25 102 29 3.5 25
10. (NI~)2SO4 2.5 84 25.6 3.3 21
precipitation
6 B. Baltimore and H. A. Barker, unpublished data.
6 One unit causes an increase, of 3.85 absorbance units per minute at 240 m~, cor-
responding to 1 micromole of mesaconate per minute under the assay conditions.
monium sulfate. After stirring for an additional 30 minutes, centrifuge

down the protein for 20 minutes at 13,000 g, drain thoroughly, and dis-
solve in a minimum volume of 10 raM, p H 6.8. Dialyze in ~ inch tubing
against 100 volumes of the same buffer for 6 hours, changing the external
solution once. The dialyzed enzyme solution is best stored in a liquid
nitrogen refrigerator in small batches to minimize the deleterious effects
of repeated freezing and thawing.
Typical data on component E purification by this procedure are given
in Table I. The overall activity yield is 2 0 - 2 5 ~ with a purification factor
of 120-170. The final product m a y contain a little B-methylaspartase
activity. If this is objectionable, it can be removed by repeating the

fractionation on brushite.
Properties of Component E
Homogeneity and Molecular Weight. 1 The best preparations are not
homogeneous and usually contain 25-50% of other proteins that can be
detected by ionophoresis or ultracentrifugation. The molecular weight of
component E is estimated to be 128,000 by ultracentrifugation; the sedi-
mentation constant (S~o,w) is 6.9 S.
Stability. ~ The activity is somewhat unstable at 0°; the half-life is
about 40 days in a pH 7 phosphate buffer at this temperature. The
enzyme is best stored in dilute (10-20 raM) phosphate buffer, pH 6.8, in
the frozen state at --15 ° or preferably at --196 ° (liquid nitrogen). Both
mercaptoethanol and cationic buffers (Tris, imidazole, triethanolamine)
greatly accelerate the inactivation.
Absorption Spectrum. Component E shows a typical protein absorp-
tion spectrum with Area. at 280 m~ and Ami, at 251 m~. The A~8o m~/A2~o
m~ ratio is 1.63. There is a small and progressively decreasing absorption
in the region between 300 and 550 m~. The slight pink color of purified
preparations is probably caused by contamination with a tightly bound
corrinoid compound. The absorbance of a neutral 0.1% component E
solution is 0.62 at 280 m~.
Cobamide Coenzyme Binding? Partially purified component E (free
of component S) binds about 0.7 mole of dcoxyadenosylcobalamin per
mole when the corrinoid concentration is 60 p~/ (about 3 times the K~
value). Addition of excess component S (7 moles per mole of component
E) increases the amount of bound corrinoid under the same conditions,
to about 1.7 moles per mole of component E.
Activators and InhibitorsY 4 Unlike component S, component E is not
activated by incubation with mercaptoethanol and is not inhibited by
exposure to iodoacetate.
Component S
Assay Method 4
Procedure. The assay solution contains glutamate-buffer-salt solu-
tion, 0.08 ml; p-methylaspartase, 0.05 ml; mutase component E (for
component S assay), 0.05 ml; a suitabIe volume (1-50 ~l) of reduced
component S (for component S assay) ; sufficient 20 mM mcrcaptoethanoI
to bring the final mercaptoethanol concentration to 1 ram (if required) ;
cobamide coenzyme, 0.05 ml; and distilled water to a final volume of
1.0 ml. The maximal volume of reduced compone~t S solution is limited
to 50 #1 so that the mercaptoethanol concentration does not exceed 1 mM.

The rate of absorbance increase at 240 m~ is determined as in the com-
ponent E assay (above). The rate is usually proportional to the amount
of component S added in the range 0-0.25 absorbance unit per minute.
However, relatively crude preparations of component S may show some
departure from linearity at higher rates. The reaction is started by the
addition of cobamide coenzyme.
Units. The unit of component S activity and specific activity are
defined as described for component E, except that component S is sub-
stituted for component E.
Purification P r o c e d u r e 4
All operations are carried out at 0-4 ° unless otherwise specified.
Step 1. Preparation of the Extract. Frozen C. tetanomorphum cell
paste (200 g) is thawed in 800 ml of 20 mM potassium phosphate buffer,
pH 7.4, and dispersed with a Teflon-glass homogenizer. The suspension
is sonicated in 60 ml batches with 2 g of grade F F F corundum powder 9
per batch for 10 minutes in a Raytheon 10 kc sonicator at full power.
The resulting suspension is centrifuged for 30 minutes at 13,000 g. The
supernatant solution, including a slimy gray layer that partly decants
with the clear liquid is used for the next step. This extract may be stored
at 0 ° overnight without significant loss of activity. Substantial losses can
be prevented by performing the steps through the beginning of the
Sephadex chromatography in rapid succession in a single day.
Step ~. Isoelectrie Precipitation. The sonic extract is diluted to a
protein concentration of 10 mg/ml with water. The solution is then
acidified to pH 4.60 by dropwise addition of about 11 ml of 5 N acetic
acid with continuous stirring during 5 minutes. A voluminous precipitate
is removed by centrifugation for 20 minutes at 10,000 g and discarded.
The pH of the clear supernatant solution is raised to 7.6 by dropwise
addition of about 3 ml of concentrated ammonia with stirring. The total
time of exposure of the extract to a pH below 5.0 should be about 60
minutes. Purification of component S in this step varies from 10- to
25-fold in 53-90% yield.
Step 3. DEAE-Celhdose Chromatography. The solution from the
previous step (2650 ml; conductivity, 1800 ppm as NaC1) is dialyzed in
several 1 inch diameter dialysis tubes against 20 liters of distilled water
for 2 hours with stirring. The final conductivity of the dialyzate should
be 1000 ppm or less (as NaC1). The solution is made 1 mM in sodium
ethylenediaminetetraacetate (EDTA), pH 7.0, and 10 mM in 2-mercapto-
ethanol. The solution is immediately allowed to flow into a 4.8 cm
9 Obtained from Braun-Knecht-Heimann Co., Division of van Waters and Rogers,
Inc., 1400 16th Street, San Francisco, California.
150] GLUTAMATE MVTASE 327
(diameter) X 12 cm column of DEAE-cellulose, which has been pre-

viously equilibrated with 10 mM potassium phosphate buffer, pH 7.5,
containing 1 mM EDTA, pH 7.0. The yellow pass-through fraction is
discarded. The column is then eluted successively with the following
potassium phosphate buffers, pH 7.5, all containing 10 mM mercapto-
ethanol and 1 mM EDTA: 200 ml of 10 raM, 400 ml of 50 mM, and 300
ml of 0.1 M. Fractions, each containing 20 ml, are collected at 2 minute
intervals. The elution pattern is determined by estimating the absorbance
of each fraction at 280 m~. Component S activity is eluted in the leading
edge of a large trailing protein peak that comes off the column with the
50 mM buffer. Fractions 19-26, which arc pink and often turbid, usually
contain most of the activity and are combined, after being assayed. The
active fractions are largely resolved from a bright yellow band (fractions
26-35) eluting with 50 mM buffer and from material elutlng with 0.1 M
buffer.
Step 4. Ammonium Sulfate Precipitation. The enzyme is precipitate(]
from the active fractions (152 ml) of the previous step by addition of
70.6 g (80% saturation) of powdered ammonium sulfate with mechanical
stirring. After stirring an additional 20 minutes, the precipitated protein
is collected by centrifugation at 15,000 g for 10 minutes, and dissolved in
15 ml of 0.1 M potassium phosphate buffer, pH 6.8, containing 10 mM
mercaptoethanol and 1 mM EDTA, to give a final volume of 20-25 rot.
The yield from the combined DEAE-cellulose and concentration steps is
usually 45-65%, and the purification factor is about two.
Step 5. Fractionation on Sephadex G-IO0. The enzyme solution from
the previous step (25 ml or less) is immediately allowed to flow into a
2.4 cm (diameter) }< 98 cm column of Sephadex G-100, which has been
equilibrated with 0.1 M potassium phosphate buffer, pH 6.8, containing
10 mM mercaptoethanol and 1 mM EDTA. The column is then eluted
with the same buffer, collecting fractions of 4 ml at 15-18 minute inter-
vals. The absorbance of each fraction is measured at 280 m~. The most
prominent absorbance peak, observed between fractions 45 and 62 with a
maximum in fraction 53, is caused by the elution of fi-methylaspartase.
This is followed by a symmetrical absorbance peak (fractions 72-89;
absorbance max!reran in fraction 88) containing component S. These
fl'actions are combined to give 72 ml of an almost colorless Component S
solution. Purification in this step varies from 7- to 10-fold; recovery of
activity is better than 90%.
Step 6. CM-CeIhdose Chromatography. The solution from the previous
step is dialyzed in 1/, inch diameter Visking tubing with stirring against
2 liters of 5 mM sodium acetate buffer, pH 4.8, containing 10 mM
mercaptoethanol and 1 mM EDTA for 8-10 hours. After dialysis the
solution is adjusted to pH 4.50 by dropwise addition of 3 N acetic acid.
328 REACTIONS LEADING TO AND FROM TItE CYCLE [50]
The final conductivity of the solution must be less than that of 2000 ppm
NaC1. The solution is allowed to flow into a 2 cm (diameter) X 12 cm
column of CM-cellulose that has been previously equilibrated with 5
mM acetate buffer, plI 4.8, containing 10 mM mcrcaptoethanol and
1 mM EDTA. The column is eluted successively with the following
buffers, all containing 10 mM mercaptoethanol and 1 mM EDTA: 100
ml of 5 mM sodium acetate, pH 4.8; and 100 ml of 50 mM acetate,
pH 5.5. Fractions of 5 ml each are collected at 2-3 minute intervals and
the absorbance is measured at 280 m#. Considerable absorbing material
elutes with the pass through and the first half of the 5 mM buffer. Com-
ponent S elutes with the 50 mM buffer as a sharp absorbance peak with
a maximum in tube 49. The fractions (47-51) containing component S
are pooled, and the pH of the solution is adjusted to 6.8 with 0.1 N
sodium hydroxide.
Step 7. Ammonium Sul]ate Precipitation. The component S solution
(23.4 ml; 1.1 mg of protein per milliliter) is concentrated by addition of
662 mg powdered ammonium sulfate per ml (90% saturated) with
stirring for a total of 60 minutes. The colorless precipitate is collected by
centrifugation at 15,000 g for 20 minutes and is redissolved in about 3
ml of 20 mM potassium phosphate buffer, pH 6.8, containing 1 mM
EDTA. This solution is dialyzed against 500 ml of the same buffer for
18 hours, with one change of the external solution, and the enzyme
solution is centrifuged if necessary to remove any precipitate. Purification
by the combined CM-cellulose and ammonium sulfate steps varies from
1.5- to 2-fold and the yield is 60-70%.
Typical data on the purification procedure are given in Table II. The
overall purification is usually 330- to 350-fold over the activity in the
sonic extracts and the yields vary from 20 to 28%. The specific activity
of the final product varies from 28 to 35 units per milligram. The product
is free of mutase component E and fl-methylaspartase.
Properties of Component S*
Stability and Storage. Component S is most stable in neutral phos-
phate buffer in the frozen state; at --10 ° or --196 ° (liquid nitrogen),
full activity is retained for at least several months. Activity is slowly
lost (about 8% per month) during storage at 0% In the presence of 10
mM mercaptoethanol, the stability is markedly decreased both at 0 °
and --10%
Homogeneity and Molecular Weight. The best preparations are
essentially homogeneous in the reduced state as judged by gel filtration,
polyacrylamide gel or starch eleetrophoresis, or ultracentrifugal analysis.
The molecular weight is 17,000 ~ 1000.
Spectrum. The absorbance m a x i m u m is at 280 m~ and the minimum

at 254 m~; there is no absorption in the visible region. The absorbance
ratio (A..so/A,_,Go m~) is 1.44. The absorbance of a neutral 0.1% solution
is 0.644 at 280 m~.
TABLE II
PURIFICATION OF COMPONENT S OF GLUTAMATE ~IUTASE ~
Total Specific
Volume activity Protein activity Yield
Step (ml) (units) b (rag) (units/mg) (%)
1. Extraction 950 2,590 28,100 0.092 100

2. Isoelectric 2,650 1,370 1,460 0.94 53
precipitation
3. DEAE-cellulose 152 ~980 ~600" l. 6 42
chromatography
4. (NI-h)2SO4 25 890 557 I (i 3a
precipitation
5. Sephadex G-100 72 970 58 17 41
chromatography
6, CM-cellulose 23.4 710 26 27 30
chromatography
7. (NH,)2SO4 4.6 650 20 32 28
precipitation
a R. L. Switzer and H. A. Barker, J. Biol. Chem. 242, 2658 (1967).
b One unit causes an increase of 3.85 absorbance units per minute at 240 m~, cot
responding to 1 micromole of mesaconate per minute under the assay conditions
Activators and Inhibitors. No metal ion requirement has been ob-

served. Some preparations are partially active after considerable exposure
to oxygen, but all preparations are m a r k e d l y activated by incubation
with mercaptoethanol or other thiol and, once reduced, are rather rapidly
inhibited b y exposure to oxygen. Sulfhydryl reagents, such as mercuri-
benzoate, arsenite, and iodoacetate, are strong inhibitors.
Complete Mutase System
Properties
Substrate Specificity. 1 The only known substrates for the reversible
reaction catalyzed b y the mutase are L-glutamate and threo-3-methyl-
L-aspartate. The apparent K,, values for the substrates are L-glutamate,
approximately 1.5 m M ; methylaspartate, approximately 0.5 mM.
Cobamide Coenzyme Specificity. Co-deoxyadenosylcobalamin or one
of its analogs is essential for the glutamate nmtase reaction. " I n c o m -
plete" co-deoxyadenosyl corrinoids, lacking a benzimidazole or purine
base in the nucleotide side chain, are inactive. Using a relatively crude
glutamate mutase preparation, apparent Km values were determined 3,~°
for the following co-deoxyadenosyl cobamide derivatives: benzimidazolyl,
0.23 ~M; 5(6)-methylbenzimidazolyl, 0.43 #M; 5(6)-aminobenzimida-
zolyl, 0.46 ~M; adeninyl, 1.2 ~M; 2,6-diaminopurinyl, 1.6 ~M; and 5,6-
dimethylbenzimidazolyl, 13 /~M. The Vma~ values for all these coenzyme
analogs are the same ±20%. The absolute affinity of co-deoxyadenosyl-
benzimidazolyl cobamide (BC coenzyme) for the mutase complex is
markedly dependent on the ratio of component S to component E in the
assay system.4 As the S/E ratio increases from 0.36 to 17.8 units per unit,
the apparent Km of the coenzyme decreases from 1.29 ~M to 0.031 ~M.
Comparable data for other coenzyme analogs "are not available.
Deoxyadenosylcobalamin is less satisfactory than several of its
analogs for use in mutase assays because the absorbance of the coenzyme
at a saturating level (80/zM) is large (2.1) at 240 mt~, the wavelength
used in the assay.
Influence o] Temperature on Rate. 11 The reaction rate increases about
3.0-fold when the temperature is increased from 25 ° to 37 °. A maximal
ra~e is obtained at 40 °, using a 30-minute incubation period; with a
shorter incubation, the optimal temperature is probably higher. The
maximal temperature is about 55 ° .
Influence o] p H on Rate21 The pH-activity curve is asymmetrically
bell shaped with an optimum at pH 8.5 and a range of pH 5.0 to 9.7. A
pH slightly below the optimum is used in the mutase assay to minimize
the absorbance of mercaptoethanol at 240 m~.
Equilibrium of the Mutase ReactionJ ~ The equilibrium constant,
K = (L-glutamate)/(threo-3-methyl-L-aspartate), at pH 8.2 and 30 °, is
10.7 ± 0.4. This corresponds to a AF ° of --1.43 kcal for the conversion
of methylaspartate to glutamate.
Ef]ect of Mutase Component Concentration on Reaction Rate. ~,~ The
rate of the mutase reaction depends on the absolute and relative con-
centrations of the two components. With a constant level of one com-
ponent, the rate increases with the concentration of the other component
until saturation is reached. The rate-component concentration curves do
not obey the Michaelis-Menten equation; the curves bend more sharply
in approaching saturation than this equation predicts. With a constant
level of one component, a maximal rate is closely approached when the
level of the second component (in activity units) is about 4 times as high.
1,,j. I. Toohey, D. Perlman, and tI. A. Barker, J. Biol. Chcm. 236, 2119 (1961).
,IH. A. Barker, V. Roozc, F. Suzuki, and A. A. Iodice, J. Biol. Chem. 239, 3260
(1964).
[51] L-CITRA.MALATE HYDROLYASE 331
[ 51 ] L- C i t r a m a l a t e H y d r o l y a s e
By C. C. WANQ and H. A. BARKER
coo-
I -OOC~c/CH3
HO--C--CH3
L ~. II + H20
CI~ _ H/C~.coo_
COO
L- (+) - C i t r a m a l a t e Mesaconate
A s s a y M e t h o d 1, 2
Principle. Citramalate hydrolyase activity is estimated by measuring
the rate of absorbance increase at 250 n ~ resulting from the conversion of
citramalate to mesaconate. The enzyme from Clostridium tetanomorphum
consists of two separable protein components (I and II). The assay for
each component is done by adding an excess of the other component.
Maximal activity of the rate-limiting component in the assay is attained
only after the two components have been incubated together ("acti-
vated") under carefully controlled conditions. Under suitable conditions
the rate of absorbance increase is proportional to the concentration of the
rate-limiting protein component.
Reagents
Tris-HCI buffers, pH 8.2, 0.5 M and pH 8.4, 0.5 M
2-Mercaptoethanol (MET), 0.5 M. Prepare weekly
Ferrous ammonium sulfate (FeAS), 10 mM. Prepare daily
Tris buffer-cysteine-FeAS assay solution. This solution contains
60 mM Tris-HCl, pH 8.2, 2.0 mM L-cysteine hydrochloride,
2.0 mM NaOH, and 0.2 mM FeAS. It is made up in a graduate
cylinder or similar vessel with a small air-surface to volume
ratio and is mixed carefully to avoid excessive oxidation of
cysteine. The temperature is adjusted to 25 °. The solution is
allowed to stand at least 15 minutes; during this time the initial
purple color fades (except at the surface) to give an almost color-
1A. It. Blair and It. A. Barker, J. Biol. Chem. 241, 400 (1966).
2C. C. Wang, Doctoral Dissertation, University of California, Berkeley, California,
1966.
less solution. The solution is stable for at least 4 hours when

excessive contact with air is avoided
DL-Citramalate, s disodium salt, 1.0 M, pH 7.0
Hydrolyase component I solution (for component II assay), 400
units per milliliter of 20 mM Tris-HC1 buffer, pH 7.0. The
preparation should have a specific activity of at least 300 units
per milligram of protein and should be free of component II
activity
Hydrolyase component I solution (for component I assay). The
solution, diluted when necessary in 20 mM Tris-HC1 buffer,
pH 7.0, should contain between 10 and 1000 units/ml
Hydrolyase component II solution (for component I assay), 400
units per milliliter of 20 mM Tris-HCl buffer, pH 8.0, and
50 mM K2S04. The preparation should have a specific activity
of at least 100 units per milligram of protein and should be
relatively free of component I activity (less than 5 ~ of the
component II specific activity)
Hydrolyase component II solution (for component II assay). The
solution, diluted when necessary in 20 mM Tris-HCl buffer,
pH 8.0, and 50 mM K2S0~, should contain between 10 and 1000
units/ml
Hydrolyase Component I
Assay for Component I
Procedure. The assay involves two distinct steps: (a) the "activation"
of the system by incubating a rate limiting amount of component I with
an excess of component II under specific conditions, and (b) the spectro-
photometric assay proper, using an aliquot of the activated enzyme
solution.
The activation of the system is conveniently done in a reaction mixture
having a volume of 100 pl, although a smaller volume may be used. On
the I00 pl scale, 10 pl of hydrolyase component II solution (for com-
ponent I assay) is added to 80 ~l of a freshly prepared solution (in a
0.5 ml test tube at 0 °) containing 10 pl 0.5M Tris-HCl buffer, pH
8.4, I0 ~I 0.5 M 2-mercaptoethanol, and 5 pl of 10 re.M"ferrous ammonium
sulfate. From 1 to 10 ~l of the component I solution to be assayed, con-
taining 0.1-1.0 unit of component I, is added and the volume is made up
to 100/zl with distilled water. The solution is gently mixed in such a way
as to avoid unnecessary aeration and immediately transferred into a piece
*H. A. Barker, Biochem. Prep. 9, 25 (1962).

[51] L-CITRA~ALATE HVDROLYASE 333
of acid-washed, soft-glass capillary tubing of 1.2-1.5 mm i.d. and 12-15

mm length. Both ends of the tubing are sealed by means of a small flame
so as to leave an air space of about 1 cm at each end. The reaction mixture
in the sealed capillary is then incubated in a 37 ° water bath for 90
minutes to activate the system. The activated solution in the sealed
capillary is then kept at room temperature until needed for assay.
Storage for several hours at room temperature does not change the
activity. To obtain a sample of the activated solution, the tip of the
capillary is broken off, and an aliquot is taken from the center of the
liquid column by means of a 10/~l graduated Hamilton syringe. 4
The spectrophotometric assay is done in a silica cuvette (1.5 ml
capacity, 10 mm light path). A 5-10 #1 aliquot of the activated enzyme
solution is introduced at the bottom of a cuvette containing 1.0 ml of the
Tris buffer-cysteine-FeAS assay solution at 25 °. The reaction is im-
mediately started by addition of 25/~I of 1 M vL-citramalate by means
of a small plastic delivery and stirring device. The reaction mixture is
gently stirred and the absorbance is read at 250 m~ in a UV spectro-
photometer, preferably provided with an automatic recording system,
against a blank having about the same initial absorbance as the sample,
usually 0.5 mM sodium mesaconate. The initial absolute absorbance of
the reaction mixture should not exceed 2.0. The increase in absorbance
at 250 m~ between 30 and 90 seconds after starting the reaction is taken
as a measure of enzyme activity. The rate should be in the range of
0.02-0.4 absorbance units per minute at 25 °. A correction must be made
for component I activity in the component II preparation used in the
assay; this will also provide a correction for small absorbance changes
resulting from nonenzymatic reactions.
Units. A unit of component I activity causes an increase of 2.26
absorbance units per minute at 250 m~, corresponding to the formation
of 1 micromole of mesaconate per minute, under the conditions of the
assay. Specific activity is defined as units per milligram of protein deter-
mined by the method of Lowry et al., 5 using crystalline bovine serum
albumin as a standard.
All operations are carried out at 0-4 ° unless otherwise indicated.
8tep 1. Preparation o / t h e Extracts. Freshly harvested or previously
frozen and thawed cell paste of Clostridium tetanomorphum strain H1,
grown on a glutamate-yeast extract medium, is used as a source of
Hamilton Co., Whittier, California.
265 (1951).
enzyme. To 500 g of cell paste is added 525 ml of 50 mM Tris-HCl

buffer, pH 7.5, and the suspension is sonicated in 60 ml portions in a
10 kc Raytheon sonicator for 15 minutes. The sonicate is centrifuged for
30 minutes at 10,000 g and then for 2 hours at 55,000 g. The precipitates
are resuspended in 400 ml of the same buffer and again centrifuged for
2 hours at 55,000 g. The supernatant solutions are combined and the
precipitate is discarded. The pH of the extract is about 7.0.
Step 2. Protamine Treatment. The protein concentration of tile extract
is adjusted to 30 mg/ml with distilled water to give a final volume of
1670 ml. To this solution 0.6 volume (1 liter) of 1% protamine sulfate
(salmine, Lilly) solution is added slowly with stirring during about 40
minutes. After stirring for an additional 30 minutes, the solution is
centrifuged at 10,000 g for 30 minutes. The precipitate is discarded.
Step 3. Ammonium Sul]ate Precipitation. Powdered ammonium sulfate
(451 g) is slowly added to the enzyme (2560 ml; 10.2 mg protein per
ml) with stirring to give a 30% saturated solution. After stirring for an
additional 30 minutes, the precipitate is removed by centrifugation at
10,000 g for 20 minutes. An additional 1221 g of ammonium sulfate is
added to bring the supernatant solution (2700 ml) to 90% saturation.
After an additional 30 minutes of stirring, the solution is centrifuged at
10,000 g for 1 hour. The supernatant solution is discarded, and the
precipitated protein is dissolved in 300 ml of 60 mM Tris-HC1 buffer,
pH 9.0.
Step 4. Heat Treatment at 55 °. The concentrated enzyme solution
(498 ml; 49 mg protein per ml) is transferred to a 500 ml volumetric
flask containing a magnetic stirring bar, and 1.8 ml of 14.1M 2-mer-
captoethanol and 0.50 ml of 0.10M ferrous ammonium sulfate are added
to give final concentrations of 50 mM and 0.1 mM respectively. The air
above the solution is displaced by O~-free argon, and the flask is closed
with a rubber stopper holding a thermometer. The flask is first incubated
in a 37 ° water bath for 2 hours to reduce the enzyme and is then trans-
ferred to a 55 ° water bath. When the solution is stirred vigorously, it
reaches a temperature of 55 ° in about 5 minutes. The flask is kept at this
temperature for another 5 minutes and then is rapidly cooled in an ice
bath with stirring. The denatured protein is removed by centrifugation at
10,000 g for 30 minutes and discarded. The supernatant solution is di-
alyzed in 1 inch diameter cellophane tubing for 24 hours against 5 liters
of 0.10M Tris-HC1 buffer, pH 7.0; the buffer is changed twice. If a
precipitate forms, it is removed by centrifugation.
Step 5. DEAE-Sephadex ~ Column Chromatography. The dialyzed en-
*DEAE-cellulose may also be used to obtain about the sa.me degree of purification
and yield by a slightly modified procedure (see footnote I).
[51] L-CITRAMALATE HYDROLYASE 335
zyme solution (440 ml; 33.6 mg protein per ml) is passed into a DEAE-
Sephadex A-50 column (5.0 cm X 30 cm), previously equilibrated with
0.10M Tris-HC1 buffer, pH 7.0. Elution of the column is started witb
2800 ml of the same buffer and continued with 2 liters 0.10M Tris-
HC1 buffer, pH 7.0, containing 0.10M K2SO~. Fractions (25 ml) are
collected at a rate of 2 ml per minute. Component I, virtually uncon-
taminated with component II, elutes with the Tris buffer (fractions 28-
90). Component II, heavily contaminated with component I activity, is
eluted by the Tris-K2SO4 solution (fractions 165-190) (see Purification
of Component II).
Step 6. Ammonium Sul]ate Precipitation. The component I fractions
from the previous step (1540 ml; 1.9 mg protein per ml) are brought to
0.83 saturation with ammonium sulfate by slow addition of 910 g of the
powdered salt while the solution is stirred continuously. After stirring for
another 30 minutes, the precipitated protein is separated by centrifuga-
tion at 10,000 g for 1 hour and dissolved in 50 ml of 20 mM Tris-HC1
buffer, pH 7.0. The protein solution (82 ml; 29.5 mg protein per ml) is
dialyzed against 2 liters of 20 mM Tris-HC1 buffer, pH 7.0, for 12
hours with two changes of the external solution. The dialyzed solution is
centrifuged at 10,000 g for 15 minutes to remove a small precipitate.
Step 7. DEAE-Sephadex Column Chromatography. The dialyzed
solution (100 ml; 21.5 mg protein per ml, pH 7.0) is applied to a 2.5
cm X 40 cm DEAE-Sephadex A-50 column, previously equilibrated with
20 mM Tris-HC1 buffer, pH 7.0. The column is eluted with 3 liters of
the same buffer. Fractions (25 ml) are collected at a rate of 1.0 ml per
minute. Component I elutes in an almost symmetrical activity peak
between fractions 73 and 96. These fractions are pooled.
Step 8. Brushite Column Chromatography. The component I fractions
from the previous step (564 ml; 0.26 mg protein per ml) are applied to a
1.0 cm X 10 cm brushite ~ column previously equilibrated with 20 mM
Tris-HCl buffer, pH 7.0. The column is then washed with 100 ml of
the same buffer. The eluate, which contains no component I activity, is
collected in 25 ml fractions at the rate of 0.33 ml per minute. Component
I is then eluted with 120 ml of 0.10M ammonium sulfate. Fractions
(2 ml) are collected at the same rate. The activity elutes in fractions
10-24 of the second eluent. These fractions (30 ml; 1.50 mg protein per
ml) are combined and carefully adjusted to pH 5.6 with 1 N HCl. The
enzyme solution is then dialyzed for 18 hours against 2 liters of 15 mM
sodium citrate buffer, pH 5.60; the external solution is changed twice.
Step 9. CM-Cellulose Column Chromatography. The dialyzed solution
~W. T. Jenkins, BiocAem. Prep. 9, 83 (1962).

(35 ml; 1.29 mg protein per ml), having essentially the same pH and
electrical conductivity as 15 mM sodium citrate, pH 5.6, is passed into a
1.0 cm X 10 cm CM-cellulose column previously equilibrated with the
same buffer. Elution of the column is started with 50 ml of this buffer and
continued with a linear salt gradient made with 50 ml of 15 mM sodium
citrate, pH 5.6, in the mixing chamber and 50 ml of 0.10 M sodium citrate
buffer, pH 6.5, in the reservoir. Fractions (2 ml) are collected at the rate
of 0.4 ml per minute. Component I activity elutes in a narrow peak
(fractions 46-50) soon after the salt gradient is started. The pooled
component I fractions (10 ml; 0.95 mg protein per ml) are concentrated
by precipitation with 0.80 saturated ammonium sulfate, and then di-
alyzed against 1 liter of 0.10M Tris-HCl buffer, pH 8.4 for 18 hours,
with one change of buffer. Typical data on component I purification are
given in Table I. The overall yield is 18-24% with a purification factor
of about 1000.
Properties of Component 12
Homogeneity and Molecular Weight. The best preparations contain
more than one protein. Disc eleetrophoresis in polyacrylamide gel (ani-
onic system) shows the presence of two major protein components of
similar mobility in about equal amounts and one or more minor com-
ponents. Two proteins are also seen in the Ouchterlony test 8 when highly
purified component I is tested against rabbit anti-component I serum. It
is not known whether only one or both proteins possess component I
activity. All component I preparations after step 5 are free of component
II, fl-methylaspartase 9 (threo-3-methyl-L-aspartate ammonia-lyase) and
citramalate pyruvate lyase, l° The molecular weight of component I is
estimated to be 46,000 ± 5000 by gel filtration using columns of Sephadex
G-100 and G-200.11
Stability. Component I is best stored at a protein concentration above
1 mg/ml in 0.1 M Tris-HC1 buffer, pH 8.4, at --10 ° or lower; the
activity is stable for months under these conditions. Freezing and thawing
does not significantly decrease the activity. The enzyme is quite stable
from pH 7 to 11. Below pH 6 it becomes increasingly labile as the pH
falls, but even at pH. 2.0 (75 mM sodium citrate buffer) 64% of the
activity remains after incubation at 25 ° for 24 hours. Component I
activity is considerably less stable at low than at high ionic strengths.
sO. Ouchterlony, Acta Palhol. Microbiol. Scand. 26, 507 (1949).
H. A. Barker, R. D. 8myth, R. M. Wilson, and H. Weissbach, J. Biol. Chem. 234,
320 (1959).
ioH. A. Barker, this volume [52].
u j . R. Whitaker, Anal. Chem. 35, 1950 (1963).
[51] L-cITrtAMXLXT~ HYDROLYASE 337
~ :E x
.~'~
l
tt~
e,
<
~q
For prolonged storage, the concentration of Tris-HC1 buffer should

be 0.1 M or higher. Ammonium sulfate and possible other salts can re-
place part of the buffer, when desirable. The enzyme is relatively stable
at 37 ° or below, but loses up to 35~ of its activity when heated at 55 °
for 10 minutes under otherwise favorable conditions. Oxygen does not
affect the stability of component I.
Absorption Spectrum. Component I shows a typical protein absorption
spectrum with Amax at 280 mt~ and Ami, at 252 mt~. The A..so,,~:A26o~,v
ratio is 1.75 for a highly purified preparation (11,800 units per milligram
of protein). The absorbance of a neutral 0.1% component I solution is
0.90 at 280 m~.
Hydrolyase Component II
Assay for Component II 1,2

This is done by the two-step procedure described in the Assay for
component I, except that an excess of component I and a limiting amount
of component II is used. In carrying out the activation procedure on a I00
~l scale, I0 ~I of component I solution (for component II assay) is added
to 80 /~l of Tris-mercaptoethanoI-ferrous ammonium sulfate solution.
From I to 40/~l of the component II solution to be assayed, containing
0.1-1.0 unit, is added, and the volume is made up to 100 #l with water.
The remainder of the procedure is identical with that used for component
I assay.
The activity unit and the specific activity of component II are
defined as for component I.
Steps 1 through 5 are the same as in the purification of component I.
Step 6. Ammonium Sul]ate Precipitation. The component II fractions
from step 5 (655 ml; 11.1 mg protein per ml) are brought to 0.60 satura-
tion by addition of 255 g of powdered ammonium sulfate. The precipitate,
collected by centrifugation at 10,000 g for 30 minutes, is dissolved in 100
ml of 20 mM Tris-HCl buffer pH 7.0. The solution is dialyzed against
2 liters of the same buffer for 12 hours with two changes of buffer.
Step 7. Brushite Column Chromatography. The dialyzed solution (225
ml; 22.2 mg protein per ml) is passed into a brushite column (4.0 cm X
39 cm) previously equilibrated with 20 mM Tris-HC1 buffer pH 7.0
and is followed by 1 liter of the same buffer. The eluate is collected in
12 ml fractions at a rate of 1 ml per minute. About 75% of the added
component II activity and 33% of the component I activity elute to-
gether in fractions 53-79. These fractions are combined and used for
[51] L-CITRAMALATE HYDROLYASE 339
further purification. This step does not significantly increase the specific
activity of component II but removes much of the contaminating compo-
nent I.
Step 8. CM-Sephadex Column Chromatography. The component II
solution (332 ml; 10 mg protein per ml) is acidified to pH 5.4 with 1 N
HC1 and a small precipitate is removed by centrifugation. The super-
natant solution is diluted (to 500 ml) with distilled water until its
conductivity is equal to that of 10 mM sodium citrate buffer pH 5.4. The
solution (4.7 mg protein per ml; 126 units/rag) is then passed into a CM-
Sephadex C-50 column (2.5 cm }( 40 era), previously equilibrated with
10 mM sodium citrate buffer pH 5.4. The column is eluted first with 250
ml of the same buffer and then with a linear salt gradient made with
500 ml of 10 mM sodium citrate pH 5.4 in the mixing vessel and 500 ml
of 0.10 M sodium citrate pH 5.4 in the reservoir. Fractions (10 ml) are
collected at the rate of 1 ml per minute. Component II activity elutes in
two well separated peaks. The first peak (A) comes off in the pass-
through fractions and the early fractions of dilute buffer (fractions 7-
56); this peak contains about 39% of the added component II activity
and is virtually free of component I activity. Peak A is used for the
further described purification. The second component II peak (B) elutes
in fractions 109-140 in the salt gradient; this peak contains about 49%
of the added component II, but is contaminated with considerable com-
ponent I activity. The peak B fraction may be put separately through
steps 9 and 10 to give a final product of somewhat lower specific activity.
Step 9. Ammonium Sulfate Precipitation. Fraction A (504 ml; 1.92
mg protein per ml) is brought to 0.50 saturation 1~ by addition of 158 g
of ammonium sulfate. The precipitate is collected by centrifugation, dis-
solved in 20 ml of 0.10M Tris-HC1 buffer, pH 9.0, and the solution is
centrifuged briefly to remove insoluble material.
Step 10. Sephadex G-200 Column Chromatography. The enzyme solu-
tion (25 ml; 19 mg protein per ml) is passed into a 3.0 cm X 100 cm
Sephadex G-200 column previously equilibrated with 0.10M Tris-
HC1 buffer pH 9.0. The column is eluted with the same buffer at a
rate of 0.20 ml per minute; the effluent is collected in 5.0 ml fractions.
Component II activity elutes in a single, ahnost symmetrical peak
(fractions 64-83) located between two prominent absorbance (280 m~)
peaks (fractions 47-68 and 80-110). Component II is purified about
9-fold in this step with a yield of about 83%. The active fractions (100
ml; 0.48 mg protein per ml) are concentrated by precipitation with 0.80
1.~When this step is applied to fraction B of step 8, 0.30 saturated ammonium sulfate
is used.
340 R~ACTIONS L E A D I N G TO A N D FROM T H E CYCLE [51]
~v
Z
O
~v
r~
O
O
~ ~ ~i ~ o.~,~ ~ ~.~
[51] L-CITRAMALATEHYDROLYASE 341
saturated ammonium sulfate and then are dialyzed against 1 liter of

0.10 mM Tris-HCl buffer pH 8.0 for 8 hours, with one change of buffer.
Typical data on component II purification are given in Table II. The
overall yield is 14~o with a purification of about 150. An additional 19%
yield with a purification of about 90 (104,000 units; 900 units/rag) can
be obtained by separately carrying the B fractions from step 8 through
steps 9 and 10. This product is also essentially free of component I activ-
ity. It should be noted that the A and B fractions show the same differ-
ence in elution pattern when they are rechromatographed on CM-
Sephadex columns as in the original separation.
Properties of Component II
Homogeneity and Molecular Weight. Polyacrylamide gel disc electro-
phoresis (anionic system) shows that a quarter or a third of the protein
in the best preparation (1600 units per mg protein) is one component;
several other components are also present. It is not known whether
component II activity is associated with the major protein component.
Heterogeneity of the preparations is also indicated by the absence of
correlation between activity and 280 m/~ absorbance in the elution pat-
tern obtained by gel filtration on the Sephadex G-200 column used in the
last step of component II purification. The A and B preparations of
component II show a reproducible difference in their elution patterns
from CM-Sephadex (see above). Apparently they contain distinct species
of component II carrying different charges.
The best component II preparations show no component I, fl-methyl-
aspartase, or citramalate pyruvate lyase activity. The absence of com-
ponent I protein from component II preparations is indicated by a nega-
tive reaction with anti-compound I serum (rabbit) in the Ouchterlony
test.
The molecular weight of component II, estimated by gel filtration
through a Sephadex G-200 column equilibrated with 0.10M Tris-HC1
buffer, pH 9.0, is ~10,000 ± 20,000. This material appears to contain 8
subunits, since gel filtration at pH 7.5 shows the presence of three addi-
tional active protein species having molecular weights of about 37,000,
70,000, and 164,000. When the pH is again raised to 9.0, these lower
molecular weight species reassociate to form active component II of the
original molecular weight.
Stability. Component II is best stored at --10 ° or lower in 0.10 M
Tris-HC1 buffer pH 8.0 at a protein concentration above 2 mg/ml.
Under these conditions the activity does not decline appreciably during
storage for 2 months at --10% At --196 °, the activity remains constant
342 REACTIONS LEADING TO AND FROM TtIE CYCLE [51]
for at least 8 months. Since freezing and thawing causes a 10-153 loss in
activity, the enzyme should be frozen and stored in small aliquots.
Component II is stable for 5 minutes at 55 ° in the presence of
mercaptoethanol and ferrous ion. At 37 °, the activity is stable for at
least 2 days when mercaptoethanol, ferrous ion, and component I are
present. Omission of either component I or ferrous ion, greatly decreases
the stability (50% loss of activity in 3 hours at 37°), whereas the further
omission of mercal)tocthanol again increases tile stability (no loss of
activity after 3 hours at 37°).
Component II is most stable at pH 7 to 8; about 80% of the activity
remains after storage for 48 hours at 4 °. Above pH 9 and below pH 6
the stability falls off rather steeply.
The stability of component II at 4 ° is increased by raising the buffer
or salt concentration; 0.1-0.5 M is favorable.
Component II should not be frozen in the presence of mercaptoeth-
anol.
Absorption Spectrum. The absorption spectrum of component II is
similar to that of component I, except that the A2som~t:Az6om~ratio is
somewhat lower (1.50), probably indicative of the lesser degree of purity.
The absorbance of a neutral 0.1% solution of the best preparation of
component II (1600 units per mg protein) is 0.95 at 280 m~.
Activators and Inhibitors. Component II is reversibly inactivated by
oxygen and, in the oxidized form, is activated by incubation with
fl-mercaptoethanol and some other sulfhydryl compounds; 30 mM mer-
captoethanol allows maximal activation in 90 minutes at 37°; 10 mM
gives about half-maximal activation under the same conditions. Ferrous
ion is required for activation. Half-maximal and maximal activation re-
quire about 20 ~M and 70 #M ferrous ammonium sulfate, respectively;
simple Michaelis-Menten kinetics are not observed. Ferrous ion cannot be
replaced by Mg *+, Mn ++, Ca +* or Co ++. Manganese ion is somewhat in-
hibitory; 5 mM MnCl~_ causes about 50% inhibition in the presence of
0.5 mM ferrous ammonium sulfate.
Properties of the Complete Citramalate Hydrolyase System s
Interaction o] the Components. Neither component alone possesses
hydrolyase activity. To obtain maximal activity in the assay the two
components must previously be incubated together under the conditions
for activation described in the assays. The observed activity depends in
a complex manner on several parameters. Without going into detail, these
relations may be briefly summarized.
The activity of each component depends upon the concentration of the
other component. Activity increases with the concentration of the second
[51] L-CITR&MALATE tIYDROLYASE 343
component until saturation is reached; this occurs when the activity of

the second component is about 4 times that of the rate-limiting compo-
nent. The absolute concentration of the saturating component should be
about 40 units/ml. The interaction of the two components is most rapid
and complete in an oxygen-free solution containing 50-200 mM Tris-
or ethanolamine-chloride buffer, pH 8-10, 30-50 mM mercaptoethanol,
and 0.1-0.5 mM ferrous ammonium sulfate. The temperature of the solu-
tion must be above 20 ° to achieve maximal activity. At 37 °, full activa-
tion takes 60-90 minutes when the component II preparation has not
previously been reduced; when it has been reduced, activation takes
15-20 minutes.
Substrate Specificity. The known substrates for citramalate hydroly-
ase are L-(+)-citramalate, 13 L-malate, mesaconate, and fumarate. At pH
8.2 and 25 °, the K~ values for these substrates are 2.4, 6.7, 0.59, and
0.95 raM, and the relative Vmax values are 100, 256, 407, and 650, respec-
tively, when component II is rate limiting. The same K~ values are
obtained under otherwise identical conditions when component I is
rate-limiting. The D-is0mers of citramalate and malate are not substrates
for the enzyme. D-Malate, succinate, and L-tartrate are competitive in-
hibitors with K~ values of 3.5, 6.9, and 13.8 raM, respectively, determined
with L-citramalate as substrate.
Influence of pH on Rate. In the assay solution, containing 2.0 mM
cysteine, 0.2 mM ferrous ammonium sulfate, and 60 mM Tris-HC1
buffer, maximal activity is obtained at pH 8.0-8.5; the activity is lower
in more acid or alkaline solution, reaching 50% of the maximal value at
about pH 7.1 and 9.3. When 2.0 mM 2-mercaptoethanol is substituted for
cysteine, under otherwise identical conditions, the pH optimum is shifted
to pH 6.5-7.0, and the absolute rate is decreased about 20%. Under
these conditions, a half-maximal rate is observed at pH 5.7 and 7.7. It
should be noted that the stability of the activity in the assay solution
is greatly diminished when mercaptoethanol is substituted for cysteine;
consequently with mercaptoethanol the rate of reaction decreases with
time.
Influence of Sulfhydryl Compounds. In the assay solution at pH 8.2-
8.4, D-cysteine, 2-mercaptoethylamine, and DL-homocysteine are as ef-
fective as L-cysteine; 2,3-dimercapto-l-propanol is about half as effec-
tive; and mercaptoethanol, reduced glutathione, sodium thioglycolate,
and 3-mercaptopropionate are ineffective. The optimal concentration of
L-cysteine is 0.5-2.0 mM; at higher and lower concentrations the activity
is significantly decreased.
,sp. A. von der Miihll, G. Settimj, H. Weber, and D. Arigoni, Chimia 19, 595 (1965).
Divalent Cation Requirement. Ferrous ion is essential in the assay

solution to stabilize the activity, even though the system has been acti-
vated in the presence of Fe+*. Maximal activity is obtained with 0.2-0.9
mM ferrous ammonium sulfate. Omission or substitution of Fc ++ by Co ++,
Ca +*, Cu ++, Mg+*, Zn ++, or Mn ++, in the assay solution results in a rapid
decline of activity during the assay. Manganese ion causes the most rapid
and extensive inhibition. In the presence of 0.2 mM Fe+*, the same
concentration of MnCl, causes a 2 0 ~ inhibition.
Equilibrium. The equilibrium constant, K = (L-citramalate)/(mesa-
conate), at pH 8.4 and 25 ° is between 4.6 and 7.0. A more precise value is
not available.
[52] Citramalate Pyruvate Lyase 1

By H. A. BARKER
COOH
I
HOC--CH3 --~ CHaCOOH "4- CH3COCOOH
f
CH2
I
COOH
Assay Method
Principle. The lyase activity can be measured most conveniently by
coupling the above reaction with the reduction of pyruvate by N A D H in
presence of excess lactate dehydrogenase. The decrease in absorbance at
340 ms is measured.
Reagents
Dipotassium L-(~)-citramalate, ~ 0.1 M, or DL-citramalate, 4 0.2M
NADH, 1.0 mM
MgC12, 10 mM
Lactate dehydrogenase, about 5 Kornberg units 5 per milliliter
I The enzyme has also been called citramalase2

* If. A. Barker, in "The Bacteria" (I. C. Gunsalus and R. Y. Stanier, eds.), Vol. II,
p. 151. Academic Press, N e w York, 1961.
*H. A. Barker and H. H. Blair, Biochem. Prep. 9, 21 (1962).
'H. A. Barker, Biochem. Prep. 9, 25 (1962).
'A. Kornberg, Vol. I, p. 441.
Divalent Cation Requirement. Ferrous ion is essential in the assay

solution to stabilize the activity, even though the system has been acti-
vated in the presence of Fe+*. Maximal activity is obtained with 0.2-0.9
mM ferrous ammonium sulfate. Omission or substitution of Fc ++ by Co ++,
Ca +*, Cu ++, Mg+*, Zn ++, or Mn ++, in the assay solution results in a rapid
decline of activity during the assay. Manganese ion causes the most rapid
and extensive inhibition. In the presence of 0.2 mM Fe+*, the same
concentration of MnCl, causes a 2 0 ~ inhibition.
Equilibrium. The equilibrium constant, K = (L-citramalate)/(mesa-
conate), at pH 8.4 and 25 ° is between 4.6 and 7.0. A more precise value is
not available.
[52] Citramalate Pyruvate Lyase 1

By H. A. BARKER
COOH
I
HOC--CH3 --~ CHaCOOH "4- CH3COCOOH
f
CH2
I
COOH
Assay Method
Principle. The lyase activity can be measured most conveniently by
coupling the above reaction with the reduction of pyruvate by N A D H in
presence of excess lactate dehydrogenase. The decrease in absorbance at
340 ms is measured.
Reagents
Dipotassium L-(~)-citramalate, ~ 0.1 M, or DL-citramalate, 4 0.2M
NADH, 1.0 mM
MgC12, 10 mM
Lactate dehydrogenase, about 5 Kornberg units 5 per milliliter
I The enzyme has also been called citramalase2

* If. A. Barker, in "The Bacteria" (I. C. Gunsalus and R. Y. Stanier, eds.), Vol. II,
p. 151. Academic Press, N e w York, 1961.
*H. A. Barker and H. H. Blair, Biochem. Prep. 9, 21 (1962).
'H. A. Barker, Biochem. Prep. 9, 25 (1962).
'A. Kornberg, Vol. I, p. 441.
[52] CITRAMALATE P Y R U Y A T E LYASE 345
Enzyme: suitably diluted in 10 mM potassium phosphate buffer,

pH 7.4
Procedure2 The reaction mixture contains 0.1 M sodium L-(-}-)-citra-
malate or 0.2 M DL-citramalate, 0.10 ml; 1 mM NADH, 0.10 ml; 10 mM
MgC12 0.1 ml; phosphate buffer, 0.1 ml; lactate dehydrogenase solution,
0.02 ml; and lastly, 0.005 to 0.04 unit of citramalate lyase in a total
volume of 1.00 ml. The reaction is carried out at 24 ° ~ 1 ° in a 1 ml
volume, 1 cm light path silica cuvette. Absorbance readings at 340 m~ are
made at 0.5 minute intervals from 0.5 to 2.5 minutes after addition of
the enzyme, and the average rate of absorbance change is estimated.
During the first 3 minutes the reaction rate remains almost constant. A
correction must be applied for N A D H oxidation in the absence of citra-
malate. Activity is proportional to enzyme concentration over the indi-
cated range.
Units. One unit of activity is defined as the amount of enzyme
catalyzing the formation of 1 micromole of pyruvate and the oxidation of
1 micromole of N A D H per minute under the assay conditions. This
corresponds to a corrected absorbanee change of 6.21 per minute at 340
m#. Specific activity is defined as units per milligram of protein meas-
ured by the method of Lowry et al. T using crystalline bovine serum
albumin as a standard.
Extraction and Storage of the Enzyme

The lyase is obtained from cells of Clostridium tetanomorphum Hl
grown anaerobically in a yeast extract-sodium glutamate medium2 Ex-
tracts are prepared by treating a cell suspension (15 g of packed cells and
30 ml of distilled water) in a 10 kc Raytheon sonic oscillator for 20
minutes at 0 ° to 5 ° and centrifuging the resulting homogenate for 10
minutes at 11,000 g. The clear supernatant solution (30-35 mg protein
per milligram) is either immediately frozen and stored at or below --10 °,
or lyophilized and stored at a low temperature as a dry powder. The
specific activity of lyophilized preparations ranges from 0.08 to 0.4 unit
per milligram, dry weight, or 0.12 to 0.6 unit per milligram of protein.
When protected from moisture and stored at --10 °, lyophilized prepara-
tions retain activity for more than a year. The specific activity of freshly
prepared extracts is 0.8--1.3 units per milligram of protein. The activity
of extracts declines rapidly, even at low temperatures. The half-life of
• H. A. Barker, Arch. Mi~robiol. 5@, 4 (1967).
265 (1951).
"H. A. Barker, R. D. Smyth, R. M. Wilson, and H. Weisabach, J. Biol. Chem. 234,
320 (1959).
346 REACTIONS LEADING TO AND FROM THE CYCLE [~2]
lyase activity in 30 mM phosphate buffer pH 7.0 at --15 °, 0 °, 25 °, and

37 ° is about 12 hours, 2.5 hours, 1 hour, and 15 minutes, respectively.
Attempts to stabilize the activity in extracts have been unsuccessful. The
catalytic properties of the lyase have been determined mainly with un-
fractionated extracts or with preparations freed of low molecular weight
impurities by precipitation with 80% ammonium sulfate, followed by
dialysis for a few hours.
Properties
Substrate Specificity. L-(+)-Citramalate 9 is the only substrate
known to be cleaved by the lyase. D-(--')-Citramalate, DL-isocitrate,
DL-malate, citrate, and mesaconate are not converted to keto acid under
conditions allowing rapid cleavage of (+)-citramalate to pyruvate and
acetate. The apparent Km of sodium L-(~-)-citramalate is 0.6 mM at low
substrate concentrations, but it appears to increase at higher substrate
concentrations. Maximal activity is obtained with 10-20 mM (-t-)-
citramalate; at higher concentrations a small inhibition is observed.
Effect oJ pH and Buffers. In 50 mM potassium phosphate buffer con-
taining 1 mM MgCI~, the activity-pH curve shows a broad maximum at
pH 7.3-7.5. At pH 6.0, the activity is 52%, and at pH 8.0 77% of that at
pH 7.4. In 60 mM Tris-HC1 buffer, the activity is maximal at pH 8.0-8.2
and is markedly lower at pH 7.5 and pH 8.8. At pH 7.7, the activity is
the same with phosplmte and Tris buffers. EDTA and pyrophosphate
buffers are strongly inhibitory.
Divalent Cation Requireme~t. A divalent cation, Mg ÷÷, Mn ÷+, or Co÷÷
appears to be essential for activity. The apparent K,~ of MgCl~ is about
0.1 mM. Calcium ion or Fe ÷÷ is unable to substitute for Mg *÷, and these
cations at 1 mM inhibit moderately in the presence of 10 #M MgClo.
Monovalent cations appear to be inert in this system.
Equilibrium. The equilibrium constant for (-t-)-citramalate cleavage
at pH 7.4 and 25 ° is about 8.3 M, corresponding to a AF ° value of --1.2
kcal.
Occurrence. The enzyme has been observed only in C. tetanornorphum
grown with glutamate as a major energy source.
Unpublished experiments of C. C. Wang and H. A. Barker have shown that the

citramalate hydro-lyase~° of C. tetanomorphum dehydrates (-~)-citramalate and
L-(+)-malate, but not their cnantiomers. Consequently it is probably that the
citramalate formed and decomposed by this organism is L-(-b)-citramalate.
Z°A. H. Blair and H. A. Barker, J. Biol. Chem. 241, 400 (1966).
[53] ~-METHYLA.SPARTASE 347
[53] ~ - M e t h y l a s p a r t a s e f r o m C l o s t r i d i u m t e t a n o m o r p h u m
IEC 4.3.1.2 L-threo-3-Methylaspartateammonia-lyase]
By MYRTLEW. HSIANG and HAROLDJ. BRIGHT
threo-~-Methyl-L-aspartate ~-mesaconate + NH3 ~- H +
Assay Method
The assay is identical in design and execution to that described by
Barker et al. 1
Principle. With amino acid as substrate, the rate of mesaconate for-
mation is measured spectrophotometrically. The absorption maximum of
mesaconate2 is at 202 m~, and ~o2 ~ is 11,600 M-lcm -1. In order to allow
the use of most types of commercial spectrophotometer, and yet retain
sufficient sensitivity, the assay is performed at 240 m~; c_~o ~ is 3850
M-~cm-1.~ Although AF ° for the reaction as written is -}-700 calories per
mole at pH 9.7,1 the stoichiometry of the reaction results in an almost
complete conversion of amino acid to mesaconate at equilibrium under
most experimental conditions. Furthermore the K,~ values of the products
(particularly that for ammonia) are larger than the K~ of fl-methyl-
aspartate2 The reverse reaction, therefore, does not interfere with the
assay. The activity of fl-methylaspartase in extracts of Clostridium
tetanomorphum is so high relative to that of other enzymes utilizing
fl-methylaspartate or mesaconate, that interference by the latter enzymes
is negligible under the assay conditions.
Reagents
Assay solution. Potassium threo-fl-methyl-L-aspartate, 4 mM, pH
9.7; ethanolamine-HC1, 50 mM, pH 9.7; KC1, 10 mM; MgC12,
1 mM. This solution should be tightly stoppered to minimize the
uptake of C02.
Enzyme. A suitable dilution is made in an ice-cold solution at pH
6.5-7.0. This solution may consist of a 10 mM buffer (potassium
phosphate or imidazole-HC1) or 0.1 to 0.5M tetramethylam-
monium chloride, the pH of which is adjusted, if necessary~ with
tetramethylammonium hydroxide.
I H. A. Barker, R. D. Smyth, R. M. Wilson, and H. Weissbach, J. Biol. Chem. 234,

320 (1959).
" H. J. Bright, unpublished experiments.
sH. J. Bright, J. Biol. Chem. 240, 1198 (1965).
348 aEACTmNS LEADING TO AND FROM THE CYCLE [53]
Procedure. One milliliter of the assay solution is placed in a silica

cuvette of 1.0 cm pathlength and 1.0 ml capacity. Ten microliters of the
enzyme solution is added on a plastic rod, and the solution is mixed
thoroughly. The rate of mesaeonate formation is then recorded at 240 m~
on a suitable spectrophotometer, the cell compartment of which is main-
tained at 25 °. With absorbance increases in the range of 0.05-0.5 rain -1,
the rate of mesaconate formation remains constant for several minutes.
the formation of 1 mieromole of mesaconate per minute under the assay
conditions described. This corresponds to a rate of absorbance change
of 3.85 cm -1 rain -~ at 240 m~. Specific activity is defined as units per
milligram of protein. Protein is determined by the method of Lowry et
al. ~ using dry bovine serum albumin as a standard.
Growth o] Clostridium tetanomorphum. Except for minor modifica-
tions, the growth and harvesting of the bacteria follow the procedures
described by Barker et al. ~
Clostridium tetanomorphum strain HI is grown in an unsterilized
medium of the following composition: Difeo yeast extract, J%~ mono-
sodium glutamate, 80 mM; potassium phosphate, 40 mM, pH 7.4; and
tap water. Just before inoculation with 30 ml of an active culture (grown
in thioglycolate nutrient broth under normal sterile conditions) per liter
of medium, 35 mg of sodium hydrosulfite powder is added per liter of
medium. The culture vessels (usually two 50 liter plastic jugs) are filled
to the neck; they do not require an anaerobic atmosphere. After incuba-
tion at 37 ° for 18 hours, the cells are harvested in a Sharpies continuous
centrifuge. The cells are washed by centrifugation, once with 20 volumes
of 20 mM sodium sulfate containing 50 mM 2-mercaptoethanol and once
with 20 volumes of oxygen-free water and are then frozen. The yield is
usually about 3 g of wet packed cells per liter of culture.
Unless otherwise specified, all steps are carried out at 0-3 ° .
Step 1. Cell Breakage. Thawed cell paste, 28 g, is suspended in 42 ml
of 50 mM potassium phosphate, pH 6.8. This solution is passed through
a French pressure cell (16,000 psi) and the resulting extract is centri-
fuged at 27,000 g for 40 minutes.
Step ~,. Heat Treatment. Three milliliters of a solution containing
20 m M MgCI~, 0.2 M potassium mesaconate, p i t 6.8, and 0.2 M NH4CI
is added to 60 ml of the supernatant solution from Step 1. The resulting
O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem.

193, 265 (1951).
[53] ~-METHYLASPARTASE 349
solution is then divided equally into two 50-ml Erlenmeyer flasks and the
flasks are gently shaken at 52 ° in a constant temperature bath for 10
minutes. The solutions are then cooled to 3 ° , combined, and centrifuged
for 30 minutes at 27,000 g. The precipitate is washed with 10 ml of 10
mM potassium phosphate, pH 6.8, and the suspension is centrifuged. The
resulting supernatant solution is then combined with that from the first
centrifugation.
Step 3. Protamine Treatment. Thirty-five milliliters of 1 ~ protamine
sulfate, pH 5.9, is stirred into 48 ml of the solution from step 2. The pH
of the enzyme solution during the addition of protamine sulfate should
be monitored and maintained at 6.8 by the additioh of 1.0 M K~HPO~
solution. After the addition of protamine sulfate is complete, the solution
is stirred for an additional 15 minutes and then centrifuged at 27,000 g
for 25 minutes. To 90 ml of the supernatant solution is added first 2.7
ml of 1.0 M potassium phosphate, pH 7.6, and then a saturating amount
of ammonium sulfate in powdered form (70.7 g/100 ml) with mechanical
stirring. After stirring for an additional 15 minutes the precipitate,
which contains the enzyme, is obtained by centrifugation at 27,000 g for
25 minutes. The precipitate is dissolved in 8 ml of 50 mM potassium
phosphate, pH 7.6.
Step 4. Sephadex G-IO0 Fractionation. G-100 Sephadex beads, 30 g,
are suspended in 1.5 liters of 50 mM potassium phosphate, pH 7.6, at
room temperature for 48 hours in a covered beaker. The solution is
decanted. The Sephadex is resuspended in 1.5 liters of 50 mM potassium
phosphate, pH 7.6, and decanted again to remove fine particles. This
operation may be repeated if necessary. The Sephadex G-100 is then
packed at room temperature to a height of 92 cm in a 2.5 X 100 cm
column. The column is placed in a cold-room and washed overnight with
about 500 ml of 50 mM potassium phosphate, pH 7.6. The enzyme solu-
tion from step 3 is then placed on the column and 50 mM potassium
phosphate, pH 7.6, is applied to the column at the rate of 25 ml per
hour. The eluent solution is collected in 5 ml fractions, and the enzyme
appears after 180 ml of buffer has passed through the column. Those
fractions with a specific activity greater than 25 units per milligram are
combined and used in step 5.
Step 5. DEAE-Sephadex Fractionation. Thirty grams of DEAE-
Sephadex A-50 (medium, powder form) is suspended in 1.5 liters of water
at room temperature for 24 hours in a covered beaker. The solution is
decanted. The DEAE-Sephadex is resuspended in water and decanted
again. This operation is repeated until no fine particles remain. The gel
is then washed successively on a Biichner funnel with 400 ml of 0.5 N
HC1, 1 liter of water, 400 ml of 0.5 N NaOH, and 1 liter of water. The
gel is suspended in 1.5 liters of 50 m M potassium phosphate, pH 7.6,

and the p H of the suspension is readjusted to pH 7.6 by the addition,
with stirring, of concentrated phosphoric acid. This suspension is de-
canted. The DEAE-Sephadex is resuspended in 1.5 liters of 50 m M
potassium phosphate, p H 7.6, and decanted again. The last operation is
repeated until no fine particles remain. The DEAE-Sephadex is then
packed at room temperature to a height of 45 cm in a 2.5 X 50 cm
column. The column is placed in a cold-room and washed overnight with
about 500 ml of 50 m M potassium phosphate, pH 7.6. The combined
fractions from step 4 (generally 60 ml) are placed on the column, after
which the column is washed with 100 ml of 50 m M potassium phosphate,
pH 7.6. The enzyme is eluted with 75 m M KC1 in 50 m M potassium
PURIFICATION OF ~-METHYLASPARTASE FROM Clostridium telanomorphum

Activity
Specific
Total activity
Volume Protein units ~ Yield (units/mg
Fraction (ml) (mg) ( )~103) (%) protein)
Cell extract 60 2680 12.55 100 4.7

Heated extract 58 434.5 10.20 81 23.4
Protamine-treated 90 376.5 9.45 75.3 25.1
extract
Pooled Sephadex G-100 60 94.6 7.58 60.4 80.1
fractions
Pooled DEAE-Sephadex 200 21.6 6.05 48.2 280.0
fractions
a A unit of enzyme is the amount required to catalyze the formation of 1 micromole
of mesaconate per minute under the assay conditions.
phosphate, pH 7.6, at a flow rate of 20 ml per hour in 10 ml fractions.
The enzyme appears after about 400 ml of the KC1 solution has passed
over the column. Those fractions of high and constant specific activity
(usually 280 units per milligram or higher) are combined and the solu-
tion is concentrated either by surrounding a dialysis bag containing the
combined fractions with dry Sephadex G-200 or by ultrafiltration using,
for example, the Schleicher and Schuell collodion bag apparatus. The
enzyme is conveniently stored in 0.5 M tetramethylammonium chloride,
pH 6.8 (10-20 mg of protein per milliliter) at 4 °, or it may be crystal-
lized from ammonium sulfate2,6
It. J. Bright and L. L. Ingraham, Biochim. Biophys. Acla 44, 586 (1960).
H. A. Barker, R. D. Smyth, H. J. Bright, and L. L. Ingraham, Vol. V, p. 827.
[53] ~-METHYLASPARTASE 351
The purification procedure, which results in a 50% yield of ap-

parently pure /3-methylaspartase is summarized in the table. We have
found the present method of purification easier to accomplish than that
originally described, 1,6 principally because of variable yields in the
alcohol fractionation of the latter procedure.
Properties
Many properties of p-methylaspartase and of the reactions that it
catalyzes were described previously;6 we shall describe here those prop-
erties which for the most part have been studied since the publication
of that article.
Occurrence. fi-Methylaspartase appears not to be widely distributed.
The enzyme has only been reported to occur in Clostridium tetanomor-
phum 1 and in the aerobe Bacterium cadaverisJ
Purity. Preparations of fl-methylaspartase from step 5 of the purifica-
tion migrate in polyacrylamide gel electrophoresis as a single protein
band having enzyme activity both at pH 6.0 and pH 8.7. 8
Storage. Tile enzyme can be stored at 0-3 ° for many months, with
little loss of activity, in solutions of high ionic strength. Since tetra-
methylammonium does not serve as a monovalent cation activator, we
have commonly stored the enzyme in 0.5M tetramethylammonium
chloride, pH 6.8, at 0-3 °.
Substrate Specificity. fl-Methylaspartase has been shown to catalyze
the elimination of ammonia from the a-L-isomers of the following hom-
ologs: aspartate, threo-fl-methylaspartate and erythro-fl-methylaspar-
tate; 1'9 /3-ethylaspartate, fl-propylaspartate, and fl-isopropylaspartate? o
Extinction Coefficient. Solutions of the enzyme from step 5 have no
absorbance except in the ultraviolet. The absorption maximum occurs at
279 m~ and ~79,~ is 56,300 M-~cm-1.8 This extinction coefficient was
computed using dry bovine serum albumin as the protein standard and
the protein determination method of Lowry et al2 This method measures
tryptophan and tyrosine, as well as peptide bonds. The use of bovine
serum albumin as a protein standard is justified by the fact that pure
/?-methylaspartase (from step 5) and bovine serum albumin contain very
similar percentages by weight of tyrosine and tryptophan2, ~
Molar Turnover Number. The molar turnover number of a typica]
~V. R. Williams and J. G. Traynham, Federation Proc. 21, 247 (1962).

M. W. Hsiang and It. J. Bright, J. Biol. Chem. 242, 3079 (1967).
H. J. Bright, R. E. Lundin, and L. L. Ingraham, Biochemistry 3, 1224 (1964)
'° M. F. Winkler and V. R. Williams, in preparation.
'~ P. F. Spahr and J. T. Edsall, J. Biol. Chem. 239, 850 (1964).
sample of enzyme from step 5 in the pH 9.7 assay is 467 sec-~ (corre-
sponding to a V~ax value of 280 micromoles per minute per milligram of
protein). This value can be increased to 650 sec -1 by a 3-minute treat-
ment of the enzyme with 0.1 mM EDTA (in the presence of all the assay
components except Mg *~) prior to the assay. 8 This increase in turnover
number (which can also be achieved by preliminary treatment of the
enzyme with thiols) reflects divalent metal ion contamination of the en-
zyme as it is normally prepared2 The highest specific activity obtained
by the present purification procedure was 390 units per milligram, which
is somewhat smaller than the value of 490 units per milligram reported
previously2 ,6 However, the latter value was based on an ultraviolet
extinction coefficient which is now thought to be 15% too large. The dis-
crepancy between the old and new specific activity values is therefore
small. The true molar turnover number, corresponding to infinite concen-
trations of Mg ÷÷, K ÷, and substrate, probably exceeds 650 sec-1 at pH 9.7.
Titration of the enzyme with p-chloromercuribenzoate 8,12 and magnetic
resonance studies ~a of the interaction of the enzyme with Mn ÷÷ indicate
that there are two active sites per enzyme molecule. The turnover number
per active site is therefore one-half of the molar turnover number.
Molecular Weight and Subunit Structure. The molecular weight of the
native enzyme under a wide variety of experimental conditions, as
measured by equilibrium ultracentrifugation, is 100,750 __+2430. 8,12 Un-
der the same conditions, S.~o,,,, as determined by conventional sedimenta-
tion velocity ultracentrifugation, is 6.05 S. Trailing boundary ultracen-
trifugation, which measures the sedimentation of catalytic activity, also
yields a value of $2~., close to 6.05 S. The molecular weight of the
catalytically active enzyme species is therefore 105.
The native enzyme is a tetramer, comprising two pairs of mono-
mers.12,14 Guanidine-HC1 splits the enzyme into two dimers of molecular
weight 5 X 104. The combination of the native enzyme with eight or
more molecules of p-chloromercuribenzoate causes the formation of four
monomers, each of molecular weight 2.5 >( 10~. The monomers appear to
be catalytically inactive, but after removal of the p-chloromercuribenzo-
ate with mercaptoethanol in a 24-hour incubation, catalytic activity can
be recovered, regardless of the number of p-chloromercuribenzoate mole-
cules initially bound to the enzyme. This behavior probably reflects the
reformation of the catalytically active tetramer.
Kinetics and Mechanism. The enzyme catalyzes the exchange of the
" M. W. Hsiang and H. J. Bright, Federation Proc. 26, 605 (1967).
18G. A. Fields and It. J. Bright, in preparation.
" M . W. Hsiang and It. J. Bright, in preparation.
[53] ~-METHYLASPKRTASE 353
fl-proton of fl-methylaspartate with solvent protons. 15'16 The properties

of this exchange reaction have led to the formulation of a mechanism in
which an enzyme-bound fl-carbanion intermediate loses ammonia,
either by ionization or displacement, in the rate-determining step of the
overall reaction. Systematic kinetic studies of the activation of the
enzyme by Mg ÷~ and Co ÷÷ showed that substrate and M ~+ add in a ran-
dom order, rapid equilibrium fashion to the enzyme2 Magnetic resonance
studies of the interaction of Mn ~ with the enzyme substantiate this
conclusion. 13 The addition of mesaconate and ammonia to EM ÷÷ is also
a random order, rapid equilibrium process. Kinetic studies of a wide
range of divalent metal activators and inhibitors showed that divalent
metal ions of ionic radius 1.00 A or larger are inhibitors, whereas those
having a radius less than 1.00 ~, are activators. 17 The turnover number
increases with decreasing ionic radius, a fact which is consistent with the
postulate that the role of the enzyme-bound divalent metal ion activator
is to interact with the fl-carboxylate group of the substrate and facilitate
the extraction of the fl-proton by an enzyme base. Indirect evidence sug-
gests that the two sulfhydryl groups required for catalytic activity, out
of a total of 20 in the molecule, represent the postulated basic groups at
two active sites on the enzyme molecule. The ionic radius of the divalent
metal activator is also important in determining the stability of the
appropriate binary and ternary complexes. Williams and Selbin reported
K~ and Vma~ values for seven divalent metal activators of the enzyme? 8
Additional evidence for the participation of a sulfhydryl group in the
catalytic mechanism has been obtained from studies of the protection
afforded by substrates against sulfydryl reagents. The dissociation con-
stant K8 for the species ES, computed from such studies with PCMB
agreed well with the value obtained from independent kinetic studies. 3
The extent of protection afforded by a series of homologs of aspartic acid
(both substrates and pseudosubstrates) against inhibition by N-ethyl-
maleimide suggested that the sulfhydryl group lies near the ;~-carbon
atom of the substrate in the enzyme-substrate complex. TM In addition,
photooxidation studies showed that the rate of sulfhydryl destruction
was very similar to the rate of loss of enzyme activity.
lJ H. J. Bright, L. L. Ingraham, and R. E. Lundin, Biochlm. Biophys. Acta 81, 576

(1964).
I~tt. J. Bright, J. Biol. Chem. 239, 2307 (1964).
1, H. J. Bright, Biochemistry 6, 1191 (1967).
laV. R. Williams and J. Selbin, J. Biol. Chem. 239, 1635 (1964).
~V. R. Williams and W. Y. Libano, Biochim. Biophys. Acta 118, 144 (1906).
[54] Aspartase
[EC 4.3.1.1 ~-Aspartate ammonia-lyase]
By VmGINIA R. WILLIAMS and DON~D J. LARTmUE

-OOC--CH2--NH3+--COO - ~- - O O C - - C H - ~ C H - - C O O - -b *NH4+
L-aspartate fumarate
The enzyme which catalyzes the reversible conversion of L-asparate
to fumarate and ammonium ion has been known for half a century. Its
existence was first postulated by Harden 1 in 1901, but Quastel and Woolf2
in 1926 established the stoichiometry of the reaction and demonstrated
that the enzyme was a deaminase rather than an oxidase. This deaminase
was given the name "aspartase" by Woolf, 3 and it will be so designated
throughout the discussion that follows.
Assay Method
Principle. Aspartase can be assayed conveniently by measuring the
production of either fumarate or ammonia. A unit of aspartase activity
is that quantity of enzyme which produces 1 micromole of fumarate (or
ammonia) per minute. Specific activity is defined as units per milligram
of protein: These are not the traditional definitions, but they are con-
sistent with the modern terminology for other enzymes.
Method 1
A modification of the spectrophotometric fumarase assay developed
by Racker 4 is used tor routine aspartase determination. This method has
the advantages of analytical sensitivity and suitability for initial rate
studies.
Reagents
Potassium L-aspartate, 0.5 M, pH 7.0
MgSO4, 30 mM
EDTA, 3 mM, adjusted to pH 7.0 with Tris
Procedure. Absorbance is measured conveniently in a double-beam
spectrophotometer equipped with a suitable recorder and constant tem-
IA. Harden, J. Am. Chem. Soc. 79, 623 (1901).
2j. H. Quastel and B. Woolf, Biochem. J. 20, 545 (1926).
~B. Woolf, Biochem. J. 23, 472 (1929).
• E. Racker, Biochim. Biophys. Acta 4, 211 (1950).
[54] ASPARTASE 355
perature housing. A temperature of 30 ° is recommended for routine

assays, since it is easy to maintain. A pair of 1 cm silica cells is used with
the reagent volumes given below.
Into each cell pipette 1.0 ml of 0.15M Tris-HC1 buffer, 0.3 ml of
0.5 M L-aspartate, 0.1 ml of 30 mM MgSO~, 0.1 mI of 3 mM EDTA, and
1.4 ml water. Mix the solutions and adjust the spectrophotometer to zero
absorbance at 240 mt~, then add to the sample cell 0.1 ml of a suitable
(lilution of the enzyme; the change in absorbance is measured as a func-
tion of time. (With crude enzyme preparations, which contain large
amounts of inert protein, the enzyme may be added to both the reference
and sample cells and the substrate omitted from the reference cell.) The
reaction rate should fall within 0.04-0.40 absorbance units per minute
for a 1 cm light path cuvette. The rate remains constant for several
minutes and is directly proportional to the enzyme concentration. The
molar extinction coefficient of potassium fumarate under similar condi-
tions is reported ~ to be 2.53 X 103 M -I cm-k Specific activity is calculated
as follows:
&A240rain-1
Specific activity = 2.53 ml umole-VX mg protein ml -~ assay solution"
Protein is determined by the method of Lowry et al., 6 using crystalline

bovine serum albumin as standard.
Method 2
Any method of ammonia measurement may be used. The simplest

procedure is direct nesslerization7 of an aliquot of reaction mixture from
method 1. It may be necessary to distill the ammonia by the Conway
technique 8 prior to nesslerization. A number of substances interfere with
direct determination, e.g., sulfhydryl compounds and some amino acids,
such as histidine and arginine. This method is less sensitive than method
l; consequently, assays should be incubated until the ammonia concen-
tration reaches 1 raM. The reaction is stopped by the addition of 0.1
volume of 20% trichloroacetic acid to the assay tubes. Precipitated
protein is centrifuged and the supernatant solution is analyzed for
ammonia. A rate curve can be obtained from assays incubated for 0, 5,
T. F. Emery, Biochemistry 2, 1041 (1963).

6 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193,
265 (1951).
P. B. Hawk, B. L. Oser, and W. It. Summerson, "Practical Physiological Chemistry,"
13th ed., p. 1329. Blakiston, New York, 1954.
P. B. Hawk, B. L. Oser, and W. H. Summerson, "Practical Physiological Chem-
istry," 13th ed., p. 886. Blakistoa, New York, 1954.
356 REACTIONS LEADING TO AND FROM THE CYCLE [$4]
I0, and 15 minutes. Since the reaction rate is not linear for longer periods,
Method 1 should be used for precise results.
This purification method yields a preparation free of enzymes which
might complicate the study of aspartic acid deamination, especially
fumarase.
Cells of Bacterium cadaveris Gale, 1944, ATCC-9760 (more appro-
priately, Enterobacter ha]nine), are grown in 6-liter batches of medium
containing 1~ yeast extract, 1% tryptone, and 0.5~ K2HPO,. Growth
from a 10~ inoculum is allowed to proceed for 48 hours at 30 ° without
aeration or agitation. The cells are harvested, washed once with 80 mM
KCI, and frozen until used. This procedure yields about 25 g of wet,
packed cells from 18 liters of medium.
Step 1. Sonication. Suspend approximately 50 g of cells (wet weight)
in 100 ml of 0.1 M potassium phosphate buffer, pH 7.0; add 1.5 ml of
0.1 M mercaptoethanol, and divide the suspension into two portions. The
cells are then disrupted by sonication in a Branson Model LS-75 Sonifier.
Maintain the temperature below 10 ° for five 1 minute sonications for
each portion of the suspension. Then centrifuge the sonicate in the No. 30
rotor of the Spinco Model L-2 ultracentrifuge at 105,000 g at 2 ° for 1
hour. Discard the precipitate.
Step ~. Protamine Sul]ate Precipitation. Determine the protein con-
tent of the clear amber solution (110 ml) 9 and adjust to 30-45 mg/ml
with 0.1 M potassium phosphate buffer, pH 7.0. Dissolve in potassium
phosphate buffer (0.1 M, pH 7.0) a quantity of protamine sulfate equal
to 15~ of the total weight of protein to form a 1 ~ solution (50-75 ml).
Make the protamine sulfate solution to 1 mM mercaptoethanol, and then
add it drop by drop to the enzyme solution, which is stirred in an ice
bath. Stir for an additional 30 minutes after the addition is complete.
During this and subsequent steps, do not allow the temperature to exceed
4 °. Centrifuge the suspension at 27,000 g for 30 minutes in a refrigerated
centrifuge and discard the precipitate.
Step 3. pH Fractionation. Adjust the supernatant solution from step 2
(about 170 ml) rapidly to pH 4.2 with 2 M acetic acid (about 15 ml).
Centrifuge the solution immediately at 27,000 g for 10 minutes and
discard the supernatant solution. Suspend the precipitate in 0.1 M potas-
o In the relatively crude fractions obtained in steps 1-5, protein may be determined
by the rapid turbidimetric method of Exton [W. G. Exton, J. Lab. Clin. Med.
10, 722 (1925)], standardized against crystalline bovine serum albumin. The Lowry°
method may also be used. Although more time-consuming, it has the advantage of
greater accuracy.
[54] ASPARTASE 357
slum phosphate buffer, pH 7.0, (containing 1 mM mercaptoethanol and

10 t ~ / M g S 0 4 ) to a total volume equal to one-third to one-half the
initial volume. Suspension of the thick, gummy precipitate is aided by
one or two short sonications. Centrifuge the solution at 27,000 g for 15
minutes and discard the precipitate.
Step 4. Ammonium Sul]ate Fractionation. To the supernatant solutior,
from step 3 add 1/20 volume of 1 M potassium phosphate buffer, pH 7.4.
Add solid ammonium sulfate, 176 mg per milliliter of enzyme solution, to
bring the salt concentration to 30% of saturation. Add the salt slowly
while stirring, and continue stirring for 15 minutes after the addition is
complete. Centrifuge the material at 27,000 g for 15 minutes, and discard
the precipitate. Add 162 mg ammonium sulfate per milliliter to the
supernatant solution to bring the salt concentration to 55% of satura-
tion. Centrifuge the suspension and discard the supernatant solution. The
precipitate can be kept frozen for a year without appreciable loss of
activity.
Step 5. Dialysis. Dissolve the frozen ammonium sulfate precipitate
from step 4 in a minimum volume of 10 mM potassium phosphate buffer,
pH 7.0, containing 1 mM mercaptoethanol and 10 ~M MgS04, and
dialyze against 400 ml of the same buffer until the dialyzate gives only
a faint color with Nessler's reagent. Change the dialyzate every 30
minutes; about 6 liters of buffer are required. The enzyme may be frozen
at this stage; however, 50% loss of activity is observed after 2 weeks of
storage.
Step 6. Column Chromatography. Either DEAE- or ECTEOLA-
cellulose may be used for the final purification step. DEAE- and
ECTEOLA-celluloses are prepared as follows: The material is suspended
in distilled water and allowed to settle for 20 minutes. The fines are
decanted. The process is repeated until the liquid above the bulk of the
material is clear after a 20-minute settling interval. The material is
washed twice with saturated KC1 and collected using a Biichner funnel.
It is then washed with distilled water until the washings are free of
chloride. The material is suspended in 10 mM potassium phosphate buffer,
pH 7.0, filtered, resuspended in the same buffer, and stored. Regeneration
is done the same way.
The cellulose exchanger is packed in a jacketed column to give a bed
size either 1 or 2 cm in diameter and 20 cm in length. Column tempera-
ture is maintained at 4 °. Packing is achieved by pouring the DEAE or
ECTEOLA suspension into the column and allowing the material to
settle. Apply nitrogen at 5 psi to the top of DEAE-cellulose columns
several times to hasten preparation and pack tlle cohmm tightly. Th(,
1 em columns are washed with 150 ml of cquilibr'tting buffer; the 2 cm
358 REACTIONS LEADING TO AND FROM THE CYCLE [S4]
columns, with 500 ml. Flow rates are maintained at 1 ml per 3-5 minutes
by adjusting the stopcock at the bottom.
The procedure employed with the larger DEAE column is as follows:
Apply 5 ml of the enzyme solution (about 100 mg protein) to the column,
followed by 95 ml of 10 mM potassium phosphate buffer, pH 7.0. Then
wash the column with 150 ml of the same buffer containing 0.2 M KC1.
Elute the enzyme with 100 ml of 10 mM potassium phosphate buffer, pH
7.0, containing 0.7 M KCI. Collec~ 10 ml fractions in test tubes containing
0.1 ml of 0.1 M mercaptoethanol. Under these conditions, the enzyme
elutes sharply with the 0.7 M KCI front. Only a 1- to 2-fold dilution of
the enzyme results. If the 1 X 20-cm column is used, all volumes are
decreased to one-fourth of those reported above.
Elute the ECTEOLA column with a linear gradient from zero to
0.5M KC1 at a constant level of 10 mM phosphate buffer, pH 7.0. Use
a refrigerated fraction collector to collect 10 ml fractions. The enzyme is
eluted at about 0.25 M KC1. Usually only one er two active fractions
are obtained; the total dilution is about 2-fold. The eluted enzyme is
unstable and should be used immediately o1" precipitated with ammonium
sulfate, which is added to saturation.
Tris-HC1 buffers of the same molarity and pH may be substituted for
the phosphate buffers with minor changes in the recoveries of the enzyme.
Data for a typical preparation are shown in the table? ° Specific
activities are determined at pH 7.0, since aspartase becomes increasingly
unstable as the pH is elevated. However, maximum rates are obtained
at pH 8.5 (in the presence of saturating levels of substratO °) and the
rate of fumarate production is 5-fold greater than that at pH 7.0. The
specific activity of the final fraction shown in the table would be
approximately 175 instead of 35 if measurements had been made at
pH 8.5.
Properties
General Protein Characteristics

Aspartase is also discussed in an earlier volume. 11 Aspartase is an
acidic protein with an isoelectric point of 4.8, determined by starch elec-
trophoresis? 2 It attaches quite tightly to DEAE-cellulose in the neutral
pI-I range. Sucrose density gradient centrifugation using eatalase as
reference shows that the enzyme has a molecular weight of approximately
180,000.1° It can be dissociated into four subunits of equal weight by
t°V. R. Williams and D. J. Lartigue, d. 3iol. Chem. 2242, 2973 (1967).

11A. I. Virtanen and N. Ellfolk, Vol. II, p. 386.
'~J. 8. Wilkson and V. R. Williams, Arch. Biochem. Biophys. 93, 80 (1961).
[54] ASPARTASE 359
r-
~ I ~
r~
E-
~ ~ o~
~ ~-;~- ._~ ~ --~ ~.~ ='
©
v
E
7.
©
F~
(?)
360 REACTIONS LEADING TO AND FROM TItE CYCLE [54]
treating native aspartase with p-hydroxymercuribenzoate. Active tet-

ramer is regenerated by treating the mercury derivative with mercapto-
ethanol. Aspartase has not been crystallized, and its amino acid com-
position is unknown.
Characteristics as a Catalyst
a. Specificity toward Substrate. The enzyme has long been regarded
as displaying absolute specificity toward L-aspartate, fumarate, and NH~.
However, Emery5 has reported that hydroxylamine can replace NH3 in
the addition reaction, forming N-hydroxyaspartic acid.
b. K',~. At pH 7.0 in Tris-HCl buffer, K',~ for aspartate is 1.5 raM;
at pH 7.0 in potassium phosphate buffer, K ' , is 20 raM. An analysis of
the pH dependence of K'~ and Vm~ suggests that imidazole and sulfhy-
dryl groups are present at the active site. is
c. pH Optimum. In Tris-HC1 buffer the pH optimum is near 8.5.~°
Most earlier reports cite a pH optimum of 7.0-7.5, attributable to the
inhibition of aspartase by secondary phosphate anion and the choice of
low substrate concentration, or both. The observed pH optimum is
influenced greatly by the concentration of substrate in the assay system.
The optimum of 8.5 was determined from a plot of extrapolated Vma. as
a function of pH.
d. Thermodyna.mic Constants. At pH 7.2, K',q for the elimination
reaction is approximately 20 mM at 39° and 10 mM at 20°. 12,14
e. Stability. Aspartase shows maximal stability between pH 6.0 and
7.5; pH should be maintained in this range during purification. Aspartase
is inactivated rapidly on cold storage unless it is frozen as the ammonium
sulfate precipitate from step 4. Storage deterioration is irreversible.
]. Organic Cofactors. Aspartase appears to have no organic cofactors
essential to catalysis. However, Scott~5 obtained significant reactivation
of extensively dialyzed preparations with either inosine monophosphate,
adenosine monophosphate, or guanosine diphosphate. Guanosine tri-
phosphate was highly inhibitory. These results suggest heterotropic inter-
actions between nucleotide and enzyme. We have obtained confirmation of
these findings. ~°
g. Metal Ion Activators. Aspartase possesses a divalent cation require-
ment of low specificity. 12,~6 The best activators are Mg** and Mn +*.
lSD. J. Lartigue, Ph.D. Dissertation, Louisiana State University, 1965; University of

Michigan Microfilm No. 65-11, 394.
"V. R. Williams and R. T. McIntyre, J. Biol. Chem. 217, 467 (1955).
i~R. M. Scott, Ph.D. Dissertation, University of Illinois, 1959; University of Michigan
Microfilm No. 59-4, 566.
~R. It. Depue and A. G. Moat, Y. Bacteriol. 82, 383 (1961).
IS4] ASPARTASE 361
Methods employed commonly to convert metalloenzymes into their

apoenzymes are not highly effective with aspartase. The metal ion is
either tightly bound or well shielded from solvent.
h. Cooperative El~ects o/ Substrate. Aspartase exhibits typical Mi-
ehaelis kinetics at pH 6.0, but substrate concentration-activity curves
deviate increasingly from the hyperbolic shape as the pH is increased.
Above pH 7.5 they are markedly sigmoidal, suggesting that the effect of
substrate is cooperative, as is the Bohr effect observed with hemoglobin.
The molecular weight of aspartase is the same at pH 6.0 and 8.0. ~°
i. Enzyme Mechanism. Although the stereochemistry of the aspartase
reaction is known, the mechanism of the elimination is uncertain. Eng-
lard 17 and Krasna TM showed that NH~ is removed from aspartate and
added to fumarate in a stereospecific manner. These workers proposed
independently that the reaction involves c/s-elimination; however, it was
shown to be trans by Gawron and Fondy29 Neither deuterium exchange
nor a deuterium isotope effect has been observed in the elimination
reaction2 ~
Distribution
Although aspartase was thought to occur only in bacteria and a few
species of higher plants, 11 Kurata 2o has reported its presence during the
ontogeny of the frog Rhacophorus schlegelii var. arborea. Salvatore et
al., 21,~ report wide distribution of aspartase activity in animal tissue,
particularly in sharks and bony fishes. The properties of bacterial aspar-
tase indicate it may be a regulatory enzyme: its synthesis is glucose-
repressed, 2~ it possesses quaternary structure, and it exhibits cooperative
effects of substrate and heterotropic interactions with various nucleotide
activators. 1° Its role in animal metabolism is unknown.
Acknowledgment
The methods described in this report were developed with the research support
of Grant GM-11016 from the United States Public Health Service and Grant GB-5017
from the National Science Foundation.
"S. Englard, J. Biol. Chem. 233, 1003 (1958).

~SA. I. Krasna, J. Biol. Chem. 233, 1010 (1058).
1'0. Gawron and T. P. Fondy, J. Am. Chem. Soe. 81, 6333 (1959).
~oy. Kurata, Exptl. Cell Res. 28, 424 (1962).
'~ F. SaIvatore, V. Zappia, and C. Costa, Comp. Biochem. Physiol. 16, 303 (1965).
:*V. Zappia, C. Pietropaolo, C. Costa, and F. Salvatore, Ab~.tr. 150th Meeting Am.
Chem. Soc., Atlantic City, Sept., 1965, p. 37e. Spaulding-Moss, Boston, Massa-
chusetts.
~3M. A. Farley and H. C. Lichstein, Can. J. Microbiol. 9, 835 (1963).
362 REACTIONS
[55] A Radioactive Assay for Malate Synthase and

Other Glyoxylate Condensing Enzymes
B y W. S. WEGENER,H. C. REEVES,R. RABIN, and S. J. AJL
Acetyl-CoA -b glyoxy|ate--~ malate -k CoA

Propionyl-CoA ~ glyoxylate ~ a-hydroxyglutarate (CoA ester?)
Butyryl-CoA -b glyoxylate --~/~-ethylmalate (CoA ester?)
Valeryl-CoA -k glyoxylate --~/~-n-propylmalate (CoA ester?)
Assay M e t h o d
Principle. The condensation of glyoxylate with acetyl-CoA to form
malate is Catalyzed by malate synthase. 1 The methods employed for
assaying this enzyme include: (a) a spcctrophotometric assay dependent
on measuring the cleavage of the thiol ester bond of acetyl-CoA in the
presence of glyoxylate; 2 (b) a fluorometric determination of malic acid
formed;3 and (c) the determination of residual glyoxylate. The latter has
been measured spectrophotometrically as the semicarbazone ~ or phenyl-
hydrazone 2 derivative, and colorimetrically as the 2,4-dinitrophenyl-
hydrazone, ~,6 the dinitrophenylformazan carboxylic acid/,8 and the
p-nitrophenylhydrazone. 9
In addition to acetyl-CoA, glyoxylate condenses cnzymatically with
propionyl-CoA, butyryl-CoA, and valeryl-CoA to form respectively,
a-hydroxyglutarate, 1° fl-ethylmalate, 11 and fl-n-propylmalateJ 2 These
reactions are catalyzed by extracts of E s c h e r i c h i a coli and P s e u d o m o n a s
aeruginosa. In contrast to malate synthase, these latter condensation
reactions cannot be demonstrated in cell-free extracts by measuring the
glyoxylate-dependent thiol-ester cleavage of propionyl-, butyryl-, or
valeryl-CoA. Preliminary evidence has been reviewed TM that the products
~D. T. O. Wong and S. J'. Ajl, J. Am. Chem. ~oc. 78, 3220 (1956).
2G. H. Dixon and H. L. Kornberg, Biochem. J. 72, 3P (1959) ; see Vol. V [86].
8j. p. Hummel, J. Biol. Chem. 180, 1225 (1949).
J. A. Olson, Arch. Biochem. Biophys. 85, 225 (1959).
T. E. Friedemann and G. E. Haugen, J. Biol. Chem. 147, 415 (1943).
*S. L. Bonting, Arch. Biochem. Biophys. 58, 100 (1955).
'D. N. Kramer, N. Klein, and R. A. Baselice, Anal. Chem. 31, 250 (1950).
*B. A. McFadden and W. V. Howes, Anal. Biochem. 1, 240 (1960).
E. Juni and G. A. Heym, Anal. Biochem. 4, 143 (1962).
1oH. C. Reeves and S. J. Ajl, J. Bacteriol. 84, 186 (1962).
~R. Rabin, H. C. Reeves, and S. J. Aj], J. Bacteriol. 86, 937 (1963).
~2K. Imai, H. C. Reeves, and S. J. Ajl, J. Biol. Chem. 238, 3193 (1963).
~ R. Rabin, H. C. Reeves, W. S. Wegener, R. E. Megraw, and S. J. Ajl, ~cience 150,
1548 (1965).
[55] RADIOASSAYFOR GLYOXYLATECONDENSINGENZYMES 363
of the condensation of glyoxylate with propionyl-, butyryl-, and valeryl-

CoA are formed not as free acids, but rather as acyl-CoA esters which
are subsequently deacylated. This would explain the apparent lack of
activity of these condensing enzymes when assayed spectrophotomet-
rically, since such esters would be expected to absorb at 232 m/~. Since
no simple methods exist for quantitation of the condensation products,
these reactions have been studied by measuring the utilization of radio-
active glyoxylate as a function of the fatty acid acyl-CoA esters. TM
Reagents
0.1 M MgC12.6 H~O
Sodium glyoxylate, (1.5 ~mole/ml)
Sodium glyoxylate-l-t~C (1.5 ~C/ml)
Acetyl-CoA, propionyl-CoA, butyryl-CoA and valeryl-CoA, 5
~moles/ml, prepared by the method of Simon and Shemin 1~
p-Nitrophenylhydrazine-H2S04 solution (freshly prepared by add-
ing equal volumes of filtered 0.1 M aqueous p-nitrophenylhydra-
zine and 20 N H2S04)
A stock solution of reaction mixture is prepared by diluting 10 ml of
buffer, 5 ml of MgC12"6 H20, 5 ml of sodium glyoxylate, and 5 ml of
sodium glyoxylate-l-14C to 50 ml with distilled water. Sufficient reaction
mixture may be prepared for several months' use.
Procedure. One milliliter of the stock reaction mixture containing 50
micromoles of buffer, 10 micromoles of MgCl~.6 H20, 0.15 micromole of
sodium glyoxylate, and 0.15 t~C of sodium glyoxylate-lJ4C is pipetted
into 12.5 X 125 mm screw-cap tubes. To appropriate tubes are added 0.5
micromole of acetyl-, propionyl-, butyryl-, or valeryl-CoA. The reactions
are initiated by the addition of enzyme extract (1-2 mg of protein) and
incubated at 37 ° for 15 minutes. A tube containing heat-inactivated
enzyme is employed to determine the initial activity of glyoxylate-lJ4C.
A second tube containing enzyme but no acyl-CoA ester is used to
calculate the nonacyl-CoA-ester dependent utilization of glyoxylate. A
control tube also should be employed to rule out nonenzymatic, acyl-CoA
dependent disappearance of glyoxylate.
After the incubation period, the reactions are terminated by the
addition of 0.05 ml of p-nitrophenylhydrazine-H~S04 and the mixture
incubated at 37 ° for 45 minutes to allow for p-nitrophenylhydrazone
formation. Ethyl acetate (1 ml) is added, the tubes are capped, and the
'~ W. S. Wegener, H. C. Reeves, and S. J. Ajl, Anal. Biochem. 11, I l l (1965).

E. J. Simon and D. Shemin, J. Am. Chem. Soc. 75, 2520 (1953).
p-nitrophenylhydrazones are extracted by vigorous agitation for 1 min-

ute on an orbital mixer. The tubes are centrifuged for 5--6 minutes at
3000 rpm to separate the aqueous and ethyl acetate phases, and portions
of the ethyl acetate layer are transferred to dry capped tubes using
capillary pipettes. To determine glyoxylate utilization, 0.10 ml aliquots
of the ethyl acetate extracts are spotted with 2.5 gg of authentic gly-
oxylic p-nitrophenylhydrazone on strips (38 ram) of Whatman No. 1
paper. Two samples may be spotted on each strip. The chromatograms are
developed for 4 hours in hydrometer cylinders (63 X 425 mm) contain-
ing 40 ml of n-butanol-water-95~b ethanol (5:1:4) using the ascending
technique. Each strip is dried for 2 hours and the glyoxylic p-nitro-
phenylhydrazone spots are located visually. These areas of the chroma-
togram are cut into 10 X 19 mm pieces, placed in 10 ml of scintillation
fluid, and counted in a liquid scintillation spectrometer, a6 The chromato-
grams should be analyzed to determine whether glyoxylate is the only
radioactive compound extracted by ethyl acetate; if so, aliquots of the
ethyl acetate extracts may be counted directly. 17
Units. One unit of malate synthase activity is defined as the amount
of enzyme which catalyzes the acetyl-CoA dependent disappearance of
1 millimicromole of glyoxylate-l-l~C in 15 minutes at 37 °. Units of
activity of a-hydroxyglutarate, B-ethylmalate, and p-n-proplymalate
synthase are defined similarly. Specific activity is expressed as units of
activity per milliliter of ethyl acetate extract per milligram of protein.
General. The assay for malate synthase described here is specific,
since glyoxylic p-nitrophenylhydrazone is isolated chromatographically,
and quantitative, since all the glyoxylate can be accounted for accurately.
Furthermore, neither the incubation period required to form the hydra-
zone, nor the time required to extract this derivative, is critical. This
technique is particularly valuable in the assaying of enzyme preparations
of low malate synthase activity since it is sensitive to changes in con-
centration of glyoxylate of less than 1 millimicromole, is
'~The scintillation fluid employed contained: POP, 2,5-diphenyloxazole, 6 g; POPOP,

1,4-bis-[2-(5-phenyloxasolylbenzene)], 300 rag; toluene, 600 ml; and absolute
ethanol, 300 ml. No calculations were made for quenching since the degree of
quenching is comparable in all systems.
"When enzyme extracts were prepared from E. coli grown under certain nutritional
conditions, the formation of an unreported metabolite was observed. This com-
pound which was formed only in complete reaction mixtures containing enzyme,
14C-glyoxylate, and acyl-CoA donor, was extracted by ethyl acetate but remained
at the origin of the chromatograms. The compound has been preliminarily identi-
fied as the p-nitrophenylhydrazone of glyoxylyl-CoA. See W. S. Wegener, H. C.
Reeves, and S. J. Ajl, Bacteriol. Proe. p. 85 (1965).
wWhen E. coli was grown for 24 hours in glucose-citrate medium with restricted
[56] POLAROGRAPHIC ASSAY FOR APPEARANCE OF COA-BH 365
The glyoxylate condensing enzymes have been assayed by the

isotopic method only in crude cell-free extracts. Stoichiometry between
glyoxylate-l*C utilization and malate-~4C formation can be demonstrated
in the malate synthase reaction. 1. However, in the a-hydroxyglutarate,
fl-ethylmalate, and fl-n-propylmalate synthase reactions, a significantly
smaller percentage of the total activity from glyoxylate-~*C can be ac-
counted for in the respective condensation products. In the crude extracts
employed, the condensation products are probably further metabolized.
oxygen, no malate synthase activity could be detected using the spectrophotometric

ame~__y. When the same extract was assayed by the isotopic method, however,
substantial activity was observed. This activity represented 20-25% of the induced
level of enzyme activity obtained by growth of E. coli on acetate. See W. S.
Wegener, H. C. Reeves, and S. J. Ajl, J. Bacteriol. 90, 594 (1965).
[ 5 6 ] P o l a r o g r a p h i c A s s a y for M a l a t e S y n t h a s e
and Citrate Synthase
[EC 4.1.3.2 L-Malate glyoxylateqyase (CoA-acetylating)]
[E(3 4.1.3.7 Citrate oxaloacetate-lyase (CoA-acetylating)]
Malate and citrate synthases catalyze, respectively, the analogous
reactions (1) and (2), in both of which acetyl-S-CoA is cleaved to
CoASH.
Acetyl-S-CoA -b glyoxylate- ~ H20 ~ malate ~- ~- CoASH W H ~ (1)
Acetyl-S-CoA -k oxaloacetate~- -b HsO --, citrate ~- ~ CoASH -b H + (2)
Several methods have been employed for the continuous assay of the
activity of these enzymes. Citrate synthase may be assayed by coupling
with malate dehydrogenase,~ but the addition of a second enzyme is
sometimes undesirable. Other procedures involve measurements of the
extinction changes accompanying either the cleavage of the S-acyl bond
in acetyl-S-CoA,2-4 or the reaction of the liberated CoASH with a
chromogenic reagent, e.g., 5,5'-dithiobis-(2-nitrobenzoic acid). 5 The
former method suffers from the disadvantage that it can be used only
1S. Ochoa, Yol. I [114].
2E. R. Stadtman, Vol. I I I [137].
s G. H. Dixon and H. L. Komberg, Vol. V [86].
~P. A. Stere and G. W. Kosicki, J. Biol. Chem. 236, 2557 (1961).
P. A. Stere, H. Brazil, and L. Gonen, Ac$a Chem. 8cand. 17, 8129 (1963).
[56] POLAROGRAPHIC ASSAY FOR APPEARANCE OF COA-BH 365
The glyoxylate condensing enzymes have been assayed by the

isotopic method only in crude cell-free extracts. Stoichiometry between
glyoxylate-l*C utilization and malate-~4C formation can be demonstrated
in the malate synthase reaction. 1. However, in the a-hydroxyglutarate,
fl-ethylmalate, and fl-n-propylmalate synthase reactions, a significantly
smaller percentage of the total activity from glyoxylate-~*C can be ac-
counted for in the respective condensation products. In the crude extracts
employed, the condensation products are probably further metabolized.
oxygen, no malate synthase activity could be detected using the spectrophotometric

ame~__y. When the same extract was assayed by the isotopic method, however,
substantial activity was observed. This activity represented 20-25% of the induced
level of enzyme activity obtained by growth of E. coli on acetate. See W. S.
Wegener, H. C. Reeves, and S. J. Ajl, J. Bacteriol. 90, 594 (1965).
[ 5 6 ] P o l a r o g r a p h i c A s s a y for M a l a t e S y n t h a s e
and Citrate Synthase
[EC 4.1.3.2 L-Malate glyoxylateqyase (CoA-acetylating)]
[E(3 4.1.3.7 Citrate oxaloacetate-lyase (CoA-acetylating)]
Malate and citrate synthases catalyze, respectively, the analogous
reactions (1) and (2), in both of which acetyl-S-CoA is cleaved to
CoASH.
Acetyl-S-CoA -b glyoxylate- ~ H20 ~ malate ~- ~- CoASH W H ~ (1)
Acetyl-S-CoA -k oxaloacetate~- -b HsO --, citrate ~- ~ CoASH -b H + (2)
Several methods have been employed for the continuous assay of the
activity of these enzymes. Citrate synthase may be assayed by coupling
with malate dehydrogenase,~ but the addition of a second enzyme is
sometimes undesirable. Other procedures involve measurements of the
extinction changes accompanying either the cleavage of the S-acyl bond
in acetyl-S-CoA,2-4 or the reaction of the liberated CoASH with a
chromogenic reagent, e.g., 5,5'-dithiobis-(2-nitrobenzoic acid). 5 The
former method suffers from the disadvantage that it can be used only
1S. Ochoa, Yol. I [114].
2E. R. Stadtman, Vol. I I I [137].
s G. H. Dixon and H. L. Komberg, Vol. V [86].
~P. A. Stere and G. W. Kosicki, J. Biol. Chem. 236, 2557 (1961).
P. A. Stere, H. Brazil, and L. Gonen, Ac$a Chem. 8cand. 17, 8129 (1963).
with low concentrations of aeetyl-S-CoA, owing to the high extinction of

this reagent, and is not too satisfactory with crude cell extracts. The
latter method exposes the enzyme to the possibility of inactivation by
the chromogen; this inactivation is particularly marked with malate
synthase. The polarographie method ~ described below is designed to
overcome these complications.
Principle. The principle of the method rests on the observation 6 that
CoASH, but not its S-aeyl derivatives, produces an anodic polaro-
graphic wave at the dropping mercury electrode. The procedure involves
the continuous monitoring of the appearance of this anodic wave, which
can be performed automatically with a recording polarograph. The
advantages of the method are the following: (a) The method is a con-
tinuous and direct one; the enzyme assayed is not coupled with a second
enzyme. (b) Since no chromogenie reagent is introduced there is no
risk of inactivation of the enzyme by such a reagent. (c) Acetyl-S-CoA
is polarographieally inert, so that assays may be performed in the
presence of any concentration of this substrate. (d) The assay works
well with crude cell extracts, and any slight turbidity introduced with
such preparations does not interfere with the measurements. (e) The
method is highly sensitive; reaction rates may be measured as low as
10-3 moles of CoASH formed per minute.
Apparatus. The reader who is unfamiliar with polarographic tech-
niques is recommended to consult one of the texts available on this
subject. 7
The reaction vessel, illustrated in Fig. 1, is a small glass cell suitable
for 1 ml of solution and having a standard tapered (B 24) ground glass
neck. A matching stopper, made of hard polythene, is supported
vertically in a fixed position and provides a gas-tight seal for the reaction
vessel. The stopper is drilled to carry the dropping mercury electrode
capillary, A, a salt bridge, B (2% agar in saturated KC1) making
contact with a saturated calomel electrode, and inlet and outlet tubes for
nitrogen arranged so that, by means of a 2-way tap, the nitrogen may
be flushed either through the body of the solution or over the surface.
The stopper also contains a small capped hole, C, through which material
may be added to the vessel while nitrogen is bubbling through the
solution. The mercury capillary is connected with tubing to a reservoir
of variable height, and the whole is filled with pure mercury. Moist
*P. D. J. Weitzman, Biochem. J. 99, 18P (1966).

' For example: W. C. Purdy, "Electroanalytical Methods in Biochemistry."McGraw-
Hill, New York, 1965; L. Meites, "Polarographic Techniques," 2nd ed. Wiley
(Interscienee), New York, 1965; I. M. Kolthoff and J. J. Lingane, "Polarography,"
2nd ed. Wiley (Interscience), New York, 1952.
[56] POLAROGRAPHICASSAY FOR APPEARANCE OF COA-SH 367
oxygen-free nitrogen is obtained by bubbling commercially supplied

oxygen-free nitrogen through two wash-bottles containing respectively
a solution of vanadous chloride or sulfate s'9 and water. This treatment
removes any last traces of oxygen. The mercury collected from experi-
ments is pooled and purified as described by Wichers. 1° The potential is
applied and the current recorded automatically with a recording polaro-
graph of high sensitivity. Potentials are measured against the saturated
calomel reference electrode. The temperature of the assay may be con-
trolled by surrounding the reaction vessel with a jacket through which
water is circulated.
A B
...._#
N2 +_
to colom¢l
electrode
FIG. 1. Diagram of reaction vessel for polarographic assay. For explanation,

see text.
Calibration o] the Mercury Electrode. It is first necessary to calibrate

a particular electrode's response to CoASH concentration, since this will
vary with the size and characteristics of the capillary. An aqueous solu-
tion of CoASH (approximately 5 mM) is prepared and the precise
concentration of free thiol is determined by reaction with 5,5'-dithiobis-
(2-nitrobenzoic acid) 11 at pH 8, and measurement of the extinction at
412 mt~ (molar extinction coefficient---- 13,600). Known amounts of the
CoASH solution are then added to a known volume of deoxygenated
Tris-Mg-EDTA buffer (see below) and a current-voltage polarogram is
recorded for each concentration. From these curves the height of the
anodic thiol wave m a y be measured at --0.3 volt for each CoASH con-
BL. Meites, "Polarographic Techniques," 2nd ed., p. 89, Wiley (Interscience), New
York, 1965.
L. Meites and T. Meites, Anal. Chem. 20, 984 (1948).
1oE. Wichers, Chem. Eng. News 20, 1111 (1942).
" G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959).
368 REACTIONS LEADING TO AND FROM THE CYCLE [SS]
centration, and a linear calibration plot constructed. The slope of this

line relates the response of the electrode in microamperes to the concen-
tration of CoASH. Once calibrated, an electrode should operate repro-
ducibly for a fixed mercury column height. The author has used an
electrode with a response of 1.63 #amp for a 1 mM CoASH concentration.
Reagents
Tris-HCl buffer, 20 raM, pH 8.0, containing 10 mM MgC12 and 1
mM EDTA
Acetyl-S-CoA, 8 mM (approximately) prepared as described by
Stadtman 2
Sodium glyoxylate or oxaloacetate, 10 mM
Procedure. Into the reaction vessel are placed 0.94 ml of buffer

solution, 0.02 ml of glyoxylate or oxaloacetate, and 0.02 ml of acetyl-S-
CoA. The vessel is attached to the stopper so that the electrode, salt
bridge, and nitrogen inlet tube all reach into the solution. Nitrogen is
bubbled through the solution for 2-3 minutes to expel all oxygen. Com-
plete deoxygenation may be confirmed by shutting off the nitrogen and
recording a current-voltage polarogram to demonstrate the absence of
the oxygen reduction waves, although, with practice, it is sufficient
simply to check the current at --0.3 volt. The instrument is set to a fixed
potential of --0.3 volt. While nitrogen is bubbling through the solution,
0.02 ml of enzyme are introduced into the body of the reaction mixture
from a drawn out pipette through the small hole in the stopper. The
nitrogen tap is then turned so that the gas now flows over the top of the
solution. After a few seconds, during which the turbulence in the
solution subsides, the rate of change of current is recorded.
The trace obtained is the familiar polarographic oscillation pattern,
and a line may be drawn through the mid-points of these oscillations.
The slope of this line gives the rate of the reaction in microamperes per
minute. Conversion to micromoles per minute is effected by reference to
the calibration factor previously determined for the electrode.
It is important to examine the enzyme preparation for any acetyl-S-
CoA deacylase activity. This may be done by repeating the above
procedure in the absence of glyoxylate or oxaloacetate. In the presence
of any measurable deacylase activity, the activity of malate or citrate
synthase is determined by difference.
It should be noted that, although magnesium is not required for
citrate syn~hase activity, its presence in the assay mixture has been
found to maintain a linear recording of the reaction rate over a longer
period than in its absence.
[57] OXALYL-COA DECARBOXYLASE 369
[57] O x a l y l - C o A D e c a r b o x y l a s e
[EC 4.1.1.8 Oxalyl-CoAearboxy-lyase]
B y J. R. QUAVLE
Oxalyl-CoA -~ formyl-CoA -t- COs
Oxalyl-CoA decarboxylase has been found in an oxalate-grown
bacterium, 1 Pseudomonas oxalaticus and Pseudomonas OD1, 2 wheat
germ and seeds of wheat, pumpkin, and bean2 The physiological signif-
icance of the reaction in bacterial metabolism is as an intermediary
reaction in the oxidation of oxalate to carbon dioxide via formate; the
role of the reaction in plant metabolism is unknown.
In bacteria, oxalyl-CoA is formed by transfer to oxalate of CoA
from either suceinyl-CoA~ or formyl-CoA;5 no evidence has been ob-
tained for the presence of a direct activation of oxalate with ATP and
CoA. In plants, however, an enzyme has been characterized which
catalyzes such a direct activation of oxalate2
The purification and properties of oxalyl-CoA decarboxylase from
Pseudomonas oxalaticus which are described here have been published
previously2
Assay Method
Principle. The assay depends on manometric measurement of the
rate of carbon dioxide production consequent to the decarboxylation of
oxalyl-CoA. In order to eliminate binding of carbon dioxide in solution
as bicarbonate and the necessity of tipping in acid before measurement
of evolved carbon dioxide, the assay is run at pH 5.5, even though
this is well below the pH optimum of 6.6.
Reagents
Sodium citrate buffer, 0.1 M, pH 5.5
0xalyl-CoA, approximately 5 mM, prepared by ester interchange
between either thiocresyl hydrogen oxalate ~ or S-oxalyl-:N-
capryloylcysteamine7 and CoA
I W. B. Jakoby, E. Ohmura, and O. Hayaishi, J. Biol. Chem. ~ , 435 (1956).
~J. R. Quayle, Biochem. J. 89, 492 (1963).
'J. Giovanelli and N. F. Tobin, Plant Physiol. 39, 139 (1964).
~J. R. Quayle, D. B. Keech, and G. A. Taylor, Biochem. J. 78, 22.~ (1961).
'J. R. Quayle, Biochem. J. 89, 492 (1963).
J. Giovanelli, Biochim. Biophys. Acta 118, 124 (1966).
' J. Koch and L. Saenicke, Ann. Chem. 6 ~ , 129.
370 REACTIONS
Thiamine pyrophosphate, 20 mM
MgCI2, 0.1 M
Procedure. The enzyme is assayed in single side-arm, micromanom-
eter cups (volume 5-7 ml) containing the following reaction mixture:
in the main compartment, 0.5 ml of citrate buffer, 1.5 micromoles of
oxalyl-CoA, 0.1 ml of thiamine pyrophosphate, 0.1 ml of MgCl~, and
water; the side arm contains the enzyme solution, and the total volume
of the flask contents is 1 ml. The flasks are flushed with O~-free N.~
unless it is known that extracts do not contain enzymes capable of
deacylating and oxidizing formyl-CoA. In the absence of such interfering
enzymes, e.g., in the later stages of purification, air may be used as the
gas phase. The enzyme is tipped in from the side arm, and the rate of
carbon dioxide evolution is measured over 15 minutes at 30% The rate,
which is linear during decarboxylation of approximately two-thirds of
the substrate, is proportional to the amount of enzyme used. Normally,
0.05 unit of enzyme is a suitable amount for assay.
Units. One unit of enzyme is defined as the amount of enzyme that
catalyzes the evolution of 1 micromole of carbon dioxide in 1 minute
under the conditions of assay. Specific activity is expressed as units of
enzyme per milligram protein.
Growth Conditions. The source of Pseudomonas oxalaticus, its main-
tenance, and method of large-scale growth on oxalate has been described
elsewhere, s
Step 1. Preparation o] CeU-]ree Extracts. Cell-free extracts may be
prepared by sonication, crushing in a Hughes press, or passage through
a French press. The preparation described below utilizes the last method.
Bacteria (10.6 g wet weight) are suspended in 40 ml of 20 m M phosphate
buffer, pH 7.0, containing about 1 mg of crystalline ribonuclease and
deoxyribonuclease (Koch-Light Laboratories Ltd., Colnbrook, Bucks.,
England). The suspension is passed three times through a French pres-
sure cell at 0 ° and the resulting extract is centrifuged at 22,000 g for 20
minutes at 2 ° . All subsequent operations are performed at 2%
Step 2. Treatment with Protamine Sul]ate. Protamine sulfate is
added to the extract in the proportion of 1 part to 10 parts of bacterial
protein (w/w). The resulting suspension is centrifuged and the precipi-
tate is discarded.
Step 3. Ammo~ium Sulfate Precipitation. Solid ammonium sulfate is
added to the supernatant solution (at pH 7.4) to give 40, 50, and 60%
'J. R. Quayle, Vol. IX [67], p. 360.
[57] OXALYL-COA DECARBOXYLASE 371
saturation. At each of these stages the suspensions are centrifuged and

the precipitated protein is dissolved in 5 ml of 0.02 M phosphate buffer,
pH 7.0.
Step 4. Removal o] Ammonium Sul]ate. The protein fraction that pre-
cipitates between 50 and 60% of ammonium sulfate saturation is poured
onto a Sephadex G-50 (medium) column (1.4 cm X 10 cm), equilibrated
previously with 5 mM phosphate buffer, pH 7.0, and the protein is
eluted from the column by washing with the same buffer. The protein,
freed from ammonium sulfate, appears in the 6-15 ml fraction of the
eluate and is subiected to ion-exchange chromatography.
Step 5. Ion-Exchange Chromatography. Diethylaminoethylcellulose
(DEAE-cellulose, Whatman DE50), 6 g, is made into a slurry in 5 mM
phosphate buffer, pH 7.0, and the slurry is adjusted to pH 7.0 by the
addition of 5 mM potassium dihydrogen phosphate. The resin is freed
from fine particles by repeated decantation in 5 mM phosphate buffer,
pH 7.0, and then packed into a chromatographic column (1.5 cm X
30 cm). The enzyme solution is poured onto the top of the column, which
is then eluted with an increasing phosphate gradient at pH 7.0. This
is formed by connecting together the bottoms of two 500-ml polythene
bottles, the first containing 400 ml of 0.1 M phosphate buffer, pH 7.0,
and the other 400 ml of 5 mM phosphate buffer, pH 7.0. The second
bottle is stirred mechanically and the outflow is fed onto the top of the
column. The levels in both bottles drop at the same rate throughout,
and the phosphate concentration of the eluent increases linearly with
volume. Fractions of eluate (4.2 ml) are collected at a flow rate of 42
ml per hour. Under these conditions the oxalyl-CoA decarboxylase is
eluted mainly in seven fractions around fraction number 30, at a phos-
phate concentration of 12 mM. These fractions are combined, and the
resulting enzyme solution is stored at --15 °. A summary of the purifica-
tion procedure is given in the table.
Properties
Specificity. Under the conditions of routine enzyme assay, oxalate,
malonate, succinate, malonyl-CoA, or succinyl-CoA are not decarboxyl-
ated at a measurable rate. The enzyme preparation is free from gly-
oxylate carboligase, formate dehydrogenase, DPNH-oxidase, oxalyl-CoA
reductase, oxalyl-CoA deaeylase, and formyl-CoA-oxalate transferase.
Coenzyme Requirement. The activity of the enzyme is dependent on
the presence of thiamine pyrophosphate; the rate of decarboxylation in
its absence is very small.
Activators and fnhibitors. A requirement for metal ions may not be
observed with freshly purified enzyme, but on storage at --15 ° a 60%
PURIFICATION PROCEDURE FOR OXALYL-CoADECARBOXYLASE
Specific
activity
Volume Activity Protein (units/rag Yield
Step (ml) (unlts=/ml) (mg/ml) protein) (~)
1. Cell-free extract 30 116 24.8 4.7 100

2. Treatment with protamine 33 112 18.8 5.9 108
sulfate
3. 40-50~ Ammonium sulfate 6.25 187 21.8 8.55 33
precipitation
50--60~ Ammonium sulfate 5.8 165 14.6 11.3 28
precipitation
4. Eluate from Sephadex column 8.5 123 10.6 11.6 30
5. Selected combined fractions 30 9.4 0.21 44.8 b 8.1
after chromatography on
DEAE-cellulose
° One unit is the amount of enzyme required to catalyze the evolution of 1 micromole
of C02 in 1 minute.
b At the peak of activity eluted, the specific activity was 62.
stimulation by 5 mM Mg ~ or Mn ÷÷ ions has been observed2 The

presence of 1 mM ethylenediaminetetraacetic acid in a magnesium-free
reaction mixture during the decarboxylation of oxalyl-CoA causes a 40%
inhibition in rate. In the absence of preincubation the enzyme is inhibited
completely by 2.5 mM p-chloromercuribenzene sulfonate but is unaf-
fected by 1 mM iodoacetate or 1 mM N-ethylmaleimide.
Reversibility. There is no evidence that the enzyme is reversible to
any significant extent.
Stability. The enzyme may be stored for a month at 3 ° with loss of
607~ of its activity, or for 2 months at --14 ° with loss of 50%
activity. It is completely inactivated after 5 minutes at 50 °.
pH Optimum. The pH optimum for the decarboxylation of oxalyl-
CoA in phosphate buffer is 6.6.
Kinetic Properties. The K~ for oxalyl-CoA, measured at pH 6.5 in
phosphate buffer at 30 °, is 1 raM.
PREVIOUSLY PUBLISHED ARTICLESFROM METHODS IN ENZYMOLOGY
RELATED TO SECTION III
Vol. I [96]. Aceto-CoA-Kinase. Mary Ellen Jones and Fritz Lipmann.

Vol. I [98]. Phosphotransacetylase from Clostridium kluyveri. E R. Stadtman.
Vol. I [100]. Deacylases (Thiol Esterase). John Gergely.
Vol. VI [40]. Acyl Phosphatase from Skeletal Muscle. Isaac Harary.
Vol. IX [50]. Purification and Resolution of the Pyruvate Dehydrogenase Comple
(Escherichia coli). Lester J. Reed and Charles R. Willms.
[53] ACETYL-COA SYNTHETASE 375
[58] A c e t y l - C o A S y n t h e t a s e
lEG 6.2.1.1 Acetate:CoA ligase (AMP)]
B y LESLIE T. WEBSTER, JR.
Acetate -t- ATP + CoA ~ Acetyl-CoA -[- 5-AMP q- PP

Assay Methods
Principle. Both indirect and direct evidence has been obtained that
enzyme-bound acetyl adenylate is an intermediate product of the acetyl-
CoA synthetase reaction. 1,~ The intermediate may be attacked by free
CoA to form acetyl-CoA or by pyrophosphate to form ATP as follows:
Ae -b ATP -t- E ~ E(Ac-AMP) q- PP (1)

E(Ae-AMP) -b CoASH ~- AcCoA -k 5-AMP -t- E (2)
The routine assay methods have utilized either the overall or the first
partial reaction. In the overall reaction, acetyl-CoA formed can be
converted chemically or enzymatieally to another product; CoA is usu-
ally added in small quantities and recycles upon transfer of the acetyl
group to aeceptor. Examples of such coupled systems are either the
formation of acetohydroxamate in the presence of hydroxylamine,s of
citrate in the presence of citrate condensing enzyme and an oxaloacetate
generating system,4 or of an acetoarylamine in the presence of acetoaryl-
amine synthase and an arylamine acceptor2 Alternatively, disappear-
ance of SH in reduced free CoA may be monitored directly.6 The first
partial reaction can be assessed by determining the rate of pyrophos-
phate-32P exchange into ATP in the absence of CoA. The usual control
for all the above systems is to omit acetate. Treatment of crude enzyme
preparations by gel filtration may remove interfering compounds of
small molecular weight.
Details of the hydroxylamine assay have been given in a previous
volume2 The ATP-pyrophosphate exchange is least suitable for crude
enzyme preparations because of the presence of ATPase and pyrophos-
phatase. The system coupled with citrate condensing enzyme and malate
~P. Berg, J. Biol. Chem. 222, 991 (1956).

~L. T. Webster, Jr., J. Biol. Chem. 238, 4010 (1963).
M. E. Jones and F. Lipmann, Vol. I [96], p. 585.
4j. R. Stern, B. Shapiro, E. R. Stadtman, and S. Ochoa, J. Biol. Chem. 193, 703
(1951).
5H. Tabor, Vol. I [lOll, p. 608.
6R. R. Grunert and P. H. Phillips, Arch. Biochem. Biophys. 30, 217 (1951).
376 REACTIONS YIELDING ACETYL-COA [58]
dehydrogenase may give erroneous low values of activity if sufficient

DPNH is not included in the assay system/Assays involving measure-
ment of the remaining reduced coenzyme A are quick and advantageous
for studying the reaction mechanism once the products have been
established; this procedure, as applied to acetyl-CoA synthetase from
beef heart mitochondria, is described below.
Reagents
Tris-HCl buffer, 833 raM, pH 8.0 at 25 °
Magnesium chloride, 37.5 mM
Nickel chloride, 0.5 mM
Dipotassium ATP, 30 mM
Potassium acetate, 15 mM
Trilithium CoASH, 8 mM
Enzyme. Dilute with 20 mM KHCOs to achieve 0.8--1.6 units/ml.
Metaphosphoric acid, 30~
Assay Procedure. To 2 small conical centrifuge tubes add 30 #l
of buffer, 20 ~l of MgCI~, 10 ~l of NiC12, 20 ~l of ATP, 30 ~1 of potassium
acetate, and 40 ~l of water (0.15 ml combined volume). Acetate is
omitted from the control tube. Fifty microliters of the CoA and 50 ~l
of freshly diluted enzyme solution are rapidly and successively added
to the contents of each tube at 37 ° (250 ~l final volume). After 3 minutes
each reaction is terminated by 60 ~l of 30~ metaphosphoric acid. De-
natured protein is removed by centrifugation, and a 0.1-0.15 ml aliquot
of the supernatant solution is assayed for sulfhydryl content by the
nitroprusside method of Grunert and Phillips2 Free sulfhydryl content
can also be determined with 5,5'-dithiobis-(2-nitrobenzoic acid), which
offers the advantages of increased stability and sensitivity.8 In the latter
case, the reaction is terminated by 60 ~l of 15% trichloroacetic acid.
Units. One unit of enzyme catalyzes the disappearance of 1 micromole
of reduced CoA per minute at 37 °. Specific activity is expressed in units
of enzyme activity per milligram of protein. Protein concentrations are
determined by the biuret reaction,' bovine plasma albumin being used
as the standard.
Acetyl-CoA synthetase has been highly purified from yeast extracts 1
and from beef heart mitochondria.1° Only the latter procedure is de-
scribed. The purification procedure is summarized in the table.
D. J. Pearson, J. Biochem. 95, 23c (1965).
'G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959).
'A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949).
zoL. T. Webster, Jr., Y. Biol. Chem. 240, 4158 (1965).
[S8] ACETYL-COA SYNTHETASE 377
PURIFICATION OF ACETYL-CoA SYNTBETASE FROM BEEF HEART MITOCtIONDRIAa
Specific
activity Total protein Activity Recovery
Step (units/rag) (rag) (units~) (%)
Mitochondrial supernatant 0.4-0.5 10,900 4,800 100

First (NH~)~SO~ 0.9 5,200 4,680 98
precipitate
Gel-(NH4)2SO4 1.6 2,800 4,480 93
precipitate
Second (NH4)~SO4 2.4 1,700 4,080 85
precipitate
First Sephadex 3.6 1,000 3,600 75
TEAE-cellulose 32.0 75 2,400 50
Second Sephadex 36.0 30 1,080 23
Crystals --~36 -- -- --
* An average preparation from 10 kg of myocardium.

b One unit of enzyme catalyzes the disappearance of 1 micromole of reduced CoA per
minute at 37 °.
All steps are carried out at 4 °. Whenever recrystallized (NH~)~S04

is added, the pH is adjusted to 8 with NH40H and the solution is stirred
for 10 minutes after the salt dissolves. The protein is maintained in 3
mM mercaptoethanol-0.5 mM EDTA, pH 8, throughout the purification.
When isolated as an (NH~)2S0~ precipitate, the enzyme is recentrifuged;
the "packing" centrifugation removes excess liquid (NH,)~S04 which
inhibits enzymatic activity. Dialysis tubing is boiled in 20 mM
K H C Q - 1 mM EDTA, rinsed with distilled water, and dried prior to
use.
Step 1. Isolation o] Mitochondria. Ice-chilled fresh beef myocardium
is ground to a medium pulp in a meat grinder. One kilogram is homoge-
nized (20 seconds at high speed and 20 seconds at low speed) with 2.8
volumes of 0.13 M KC1 in a 1-gallon Waring blendor. Cellular debris and
nuclei are removed by centrifugation at 1200 g for 10 minutes. The
mitochondria are isolated from the supernatant solution by constant-flow
centrifugation (Lourdes CFR-1 rotor, 28,000 g with a flow rate of 40-
60 ml/minute). By this procedure, 5 kg of myocardium can be proc-
essed in about 5 hours with an estimated yield of 180-300 g, wet weight,
of packed mitochondria. The particles are diluted to a volume of 1700
ml with 0.13 M KCI and suspended by brief homogenization. The mix-
ture is poured into four l-liter plastic bottles and frozen at --20%
Step ~. Preparation o] Crude Mitochondrial Supernatant Solution. On
a subsequent day, the mitechondrial suspension is thawed rapidly by
agitation in a water bath at 45 ° and the pH is adjusted to 8 with
NH4OH; the mixture is again stored at --20% The thawing and freezing
process is repeated once again at pH 8. Frozen preparations at this stage

lose little activity in a year. For purification, suspensions derived from
10 kg of ground beef myocardium are thawed for the third and final time
and centrifuged at 4 ° for 50 minutes at 20,000 g. The resulting crude
mitochondrial supernatant solution (3-4 mg of protein per milliliter)
should be quite clear; cloudy preparations usually possess poor specific
activities.
Step 3. First Fractionation with (NH4)2804. Solid (NH~)~SO~ is
added to achieve a concentration of 0.21 g per milliliter of solution.
After centrifugation (20,000 g for 15 minutes) the small precipitate is
discarded and the supernatant solution is treated with 0.235 g of
(NH,)~S04 per milliliter of original solution. The suspension is centri-
fuged 15 minutes at 20,000 g and the supernatant solution is discarded.
Step 4. Fractionation with Alumina Cy Gel. The precipitate is dis-
solved to achieve a protein concentration of 8 mg/ml in 20 mM KHC03.
For a typical preparation, 4% of the enzyme volume is added as
saturated liquid (NH4)~S04 (pH 8.3, 4 °) followed by 15% of the enzymc
volume as alumina C-r gel (15-20 mg of solids per milliliter). The gel
suspension, freshly adjusted to pH 8 with NH4OH, is added slowly with
rapid stirring; stirring is continued for 5 minutes after addition is
completed. The resulting suspension is centrifuged immediately at 6000 g
for 5 minutes, the precipitate is discarded. Recrystallized (NH4)2SO~
(0.395 g per milliliter supernatant solution) is added, and the enzyme
is precipitated by centrifugation at 20,000 g for 15 minutes. The amount
of gel added depends upon its age and is adjusted so that 55-60~ of the
protein is recovered in the final precipitate.
Step 5. Second Fractionation with (NH~)2S04. The gel-(NH4)~S04
precipitate is dissolved and diluted to 8 mg of protein per milliliter in 20
mM KHC03. Saturated liquid (NH4)2S04 (pH 8.3, 4 °) is added to
achieve a ratio of 0.64 ml per milliliter of protein solution. After removal
of the precipitate by centrifugation, 0.17 g of solid (NH~)2S04 is added
per milliliter of the supernatant solution. The precipitate is dissolved in
a minimal volume of 20 mM KHC08 and dialyzed against the same
buffer for 1 hour.
Step 6. First Chromatography on Sephadex G-IO0. The dialyzed
protein is diluted to 40 mg of protein per milliliter and pipetted onto a
column (45 X 2.6 cm) of Sephadex G-100 equilibrated with 20 mM
KHC08. The protein is eluted with the same solution at a flow rate of
not less than 0.5 ml per minute, and fractions of 6-8 ml are collected.
Most of the enzymatic activity appears slightly after the protein peak
both before and overlapping a red pigment. Fractions having specific
activities increased by 1.8-fold or more are combined.
Step 7. Chromatography on TEAE-Cellulose. The combined Sephadex
[58] ACETYL-COA SYNTHETASE 379
fractions are diluted with 20 mM KHC08 to a protein concentration of

5 mg/ml and allowed to drip on a column (36 X 2.2 cm) of TEAE-
cellulose equilibrated with 20 mM KHC0a; the flow rate is adjusted to
20 ml per hour. A linear gradient is set up between 200 ml of 20 mM
KHC03 in the mixing bottle and 200 ml of 20 mM KHC03-0.3 M KC1
in the reservoir; the flow rate is maintained at 20 ml per hour. The
enzyme is eluted in several 5-7 ml fractions approximately 300 ml after
the gradient is started. Its position can be located more exactly by the
presence of overlapping greenish-yellow or red contaminating pigments.
Fractions having specific activities of greater than 27 units/mg are
pooled and the protein is precipitated by adding 0.65 g of solid
(NH~) ~S04 per milliliter of solution. After centrifugation the precipitate
is stored at --20 ° or taken up in a minimum volume of 20 mM KHC0~
and dialyzed against the same solution for 1 hour.
Step 8. Second Chromatography on Sephadex G-IO0. The dialyzed
protein (70--120 mg) is diluted with 20 mM KHC03-0.12 M KC1 to a
protein concentration of 35 mg/ml and placed on a freshly prepared
column (22 X 1.2 cm) of Sephadex G-100 equilibrated with 20 mM
KHC03-0.12 M KC1. Elution is carried out with the same solution at
a flow rate of 0.6 ml per minute, and 2 ml fractions are collected.
Enzymatic activity is eluted after the protein peak in nonpigmented
fractions having specific activities of up to 36.7 units/mg. The enzyme
is precipitated by adding 0.65 g of solid (NH4)~S04 per milliliter of
enzyme solution. The precipitate is packed and stored at --20 ° or
subjected to the crystallization procedure.
Step 9. Crystallization. The precipitate is suspended and stirred for 10
minutes in 0.50 saturated (NH4)2SO4 (pH 8.3, 4 °) at a protein concen-
tration of 10-20 mg/ml. Undissolved protein is then removed by centrif-
ugation at 27,000 g for 15 minutes. The supernatant solution is kept at
4°; after a period of up to 4 days, a chalky white material precipitates
in crystalline or amorphous form. The enzyme is crystallized with greater
consistency and speed if seed crystals are added to the clear supernatant
solution immediately before storage.
Properties
Stability and Purity. The enzyme becomes quite unstable after the
second exposure to Sephadex (step 8) but the usual TEAE-(NH4)2S04
precipitate (step 7) is suitable for most purposes and can be stored at
--20 ° with about 10-40% loss of activity in the first month. The best
preparations of acetyl-CoA synthetase (step 9) have no detectable
ATPasc, pyrophosphatase, acetyl-CoA deacylase, or acetyl adenylate
splitting activity, and contain only traces of butyryl-CoA synthetase.
However, pyrophosphatase activity is present in the TEAE-(NH,)~S0~

fraction.
Physical Characteristics. Physical studies have been unsatisfactory
because of aggregation shown by the unstable enzyme. A molecular
weight near 30,000 is indicated for the active monomer by sedimentation-
equilibrium studies. Isolation of enzyme-bound acetyl adenylate under
equilibrium conditions also indicates a molecular weight in the same
range on the basis of a 2:1 stoichiometry between adenylate and enzyme.
pH Optimum and Equilibrium. The beef heart enzyme has a pH
optimum ranging from 7.8 to 8.4; over 50% the activity remains at pH
6.8 or 9.2. The equilibrium constant for acetyl-CoA formation in the
overall reaction, as determined with partially purified enzyme, is 0.86
at pH 7.5--8.5.11
Kinetics. Optimal activity in the overall reaction is obtained with
each substrate at 3- to 4-fold its apparent K~ concentration. At pH 8.0,
apparent K~'s for substrates in the forward reaction are: 0.2 mM for
acetate, 0.9 mM for ATP, and 0.4 mM for reduced CoA.
Cation Requirements. The enzyme shows a double requirement for
divalent cations in the overall reaction. 1~ Metal ions in one group (Mg**,
Mn *+, Fe ÷*, Co *+, or Ca**) are required only in the first partial reaction
(Reaction 1) and have high apparent K,~'s near that of ATP (0.9 mM).
The second divalent cation requirement for acetyl-CoA synthetase can
be shown only after bound metal is removed from the enzyme. Under
these conditions, the overall and both partial reactions are stimulated by
Ni +*, Cd ~, Fe ++, or Cu ++ in concentrations only slightly exceeding that of
the enzyme.
The beef heart enzyme also displays an absolute requirement for a
number of monovalent cations in the overall and both partial reac-
tions.iS, 1, This requirement is satisfied by Rb ÷, NH, ÷, Tris ÷, K ÷, Na ÷, and
Li ÷, and apparent K~'s for all these activations are in the 1-3 mM range;
at 10-200 raM, Na ÷ and Li ÷ inhibit the reaction whereas the other
cations activate maximally.
Inhibitars. Substrates may inhibit the beef heart enzyme at appro-
priately high concentrations, and 2 mM pyrophosphate produces nearly
50% inhibition of the overall reaction, a-Phenylbutyrate, an inhibitor of
fatty acid and cholesterol biosynthesis, depresses the activity of the yeast
enzyme from 70 to 80%. 15
Substrate Specificity. Glutathione or cysteine do not substitute for
CoA as acceptors. ATP is not replaced by GTP, CTP, ITP, or UTP:
1, p. Hele, J. Biol. Chem. 206, 671 (1954).
~L. T. Webster, Jr., J. Biol. Chem. 240, 4164 (1965).
'~R. W. Von Korff, J. Biog. Chem. 203, 265 (1953).
1, L. T. Webster, Jr., J. Biog. Chem. °,41, 5504 (1966).
R. Masters and D. Steinberg, Biochim. Biophys. Acta 27, 592 (1958).
[59] PHOSPHOTRANSACETYLASE FROM C. kluyveri 381
deoxy ATP at 2.4 mM affords 8670 the activity obtained with ATP. In
addition to acetate, the beef heart enzyme can activate acrylate or
propionate. The latter substrates have about the same Vma. as acetate
but higher apparent K~'s ( ~ 10 mM as compared to 0.2 raM). Fluoro-
acetate is activated by enzymes from rabbit kidney TM and pigeon liver. 17
Addendum
Since this chapter was written, unsatisfactory results have been obtained with
Sephadex G-100 in the newer bead form. Better yields of enzymatic activity are
noted when Bio-Gel P-150 is substituted for Sephadex and the enzyme is protected
with NiCh and chemicallysynthesized acetyl adenylate.
17A. Marcus and W. B. Elliott, J. Biol. Chem. 218, 823 (1956).
[59] P h o s p h o t r a n s a c e t y l a s e f r o m C l o s t r i d i u m kluyveri
lEG 2.3.1.8 Aeetyl-CoA:orthophosphateacetyltransferasel
B y HELMUT R. KLOTZSCH
Acetyl phosphate + HS-CoA ~ acetyl-S-CoA + phosphate
The enzyme was first isolated from Clostridium kluyveri in 1952 by
E. R. Stadtman.' In 1961, Bergmeyer et al. ~ reported on the crystalliza-
tion of the enzyme, which was derived from a specially grown C.
kluyveri? ~
Assay Method
The rate of production of acetyl-CoA can be measured directly by the
increase of optical density at 233 nm. Acetyl-CoA has a higher absorbency
coefficient than CoA (A, -----4.44 X 106 cm2/mole).
Reagents
Tris-HC1 buffer pH 7.4, 0.1 M
Glutathione (reduced form) in Tris-HC1 buffer, 30 mg/ml
CoA, aqueous solution, 5 mg/ml
Acetyl phosphate, potassium-lithium salt, aqueous solution, 40
mg/ml
Ammonium sulfate, aqueous solution, 1 M
Procedure. The assay is carried out at 25 °. Enzyme solution diluted

'E. R. Stadtman, J. Biol. Chem. 196, 196, 527 (1952).
H. U. Bergmeyer, H. R. Klotzsch, and G. Lang, Angew. Chem. 73, 807 (1961).
--aGerman patent 1,175,191.
[59] PHOSPHOTRANSACETYLASE FROM C. kluyveri 381
deoxy ATP at 2.4 mM affords 8670 the activity obtained with ATP. In
addition to acetate, the beef heart enzyme can activate acrylate or
propionate. The latter substrates have about the same Vma. as acetate
but higher apparent K~'s ( ~ 10 mM as compared to 0.2 raM). Fluoro-
acetate is activated by enzymes from rabbit kidney TM and pigeon liver. 17
Addendum
Since this chapter was written, unsatisfactory results have been obtained with
Sephadex G-100 in the newer bead form. Better yields of enzymatic activity are
noted when Bio-Gel P-150 is substituted for Sephadex and the enzyme is protected
with NiCh and chemicallysynthesized acetyl adenylate.
17A. Marcus and W. B. Elliott, J. Biol. Chem. 218, 823 (1956).
[59] P h o s p h o t r a n s a c e t y l a s e f r o m C l o s t r i d i u m kluyveri
lEG 2.3.1.8 Aeetyl-CoA:orthophosphateacetyltransferasel
B y HELMUT R. KLOTZSCH
Acetyl phosphate + HS-CoA ~ acetyl-S-CoA + phosphate
The enzyme was first isolated from Clostridium kluyveri in 1952 by
E. R. Stadtman.' In 1961, Bergmeyer et al. ~ reported on the crystalliza-
tion of the enzyme, which was derived from a specially grown C.
kluyveri? ~
Assay Method
The rate of production of acetyl-CoA can be measured directly by the
increase of optical density at 233 nm. Acetyl-CoA has a higher absorbency
coefficient than CoA (A, -----4.44 X 106 cm2/mole).
Reagents
Tris-HC1 buffer pH 7.4, 0.1 M
Glutathione (reduced form) in Tris-HC1 buffer, 30 mg/ml
CoA, aqueous solution, 5 mg/ml
Acetyl phosphate, potassium-lithium salt, aqueous solution, 40
mg/ml
Ammonium sulfate, aqueous solution, 1 M
Procedure. The assay is carried out at 25 °. Enzyme solution diluted

'E. R. Stadtman, J. Biol. Chem. 196, 196, 527 (1952).
H. U. Bergmeyer, H. R. Klotzsch, and G. Lang, Angew. Chem. 73, 807 (1961).
--aGerman patent 1,175,191.
382 REACTIONS YIELDING ACETYI.rCOA [59]
to a suitable concentration (10-40 ml) is added to a quartz cuvette (l

cm light path) containing: buffer, 2.62 ml; glutathione, 0.05 ml; CoA,
0.10 ml; acetyl phosphate, 0.20 ml; ammonium sulfate, 0.03 ml; (total
volume, 3 ml). Readings of optical density (wavelength, 233 nm) are
taken at 1-minute intervals.
Units. In accordance with the IUB, 1 unit of enzyme is defined as the
amount of enzyme which catalyzes the formation of 1 #mole acetyl-S-
CoA per minute under above conditions. Protein is determined by the
Biuret method2 Specific activity is expressed in units/rag of protein.
Ammonium sulfate is determined by titration with BaC12 with alizarin-S
as indicator.~,5
Cultivation o] C. kluyveri
Medium
SOLUTIO~ 1. Potassium hydroxide, 6000 g, and sodium hydroxide, 2000
g, are dissolved and diluted with tap water to 1000 liters. Crotonie acid,
17,209 g, is then added.
SOLUTION2. A solution is made up of 750 g (NH4)~HP04, 10 g CaCl~,
200 g MgS04 X 7 H20, 2 g Na2Mo04, 2 g MnC12, 0.5 g FeS04 X 7 H~O,
0.1 g p-aminobenzoic acid and 5 mg biotin. The pH is adjusted to 7.0
with 50% (w/w) potassium carbonate using methylene blue as indicator
until solution is slightly blue.
Solution 2 is poured into solution 1 with constant mechanical stirring.
Sodium dithionite is added just to the extent that the entire medium
becomes colorless. Temperature is adjusted to 35-37 ° .
Approximately 200 liters of the C. kluyveri 5a culture in similar
medium is used to inoculate the main batch.
During the course of fermentation a shift of the pH toward acid is
observed. This "production of acid" is a function of the consumption of
crotonate. The pH is maintained during the entire course of fermentation
at 6.8-7.0 by means of concentrated ammonium hydroxide. By the time
85~'o of the original amount of crotonic acid is used up, approximately
5 liters of ammonium hydroxide have been added to maintain the pH.
This turnover is accomplished within approximately 72 hours, at which
time the C. kluyveri ceils are harvested by high-speed centrifugation
(Padberg, 40,000 g). The paste is washed with isotonic saline and lyophi-
8G. Beisenherz, H. J. Boltze, Th. Buecher, R. Czok, K. H. Garbade, E. Meyer-
Arendt, and G. Pfleiderer, Z. Natur/orsch. 8b, 555 (1953).
~J. S. Fritz and M. Q. FreeIand, Anal. Chem. 26, 1953 (1954).
5It. U. Bergmeyer, G. ttolz, E. M. Kauder, It. Moe]lering, and O. Wieland, Bio-
chem. Z. 333, 471 (1961).
~*Gratefully received from Dr. C. W. Schuster, Department of Bacteriology, Uni-
versity of California, Berkeley, California.
[59] FROM C. kluyveri
PHOSPtIOTRANSACETYI,ASE 383
lized. By using the above technique, it was possible to produce 500-

600 mg lyophilized material with a phosphotransacetylase activity of
approximately 8000 units/g.
In a number of experiments using the original medium according to
Stadtman and Burton, 6 which substitutes crotonic acid with ethanol and
acetate, it was not possible to obtain C. kluyveri in comparable yield of
comparable activity.
It was found essential to start the purification of the enzyme with ma-
terial of approximately 8000 units/g in order to succeed with the follow-
ing purification steps resulting in crystallized phosphotransacetylase.
Purification of the Enzyme

Step I. Preparation of the Extract. Suspend 1000 g of C. kluyveri dry
powder in 10 liters potassium phosphate buffer, 0.01 M, pH 8.0. Tem-
perature is adjusted to 38 ° and the suspension is stirred for 4 hours at
this temperature. Suspension is centrifuged at 14,000 g for 20 minutes.
The clear grayish-yellow to brown supernatant contains the enzyme. The
residue is discarded.
Step 2. Alcohol Fractionation. The extract is cooled to 0 ° and an
equal volume of 96% (v/v) ethanol (--10 °) is added with constant
mechanical stirring within approximately 20 minutes. During this opera-
tion the temperature of the solution should decrease to --5 ° . The mixture
is centrifuged at 14,000 g for 20 minutes, maintaining its temperature at
--5 °. The precipitate is discarded. The slightly opaque supernatant is
adjusted to an alcohol content of 5 8 ~ (v/v) by the addition of --10 °
alcohol within 20 minutes. The precipitate is collected by means of
centrifugation at 14,000 g for 20 minutes. The supernatant is discarded.
The precipitate is dissolved in cold doubly distilled water; a slight
turbidity is removed by centrifugation. The solution contains the enzyme.
Step 3. Ammonium Sulfate Fractionation. The enzyme solution is
diluted to twice the volume of the original extract with cold doubly dis-
tilled water. Solid ammonium sulfate is added slowly to a final concen-
tration of 3.0 M. The precipitate is spun down for 10 minutes at 20,000 g
and dissolved in distilled water to yield a protein concentration of
approx. 7 mg/ml. Solid ammonium sulfate is added to the solution to a
concentration of 1.9 M. The precipitate of enzyme protein is centrifuged
and discarded. The supernatant is adjusted to a final ammonium sulfate
content of 3.0 M. The precipitate is collected at 20,000 g, dissolved in cold
distilled water, and dialyzed for 3 to 4 hours at approximately 2 ° against
slowly flowing distilled water.
Step 4. DEAE-Chromatography. The dialyzed enzyme is absorbed on
a DEAE-ccllulose column of approximately 30 mm diameter and 300 mm
E. R. Stadtman and R. M. Burton, Vol. I, p. 518.
height which is equilibrated with 0.05 M potassium phosphate, pH 7.6.

The column is washed with 1 liter 0.05 M potassium phosphate, p H 7.6,
and then eluted with the same buffer to which sodium chloride to a final
concentration of 0.1 M has been added. Active fractions are combined
and precipitated with ammonium sulfate to 3.0M. The precipitate is
collected at 35,000 g; the supernatant is discarded.
Step 5. Crystallization. Precipitate is suspended in cold 2.8 M am-
monium sulfate solution. Cold distilled water is added dropwise until all
protein is dissolved. Cold saturated ammonium sulfate solution, which is
adjusted to p H 8 with ammonium hydroxide, is now added slowly and
with constant gentle stirring. At the final concentration of 2.7M am-
monium sulfate all enzyme protein is crystallized; the suspension displays
a typical "silkiness."
Step 6. Recrystallization. The precipitate of the first crystallization is
subjected to 2 recrystallizations as described before.
TABLE I
SUMMARY OF PURIFICATION PROCEDURE
Total
volume Units Units Protein
Step (ml) (X 106) (%) (rag) Units/rag
Extract 7,400 86.5 100 79,000 110

Supernatant of 1st alcohol pptn. 13,500 35.6 41 28,400 125
Solution of 2nd alcohol pptn. 12,200 26.3 30 7,900 332
Solution of (NH4)2SO4pptn. 1,100 26.3 30 7,900 332
Supernatant of 1.9 M (NH4):SO~ 1,065 20.3 23.5 1,430 1,420
fractionation
Dialyzate 183 11.3 13 1,330 850
DEAE eluate 815 10.3 12 250 4,140
(NH4)2SO, pptn. 176 10.3 12 250 4,140
1st Crystallization 140 10.3 12 140 7,350
2rid Crysatllization 103 10.0 11.5 110 9,100
3rd Crystallization 106 7.0 8 77 9,100
Properties. Under the conditions described above the enzyme crystal-

lizes in fine needles of approximately 5 # length. Aqueous solutions of
the enzyme have only one peak of absorbency at 275 nm.
Stability. As a suspension in 2.7-3.4 M ammonium sulfate the enzyme
is stable for several months at 0-4 ° without loss of activity. Ammonium
sulfate suspensions withstand temperatures of 30 ° for approximately 1
week without considerable loss of activity. Diluted aqueous solutions of
the enzyme are unstable at any temperature but retain most of their
activity when kept frozen for several months.
[59] FROM C. kluyveri
PHOSPHOTRANSACETYLASE 385
TABLE II
TEMPERATURE INFLUENCE ON PHOSPHOTRANSACETYLASE ACTIVITY a
After 5 mimltes at:
Orig. 33° 40° 45° 50° 60 °
Percent activity 100 100 100 85 52 2

a 1.5 mg enzyme/ml of 0.15 M ammonium sulfate solution, pH 6.
pH and Temperature. Optimal activity is found in Tris buffer between

p H 7.0 and 7.7 with m a x i m u m at 7.4. Between 22 ° and 40 ° the reaction
is only slightly influenced by temperature.
Kinetics. T h e following michaelis constants have been determined:
for CoA
K~ = 5.6 X 10 -4 (1.05 X 10-4 ~I acetyl phosphate)
for acetyl phosphate:
K~ = 6.6 X 10-4 (7.4 X 10 -4 M CoA).
All experiments on activators and inhibitors have been conducted with

an enzyme solution which was not completely dialyzed. However the
final a m m o n i u m sulfate concentration in the assays was less than 2 X
10 -7 M a m m o n i u m sulfate and can therefore be neglected.
Activators and Inhibitors. For optimal activity the enzyme requires
the presence of NH,* ions. Optimal activity is obtained in the presence
of 7 X 10 -~ M a m m o n i u m sulfate, higher concentrations result in slight
inhibition. Similar effect can be yielded with a m m o n i u m chloride. The
ions of N a +, K +, and M g ~÷ (as chloride) cannot substitute for NH4 +.
TABLE III
INFLUENCE OF 0.1 m M INHIBITORS ON ENZYME ACTIVITY a
Activity
Inhibitor (%)
Control 100
MnCI~ 87
CuSO4 43
KCN 89
a,a'-Dipyridyl 37
p-Chlormercuribenzoate 2
• Determined under the conditions as described in "Assay Method."
Equilibrium. The equilibrium constant

Ac-S-CoA × phosphate
K=
CoA × acetyl phosphate
was determined starting with various concentrations of acetyl-CoA and
phosphate. Results are shown in Table IV. Except for the concentrations
stated, conditions were as described in "Assay Method."
T A B L E IV
EQUILIBRIUM CONSTANT
~Mole ~Mole ~Mole ~ O.D.

acetyl-CoA phosphate CoA 233 nm K
0.2 0.2 -- 0.021 169

0.2 0.2 -- 0.022 148
0.2 1.0 -- 0. 048 154
0.5 0.2 -- 0. 036 147
0.2 1.0 0.15~ 0.015 118
Specificity. The enzyme reacts specifically with CoA-SH, it has no

activity with oxidized CoA nor with desamino-CoA/,8 The turnover rate
with dephospho-CoA is approximately 0.5% of what can be obtained
with CoA-SH. Since this rate does not change during the final purifica-
tion steps (chromatography, crystallization, and recrystallization) we
assume that this phenomenon is an inspecifity rather than a contaminat-
ing activity.
G. Michal and H. U. Bergmeyer, Biochim. Biophys. Acta 67, 599 (1963).

e G. Michal, in " M e t h o d e n der enzymatischen Analyse" (H. U. Bergmeyer, ed.),
p. 517. Verlag Chemie, Weinheim, 1962.
[60] CARNITINEACETYLTRANSFERASEFROM PIGEON BREAST 387
[60] C a r n i t i n e A c e t y l t r a n s f e r a s e f r o m P i g e o n B r e a s t M u s c l e
[EC 2.3.1.7 Acetyl-CoA:carnitine O-aeetyltransferase]
By J. F. A. CIJASE
O-Acetyl-(--)-carnitine + CoASH ~ (-)-carnitine + acetyl-Cok
Assay Methods
Method A: The Direct Assay

Principle. Enzyme preparations of specific activity in excess of 1 unit
per milligram of protein per milliliter (see below) are assayed most satis-
factorily by direct spectrophotometric observation of the reacting sub-
strafes. At 232 m~, there is an increase in molar extinction of 4.5 )< 103
cm-1 on the acetylation of CoASH,1 whereas carnitine and acylcarnitine
solutions do not absorb at this wavelength.2 The catalyzed reaction may
be followed in either the forward or reverse direction, as it is readily
reversible (Keq = 0.6).s
Reagents
Neutral EDTA, 0.1 M
CoASH, 10 mg/ml. The solid coenzyme is dissolved freshly in water
Acetyl-DL-carnitine hydroehloride, 0.1 M, prepared according to
Fraenkel and Friedmann' and dissolved in water
Enzyme: 0.02-0.1 unit of carnitine acetyltransferase is a suitable
amount for assay. This corresponds to about 0.2--1.0 /~g of the
crystalline enzyme from pigeon breast muscle,~ and gives an
increase in extinction at 232 m~ of 0.05-0.25 per minute in the
system described below
Procedure. The assay system contains 0.2 ml of Tris-HC1, 0.005 ml of
EDTA, 0.05 ml of CoASH, enzyme, and water in a final volume of 1.95
ml. This mixture, in a cell of 10 mm light path, is equilibrated at 25 ° in
a spectrophotometer with a temperature-controlled cell housing. On the
addition of 0.05 ml of acetylcarnitine, the extinction of the solution at
1E. R. Stadtman, Vol. I [137].
2D. J. Pearson, Biochem. J. 95, 23c (1965).
3I. B. Fritz, S. K. Schultz, and P. A. Stere, J. Biol. Chem. 238, 2509 (1963).
G. Fraenkel and S. Friedmann, Vitamins Hormones 15, 73 (1957).
6j. F. A. Chase, D. J. Pearson, and P. K. Tubbs, Biochim. Biophys. Acta 96, 162
(1965).
232 m~ increases linearly with time for 1-2 minutes. Bovine serum
albumin was included in another description" but is now omitted as it
has no effect on the activity or stability of the enzyme.
Units. One unit of enzyme is t h a t amount which catalyzes the
acetylation of 1 micromole of CoASH per minute in the above system.
Protein is determined spcctrophotometrically at 260 and 280 m~ accord-
ing to Layne. 6 Specific activity is expressed as units per milligram of
protein.
Method B: Carnitine Acetyltrans]erase in Crude Tissue Extracts

Crude extracts contain too much 232 m~ absorbing material for the
direct assay to be applicable. Carnitine acetyltransferase activity may
be detected in such preparations in a coupled system2, 7
Principle. Acetyl-CoA, formed from acetylcarnitine and CoASH, is
removed to form citrate in the presence of malate, NAD, and excess
citrate synthase and malate dehydrogenase, the reduction of NAD being
followed at 340 m~.
Acetylcarnitine q- CoASH ~ carnitine -b acetyl-CoA
Acetyl-CoA -~ oxaloacetate -~ citrate ~ CoASH
Malate -t- NAD ~ oxaloacetate T NADH~
Acetylcarnitine -[- malate -~ N A D --~ carnitine ~ citrate -{- NADH~
Reagents
L-Malate, 1.0 M, p H 8.0
NAD, 10 raM, p H 6.0
NaCN, l0 m M
Citrate synthase: a crystalline suspension of the enzyme from pig
heart, s ca. 5 mg of protein/ml (or see footnote 8a)
Malate dehydrogenase: an ammonium sulfate suspension of the pig
heart enzyme,' ca. 5 mg of protein/ml
6E. Layne, Vol. III [73].
N. R. Marquis and I. B. Fritz, J. Biol. Chem. 240, 2193 (1965).
'P. A. Srere and G. W. Kosicki, J. Biol. Chem. 236, 2557 (1961).
,a It may be noted that a substantial copurification of carn/tine acetyltransferase and
citrate synthase occurs during steps 1--4 of the purification procedure. Most of the
citrate synthase, which is present in an amount comparable to that of the carnitine
enzyme in the eluate from step 4 is, however, left in solution after the calcium
phosphate gel treatment. If desired, it may be adsorbed by the further addition of
5 ml of gel per I00 ml of eluate. Citrate synthase may then be eluted and crystal-
lized as described for the pig heart enzyme,' when it is suitable for use in assay
method B.
' Commercial preparations are available, or see this volume [18-21].
[50] CARNITINE ACETYLTRANSFERASE FROM PIGEON BREAST 389
Tris-HC1, pH 7.8, EDTA, CoASH, acetyl-DL-carnitine and carni-

tine acetyltransferase solutions as for assay method A
Procedure. Combine Tris-HC1, 0.2 ml; EDTA, 0.05 ml; L-malate, 0.05
ml; NAD, 0.05 ml; NaCN, 0.2 ml (if the preparation shows NADH.~
oxidase activity); CoASH, 0.25 ml; malate dehydrogenase, 0.005 ml;
citrate synthase, 0.01 ml; carnitine acetyltransferase, 0.04-0.20 unit; and
water to a volume of 1.95 ml. Equilibrate the mixture in a spectrophotom-
eter at 25 ° as described in method A. On the addition of 0.05 ml of acetyl-
carnitine, an increase in extinction at 340 m~ is observed. For the cal-
culation of specific activity, it is assumed that the extinction coefficient
for NAD reduction at this wavelength is 6.22 X 103 cm-1. Specific activi-
ties may then be expressed in a manner analogous to that given for
method A.
This procedure gives erroneously low estimates of enzymatic activity
because less than 1 equivalent of NADH~ is produced per equivalent of
acetyl-CoA formed,2,1° but it is adequate for comparative purposes.
Other Methods
Assays that have been devised to study the reverse reaction between
acetyl-CoA and (--)-carnitine include a relatively insensitive hydrox-
amate procedure to follow acetylcarnitine formation,~1 and the use of the
thiol reagent 5,5"-dithiobis-(2-nitrobenzoic acid) (DTNB) to follow
CoASH release2,12 The latter technique has the disadvantage that DTNB
slowly inactivates carnitine acetyltransferase2 A very sensitive isotope-
exchange assay has also been described TM with its application to detection
of the enzyme in nervous tissue.
This procedure is essentially the same as published elsewhere2 All
operations were conducted in a cold-room at 4 ° unless otherwise
indicated.
Pigeon breast muscle, excised as soon as possible after death, m a y be
stored frozen for several months without loss of extractable activity.
Step 1. Extraction. Stored muscle is thawed, sliced, and homogenized
for I minute in a Waring blendor with 3 volumes of cold (--5 °) 2 0 %
ethanol containing 0 . 4 M KCI. The homogenate is centrifuged for 10
minutes at 23,000 g, filtered through muslin to remove fat, and dialyzed
IoW. Buckel and If. Eggerer, Biochem. Z. 343, 29 (1965).
"S. Friedmann and G. Fraenkel, Arch. Biochem. Biophys. 59, 491 (1955).
12I. B. Fritz and S. K. Sehultz, J. Biol. Chem. 240, 2188 (1965).
~R. E. McCaman, M. W. McCaman, and M. L. Stafford, J. Biol. Chem. o,41,
930 (1966).
against two changes of 2 m M potassium phosphate buffer, pH 7.5, con-

taining 0.5 mM EDTA.
Step ~. Ammonium Sul]ate Fractionation. Any precipitate formed on
dialysis is discarded, and solid ammonium sulfate is added to give 50~
saturation (312 g/liter). After 30 minutes, the precipitate is collected by
centrifugation and discarded. Further ammonium sulfate (82 g/liter) is
added to the supernatant to give 62.5% saturation. The protein precipi-
tated contains over 90% of the enzyme activity; it is dissolved in 0.1 M
phosphate, pH 7.5, and dialyzed as before.
Step 3. Acetone Fractionation. The dialyzate is cooled to 0 ° and 0.47
of its volume of acetone is added slowly, with stirring; the temperature
of the mixture is lowered progressively to --7% After 30 minutes, the
precipitate is centrifuged, dissolved in 0.1 M phosphate, pit 7.5, and
dialyzed as in step 1. It has been found repeatedly that attempts to
redissolve the acetone pellet in dilute buffer (e.g., 10 raM) result in
almost complete loss of enzyme activity, and this should be avoided.
Step ~. Fractionation on DBAE-Cellulose. The dialyzed preparation is
applied to a column of DEAE-cellulose (35 g of DEAE per gram of
protein) equilibrated with 2 mM phosphate, pH 7.5. The column is
washed with 2 column volumes of 10 mM phosphate, pH 7.5, followed by
3 volumes of 15 mM buffer. This removes about half of the protein; no
transferase activity should be present in the 10 mM eluate, and only a
trace in the 15 mM fraction. The enzyme is eluted with 4 column
volumes of 25 mM phosphate, pH 7.5.
Step 5. Calcium Phosphate Gel. One milliliter of calcium phosphate
gel14 (34 mg/ml, dry weight) is added per 100 ml of 25 mM phosphate
eluate; this absorbs all the enzyme. The gel is washed 3 times with 0.1 M
phosphate, pH 7.5, and the enzyme is eluted in 0.4 M phosphate, contain-
ing 10% ammonium sulfate2 ~
Step 6. Fractionation on Sephadex G-IO0. Ammonium sulfate (55 g)
is added to each 100 ml of gel eluate, and the precipitate is dissolved in a
minimal volume of 0.1 M phosphate, pH 7.5. Fractions (2-4 ml) of this
solution are applied to a column of Sephadex G-100 (4 cm X 21 cm) con-
nected to a reservoir of the same buffer. The first 45 ml of eluate contains
no protein and is discarded; thereafter, 5 ml fractions are taken and
assayed for protein (absorption at 280 m~) and enzymatic activity. Two,
slightly overlapping, protein peaks emerge. The first, after 65 ml of
eluate have been collected, contains enzymatically inactive colored ma-
terial; the second, at ll0 ml, corresponds with the enzyme activity. No
further protein emerges after 150 ml, and a pool is made of fractions con-
t4D. Keilin and E. F. Hartree, Proc. Roy. Soc. Lo~zdon B124, 397 (1938); see al.~o
Vol. I [11].
[60] CARNITINE ACETYLTBANSFERASE FROM PIGEON BREAST 391
raining 90-95% of the total activity. I m p u r e material appearing at low

elution volumes is discarded.
Step 7. Crystallization. The pooled fractions from step 6 are con-
centrated b y adding a m m o n i u m sulfate to about 90% saturation (65
g/100 ml) and the precipitate is dissolved in 0.1 M phosphate, p H 7.5,
to give a solution containing about 10 mg of protein per milliliter. This is
cooled to 0 ° and solid a m m o n i u m sulfate is added until a faint turbidity
appears. Any traces of denatured brown material t h a t precipitate are
discarded, and the solution is then stored at 4 °. Crystals of carnitinc
acetyltransferase appear overnight and continue to grow for several days
in the form of fine needles? Recrystallization is cffectcd by a repetition
of this process and is encouraged b y "seeding" the incipiently turbid
solution with preformed crystals. The specific activity of the recrystal-
lized enzyme is 118-119 units/rag, a value which is unaltered by further
recrystallizations.
The purification procedure is summarized in the table for a prepara-
tion starting from 900 g of pigeon muscle and yielding 15 mg of crystal-
line enzyme. No difficulties have been encountered in reproducing these
results so long as the warning given in step 3 is heeded. The method m a y
be scaled-up at least 4-fold with a proportionate increase in yield.
PURIFICATION PROCEDURE FOR CARNITINE ACETYLTRANSFERASE

FROM PIGEON BREAST ~IUSCLE
Specific
Total activity
Volume Units/ activity Protein (units/rag Yield
Step and fraction (ml) mla (units b) (mg/ml) protein) (%)
1. Centrifuged extract 2660 3.1 8250 39 0 0.18 100

2. 50--62.5% Ammonium 170 43.7 7430 64.0 0.68 90
sulfate fraction
3. 0-32% Acetone fraction 140 36.5 5130 24.2 1.51 62
4. DEAE-cellulose 1970 1.08 2130 0.11 9.8 26
25 mM eluate
5. Calcium phosphate 102 17.0 1734 0.68 25.0 21
gel eluate
6. Sephadex G-100 eluate 50 32.8 1640 0.73 45.0 20
7. Crystallization
First crystals 1.7 717 1219 6.6 108 15
First recrystallization 1.35 438 592 3.7 118 --
Second recrystallization 1.75 334 584 2.8 119 --
" Activity measurements for steps 1 and 2 were made using the coupled assay (method
B). Thereafter the direct assay (method A) was employed.
b One unit of enzyme is the amount that catalyzes the acetylation of 1 micromole of
CoASH per minute in the assay conditions.
Properties
Stability. The crystalline enzyme is stored conveniently as a suspen-
sion in 60-70% saturated ammonium sulfate, when it is completely stable
at 4 °. Dilute solutions (10-100 ~g/ml) in 0.1 M phosphate, pH 7.5, also
may be stored in the refrigerator; they lose only 10-12% of their activity
in 2 months.
Effects o] pH. Carnitine acetyltransferase shows a broad pH optimum
between 7.0 and 8.0 for both forward and reverse reactions2 ,15 It is stable
overnight at pH 5.5, but becomes rapidly and irreversibly inactivated
above pH 8.6.8
Purity. Recrystallized carnitine acetyltransfcrasc is colorless in solu-
tion and is homogeneous in the ultracentrifuge, on chromatography on
Sephadex G-100, and on electrophoresis on cellulose acetate paper. 1~ It
is also free of acetyl-CoA hydrolase, citrate synthase, malate dehydro-
genase, and palmitoylcarnitine transferase activities.
Molecular Weight. A value of 58,000 ± 3000 is indicated by sedi-
mentation equilibrium 1' and gel filtration18 methods.
Inhibitors. The enzyme is inhibited by a range of reagents (iodo-
acetamide, p-chloromercuribenzoate, DTNB, N-ethylmaleimidc) which
are somewhat specific for protein thiol groups2,12,1~ Divalent cations are
also inhibitory.
Specificity. The enzyme is highly specific for (--)-carnitine and CoA,
(--)-norearnitine and 3'-dephospho-CoA being the only known analogs
which are also substrates2 ,1~,15 (-k)-Carnitine is a competitive inhibi-
tor (K~--173 ~ / ) 1 ~ for (--)-carnitine and its derivatives. 12'1~ Group
transfer between CoASH and (--)-carnitine is catalyzed with n-acyl
groups containing up to 10 carbon atoms, and Vm~ for the reaction falls
off with increasing chain length2 ,~° Palmitoyl-CoA is not a substrate, 3
but acts as a potent competitive inhibitor with respect to both (--)-car-
nitine and acetyl-CoA (K~ -~ 0.43 p.M).2°
Affinity for Substrates. Michaelis constants for substrates of pigeon
breast muscle carnitine acetyltransferase at pH 7.8 and 30 ° have been
found to be as follows: K,,, CoASH ~-- 37 ~M; K~, acetyl-CoA : 34 p.M;
K~, (--)-carnitine----120 pit/; Kin, acetyl-(--)-carnitine-----350 ~M. ~9
Similar results have been reported for the enzyme from other sources, ~,~3
and it ~ppears that these K,~ values represent dissociation constants (K~)
for the enzyme-substrate complexes involved. 19,'~°
~J. F. A. Chase, Biochem. J. 1{}4, 503 (1967).
'~J. Kohn, Nature 181, 839 (1958).
~TD. A. Yphantis, Ann. N.Y. Acad. Sci. 88, 586 (1960).
~sp. Andrews, Biochem. J. 91, 222 (1964).
~gj. F. A. Chase and P. K. Tubbs, Biochem. J. 99, 32 (1966).
~J. F. A. Chase, Biochem. J. 104, 510 (1967).
[60] CARNITINE ACETYLTRANSFERASE FROM PIGEON BREAST 393
Turnover Number. Taking the molecular weight of pigeon breast

muscle carnitine as 58,000, it may be calculated from the experimentally
determined value of V~z [the maximum velocity at infinite concentra-
tion of both CoASH and acetyl-(--)-carnitine] that the turnover number
of the enzyme at pH 7.8 and 30 ° is 29,000 moles of substrate transformed
per mole of enzyme per minute. The corresponding value for the back
reaction between acetyl-CoA and (--)-carnitine is 23,000.
Distribution. Carnitine acetyltransferase is distributed widely in
animal tissues 7,2~,22 and is also present in the yeast Saccharomyces carls-
bergensis. 23 In general, it is found in highest concentration in tissues (e.g.,
heart and skeletal muscle) which show a high rate of fat oxidation, and
in sperm.
,1A. M. Th. Beenakkers and M. Klingenberg, Biochim. Biophys. Aeta 84, 207 (1964).
~N. R. Marquis and I. B. Fritz, J. Biol. Chem. 240, 2197 (1965).
U Personal communication from Miss Ann Light, University of Bristol, Bristol,
England.
PREVIOUSLY PUBLISHED ARTICLES FROM METHODS IN ENZYMOLOGY
RZLAVZO TO Szczlo~ IV
Vol. III [04]. Chromatographic Analyses of Organic Acids. J. E. Varner.

Vol. III [66]. Determination of a-Keto Acids. Theodore E. Friedemann.
Vol. III [67]. Preparation and Assay of Oxalacetic Acid. Samuel P. Bessman.
Vol. III [69]. Assay of Tricarboxylic Acids. Joseph R. Stern.
Vol. HI [70]. Isolation and Assay of Suceinate and Fumarate. Harris Busch.
Vol. III [71]. Isolation and Assay of L-Malate. Seymour Korkes.
Vol. IH [72]. Itaconic Acid and Related Compounds. Helge Larsen.
Vol. I I I [132]. Assay of Coenzyme A. G. David Novelli.
Vol. IV [7], Fluorescence Techniques for the Enzymologist. Donald J. R. Laurence.
Vol. IV [17]. Micromethods for the Assay of Enzymes. Oliver H. Lowry.
Vol. IV [18J. Histochemical Methods for Enzymes. George Gomori.
Vol. IV [24]. Isotopic Experimentation with Intermediates of the Tricaxboxylic Acid
Cycle. H. E. Swim and M. F. Utter.
Vol. IV [25]. Synthesis and Degradiation of Isotopically Labeled Glycolic, Glyoxylic,
and Oxalic Acids. Katherine F. Lewis and Sidney Weinhouse.
Vol. VI [Ul]. Measurement of Pyridine Nucleotides by Enzymatic Cycling. Oliver
H. Lowry and Janet V. Passonnean.
Vol. X [74]. The Fluorometrie Determination of Mitoehondrial Adenine and Pyridine
Nucleotides. R. W. Estabrook, J. R. WiUiamson, R. Frenkel, and P. K. Maitra.
Vol. X [75]. ARmy of Nueleotides and Other Phosphate-Containing Compounds by
Ultramicro Scale Ion-Exchange Chromatography. Hans W. Heldt and Martin
Klingenberg.
VoI. X [104]. Means of Terminating Reactions. Martin Klingenberg and Erich Pfaff.
[61] GAS CHROMATOGRAPHY 397
[61] S e p a r a t i o n of Citric Acid Cycle a n d R e l a t e d C o m p o u n d s

b y (]as C h r o m a t o g r a p h y
By NANCY W. ALCOCK
Introduction
Determination of the following acids is frequently required.
COOH ~C--COOH ~C--COOH H~C-- C O O H

i I
HCOH HOC-- C O O H HC-- C O O H HOCH
I
C~ ~C--COOH HOC--COOH COOH
I
H
D-Lactic acid Citric acid Isocitric acid r.-Malic acid
C OOH H2 C - - C O O H
I I
CH C--COOH HC-- C O O H
II II
CI~ HC--COOH HOOC--CH
COOH
Fumaric
Succinic acid cis-Aconitic acid
acid
COOH
I
COOH CI-~
I L
C---O CI-L O--C--COOH H
i |-- L J
CH s C--O H~C--COOH O---C--COOH
I
COOH
Pyruvic Oxaloacetic Glyoxyli c
acid a - O x o g l u t a r i c acid acid acid
A simple technique for the simultaneous determination of all these
acids in both plant and animal tissues would be a very powerful tool in
studying intermediary metabolism. Gas liquid chromatography has the
potential for such studies. Although a single technique has not yet been
described to include all the acids listed, recent publications indicate that
398 SEPARATION AND ASSAY METHODS [51]
it is possible to form derivatives involving the carboxylic groups 1-17 and

in some cases the hydroxyl groups as well. 14,16 While difficulties have
occurred frequently with the preparation of stable derivatives of the
keto acids by simple methylation techniques, suitable derivatives of these
acids have now been reported; 14,~ these are sufficiently volatile for gas
chromatography. If coupled with an appropriate fraction collector and
collection system 18,19 radioactivity of the individual compounds in the
effluent from the gas liquid chromatography column also may be deter-
mined. One of the problems associated with ch~'omatography is the
identification of a compound detected; ideally this entails characteriza-
tion of the peak position and also confirmation of the structure of the
derivative detected in the column effluent. Recently Dalgleish e t al. ~ have
described a gas liquid chromatographic technique in which derivatives
of a wide range of metabolites, including a number of citric acid cycle
members, have been separated. The exact position of the peak produced
by an individual compound was calculated by relating it to the positions
of adjacent peaks produced from internal standards added to the
mixture of derivatives prior to separation. Their technique will be dis-
cussed in more detail below. The identification of individual compounds
was confirmed by Dalgleish e t al. ~6 by combining gas chromatography
with mass spectrometry.
Procedures which have been described for the preparation of suitable
derivatives of the acids will be outlined, as well as the separation of the
derivatives by gas liquid chromatography. The techniques, however,
have had limited application to biological materials. Methods of prepar-
~L. D. Quin and M. E. Hobbs, Anal. Chem. 30, 1400 (1958).
2j. R. Lessard, R. A. Briggs, and J. V. Scaletti, Canad. J. Plant Sci. 41, 507 (1961).
sC. J. Mirocha and J. E. DeVay, Phytopathology 51, 274 (1961).
4 G. G. Esposito and M. H. Swann, Anal. Chem. 34, 1048 (1962).
C. Kowala, Z. H. Kranz, and K. E. Murray, Australian J. Chem. 54, 832 (1962).
C. W. Gehrke and D. F. Goerlitz, Anal. Chem. 35, 76 (1963).
TA. Kuksis and P. Vishwakarma, Canad. 1. Biochem. Physiol. 41, 2353 (1963).
SH. H. Luke, T. E. Freemanu, and L. B. Kier, Anal. Chem. 35, 1916 (1963).
*N. E. Sharpless, J. Chromatog. 12, 401 (1963).
*~T. S. Rumsey, C. H. Noller, J. C. Burns, D. Kalb, C. L. Rhykerd, and D. L. Hill,
J. Dairy Scl. 47, 1418 (1964).
" N . W. Alcock, Anal. Bioehem. 11, 335 (1965).
D. T. Canvin, Canad. 1. Biochem. 43, 1281 (1965).
1~M. Gee, Anal. Chem. 37, 926 (1965).
1, Z. Horii, M. MaNta, and Y. Tamura, Chem. & Ind. (London) 34, 1494 (1965).
~"G. G. McKeown and S. I. Read, Anal. Chem. 37, 1780 (1965).
**C. E. Dalgleish, E. C. Homing, M. G. Horning, K. L. Knox, and K. Yarger,
Biochem. J. 101, 792 (1966).
~F. L. Estes and R. C. Bachmann, Anal. Chem. 38, 1178(1966).
I*A. Karmen, L. Giuffrida, and R. L. BOwman, J. Lipid Res. 3, 44 (1962).
" A . Karmen, I. McCaffrey, and R. L. Bowman, J. Lipid Res. 3, 372 (1962).
ing partially purified extracts from such materials will be described else-
where in this volume. These, in combination with gas chromatography,
should make the analysis of tissues for the citric acid cycle compounds
a simpler task.
General Principles of M e t h o d s U s e d
Horning et al. ~° give a detailed description of the principles of gas
chromatography. Since the free acids of the citric acid cycle are not
sufficiently volatile for gas chromatography, suitable derivatives must
first be prepared. The methods employed have converted the carboxyl
groups either to methyl esters, ~,4-8,1°-13,15-17 to ethyl esters 3 or to tri-
methylsilyl esters24,18 In addition, hydroxyl groups have sometimes been
converted to trimethyl-silyl ethers24,16 With special treatment prior to
the trimethylsilylation, stable compounds which produced a single peak
on gas chromatography have been formed from the keto acids24
Dalgleish et al. 1~' succeeded in obtaining single peaks for methyl esters/
trimethylsilyl ethers of the keto acids, but using their procedure multiple
peaks arose when the carboxyl group was not methylated prior to tri-
methylsilylation of the acids.
Characteristics of Gas Chromatographic Apparatus Required

The prepared derivatives have been chromatographed using columns
with either selective or nonselective liquid phases on various supports.
Details of these are shown in Table I. Owing to the wide range in
volatility of the derivatives of the separate acids, temperature program-
ming is desirable; alternatively two separate chromatograms may be
run, one at a low temperature to separate the highly volatile components,
and the other at a higher temperature to separate less volatiIe compo-
nents with relatively greater retention times. Detection of the separate
components of the effluent has been made by a number of different
detector types (Table I). Separate temperature controls should be avail-
able for heating and monitoring both the injection port and the detector
of the apparatus used. Numerous commercial instruments have the re-
quired specifications.
General Methods for the Preparation of Derivatives
1. METHYL ESTERS
A variety of different reagents have been used for the preparation
of methyl esters. A procedure suitable for the nonketo acids is described
E. C. Homing, W. J. A. VandcnHeuvel, and B. G. Creech, Methods Biochem. Anal.

11, 69 (1963),
,<
o~-->i
O
~ii
~'
~6 ~9 ~ a4~ c6
T-, , ~
£
-$
~8
e, a~
in detail in this section (see Recommended Procedures). Diazomethane

was the esterifying agent used by most investigators. 1,*, 5,7,8.12,15-,7 Meth-
anol borontrifluoride/,8,~1 methanol hydrogen chloride, 8 methanol sul-
furic acid, ~°,is methyl iodide in the presence of the silver salt of the acid, 6
lithium methoxide, + and hydrogen chloride in methanol nu thionyl
chloride 1~ have also been used.
Despite the different conditions used for the methylation of the
various acids, no single method has been described which has unequiv-
ocably esterified all the acids simultaneously. While the efficiency of
particular procedures may vary with the conditions used, the esterifica-
tion of succinic acid, citric acid, and malic acid has been carried out
without difficulty. ~-8,~°-~2,15-~7 Problems have arisen with unsaturated
acids, presumably due to reaction across the double bond; with carefully
controlled conditions 15 these have been overcome. The attempts at simple
esterification of the keto acids have failed in general. The numerous re-
ports of unstable compounds giving rise to multiple peaks (Table II)
indicate the difficulties encountered. Even using diazomethanc a mild
methylating agent--Alcock (unpublished data) was unable to obtain re-
producible single peaks on chromatographing the products from the keto
acids. Simmonds et al. +-~ studied the diazomethylation reaction with
pyruvic acid, a-oxoglutaric acid, and oxaloacetic acid and found that
more than one derivative existed even when the reaction had been al-
lowed to proceed only for a few minutes; this suggests a very unstable
product.
A summary of the methods used for the successful esterification of the
acids and the problems recognized in particular cases is presented in
Table II. The difficulties are discussed below.
P r o b l e m s A s s o c i a t e d w i t h E s t e r i f i c a t i o n of I n d i v i d u a l A c i d s
F u m a r i c Acid. Using diazomethane at 25 ° McKeown et al. 1~ found a
product assumed to be a pyrazoline which could be formed by addition
across the double bond. The derivative, when chromatographed, had a
much longer retention time than the pure dimethyl ester. It is possible
that the failure of Quin et al. 1 to obtain a peak when their product of
diazomethylation was chromatographed was due to pyrazoline formation;
the chromatogram may not have been run long enough for the compound
to be eluted. Esposito et aI. + were unable to obtain the dimethyl ester
from fumaric acid using lithium methoxide; they attributed this to a
rearrangement involving the double bond and the dicarboxylic groups.
However, Mc'.Keown et al. ~ had no difficulty in obtaining the dimethyl
ester using diazomethane at --70 °.
c i s - A c o n i t i c Acid. McKeown et al. 15 found evidence of pyrazoline
formation from c/s-aconitic acid in the presence of diazomethane at 25 °,

but the pure trimethyl ester was formed without difficulty at --70 °.
However, since the cis and trans isomers both gave the same peak on
chromatography, it was concluded that spontaneous isomerization had
occurred. Kuksis et al. r obtained two peaks identified as cis- and trans-
trimethyl esters following diazomethylation of c/s-aconitic acid, but
using methanol borontrifluoride all the c/s-aconitic acid was converted
to the t r a n s - t r i m e t h y l ester. A second peak obtained following the pro-
cedure of Alcoek 11 (in which methanol borontrifluoride was used as the
esterifying agent) was thought to be the trans isomer. Luke s also identi-
fied both cis and trans isomers when either of the two acids was esterified
with diazomethane.
a - O x o g l u t a r i c Acid. On treating ~-oxoglutaric acid with methanol
borontriftuoride Alcock I1 found two peaks on the chromatogram. Quin
et al. ~ using diazomethane observed that the reagent was consumed, but
there was no peak following chromatography. However, Kuksis et al. 7
and Kowala et al2 in some instances found a single peak for a-oxoglu-
tarate following treatment with diazomethane; Canvin TM on the other
hand, found several peaks.
Oxaloacetic Acid. Kowala et al2 reported only one peak for oxalo-
acetate although, using the same methylating agent--diazomethane--
Kuksis et al. 7 found three peaks; the latter authors assumed the three
peaks to have arisen from the keto form of the acid and from the cis
and trans isomers of the enol form. No peak was obtained by Alcock 11
following treatment with methanol borontrifluoride. Estes ~7 reported a
broad flat peak following diazomethylation.
G l y o x y l i c Acid. Both Canvin ~2 and Quin et al. 1 using diazomethane,
have found multiple peaks from the product of reaction with glyoxylic
acid. A single peak was reported by Rumsey et al. ~° using methanol-
sulfuric acid.
P y r u v i c Acid. 1Rumsey et al. 1° found two peaks for pyruvic acid
following a methylation procedure using methanol-sulfuric acid.
Isoct'tric Acid. McKeown et al. ~ esterified DL-isocitric acid lactone
with diazomethane and obtained a single peak; with methanol-sulfuric
acid a trace of a second product thought to be trimethyl isocitrate was
found.
2. ETHYL ESTERS
The only authors to prepare the ethyl ester of any of the citric acid
cycle acids were Mirocha et al., ~ who prepared diethyl malate, diethyl
fumarate, and diethyl succinate. Diethyl malate was poorly recovered
during extraction into n-heptane due to its solubility in water. The mere-
404 S~PA~ATmN AND ASSAY METHODS [51]
$.
L)
:>
rD
¢J
[61] OAS CHROMATOGRAPHY 405
~.~ °
°~ ~
O© 0
°°
o'~
°~_~
ber most thoroughly investigated--fumaric acid--gave 78~'o recovery

from the pure acid; however, on addition to biological materials the
recovery was 102-104%. Preparation was with 4% (v/v) sulfuric acid in
absolute alcohol. After refluxing for 1 hour, water was added and the
esters extracted into n-heptane.
3. TRIMETHYLSILYLDERIVATIVES
Horii et al. 14 treated citric acid, cis-aconitic acid, fulnaric acid, suc-
cinic acid and malic acid directly with trimethylchlorosilane and hexa-
methyldisilazane in pyridine solution. The procedure immediately gave
trimethylsilylation of the COOH and the OH groups, and a sample of the
resulting solution could be applied directly for gas chromatography.
a-Oxoglutaric and oxaloacetic acids required prior treatment with
hydroxylamine hydrochloride; trimethylsilylation of the oxime deriva-
tives then gave a stable compound, judging by the appearance of a single
peak on the ehromatogram. Pyruvic acid should react similarly to the
other keto acids with the reagents used by Horii et al. 14 Dalgleish et al. 1~
methylated the C 0 0 H group of a number of citric acid cycle compounds
using diazometbane and subsequently any OH groups present were
converted to trimethylsilyl ethers; the potential OH groups arising from
the possible enolization of keto acids were also subjected to trimethyl-
silylation. Where trimethylsilylation alone was used by Dalgleish et al.,
multiple peaks were obtained from the keto acids (see Table II).
4, METHYL ESTER//PRoPIONYL DERIVATIVES

Dalgleish et al. 1~ also obtained derivatives of a number of compounds
in which the carboxyl group(s) were converted to methyl esters using
diazomethane and any OH groups were propionylated. The only member
of the citric acid cycle to be treated in this manner was citric acid.
Recommended Procedures
I. PROCEDURE R E C O M M E N D E D FOR PREPARATION A N D SEPARATION OF

M E T H Y L ESTERS OF T H E N O N K E T O ACIDS
a. P r e p a r a t i o n
Reagents
Ethereal diazomethane prepared from Diazald (Aldrich Chemical
Co., Milwaukee, Wisconsin) as required
Acids: the purest commercially available acids should be used
Solvents: distillation prior to use is recommended
Methylation. ~5 Dissolve 10 mg quantities of the acids in 1 ml of

methanol. Cool to --70 ° in a dry ice-acetone bath. Slowly add diazo-
methane in ether, also at --70 ° until a slight excess is present as indi-
cated by the yellow color of the diazomethane. Immediately destroy the
excess diazomethane by carefully adding 10% acetic acid in methanol
dropwise. Evaporate to a small volume and then transfer to a 10 ml
volumetric flask and make the volume to 10 ml with ether. An aliquot
of the sample can then be injected directly into the gas liquid chromato-
graphic column.
b. Separation o] Methyl Esters by Gas Chromatography ~
Gas chromatographic apparatus with temperature programming is
desirable. Alternatively, the methyl esters may be separated by running
two separate chromatograms, one at a low temperature (about 90 °)
® ~ ~"
I O.*
~ =o
I t~
'--
¢'~
• g
0 2 4 8 10 12 14 15 18 20 26 28
Retention time (minutes)
Fla. 1. Separation of methyl esters of lactic, fumarie, succinic, malic, aeonitic, and
citric acids [N. W. Alcock, Anal. Biochem. 11, 335 (1965)]. Stationary phase: 5%
diethylene glycol adipate on Chromasorb W (mesh 30-60); column: glass, length
8 feet, i.d. ~ inch; apparatus: F & M Model 400 gas chromatographic apparatus;
detector: hydrogen flame. Conditions: helium flow rate, 45 ml/minute; hydrogen
flow rate, 60 ml/minute; air flow rate, 300 ml/minute; column temperature, 88-165 °
(program rate 7.5°/minute; this was commenced 7 minutes after the injection of the
sample) ; detector temperature, set at 230 ° ; flash heater temperature set at 230 °.
and the second at a higher temperature (about 150°). Several different
detector types have proved satisfactory for the detection of the esters as
shown in Table I. The apparatus used for the separation of the methyl
esters shown in Fig. 1 was a Model 400 F & M gas chromatography
unit, with a hydrogen flame detector. The conditions were as shown in
Fig. 1.
Column and Packing. An 8 foot column (glass), ¼ inch internal
diameter (i.d.), filled with 5 ~ diethylene glycol adipate on Chromasorb
W, mesh 30-60, separated the methyl esters prepared by Alcock. 11 The
column packing was prepared as follows: 5 g of diethylene glycol adipate

(Applied Science Laboratory, State College, Pennsylvania) was dis-
solved in approximately 150 ml of ethyl acetate in a 500 ml beaker.
Ninety-five grams of acidwashed Chromasorb W (Applied Science
Laboratory, State College, Pennsylvania) was added to the solution of
diethylene glycol adipate in ethyl acetate and the mixture was stirred
gently. The ethyl acetate was evaporated by heating gently on a hot
plate. The packing was finally air dried in an oven at 80 ° . The column
was packed by attaching one end (previously plugged with a glass wool
plug) to a suction pump, and filling it with the aid of a tapered funnel.
Uniform packing was achieved by agitating the column with a mechan-
ical vibrator during filling. After plugging the open end with a glass
wool plug, the column was placed in position in the oven of the gas
liquid chromatographic apparatus and conditioned by heating to 200 °
for 24 hours, with carrier gas flowing through at a rate of 20 ml/minute.
Although isocitric acid was not methylated nor its methyl ester separated
by Alcock11 by extrapolation from the work of McKeown, who used a
column containing 570 polyester stationary phase, its separation should
be possible with the conditions described.
Conditions ]or Chromatography. By means of the 8 foot column
packed with 5% diethylene glycol adipate on Chromasorb W described
previously, the products of methylation of lactic acid, citric acid, c/s- and
trans-aconitic acids, succinic acid, fumaric acid, and malic acid were
separated and eluted from the column within 30 minutes.
The order of elution of peaks is shown in Fig. 1 and is in agreement
with that found by other workers.
2. PROCEDURE R E C O M M E N D E D FOR PREPARATION A N D SEPARATION OF

TRIM ETHYLSILYL DERIVATIVES
a. Preparation 14,16
Reagents
Hexamethyldisilazane (Applied Science Laboratory, State College,
Pennsylvania)
Trimethylchlorosilane (Applied Science Laboratory, State College,
Pennsylvania)
Acids: the purest available commercial preparation of the respec-
tive acids
Pyridine: purest available commercial preparation
Hydroxylamine hydrochloride: purest available commercial prepa-
ration
Methanol: purest available commercial preparation
410 SEPARATION AND ASSAY METtIODS [61]
Procedure ]or Nonketo Acids. (i) TRIMETHYLSILYLESTERS AND TRI-

METHYLSILYLESTERS/TRIMETHYLSILYLETHERS?4 Place 10 mg of the acids
or a mixture of acids in a ground glass-stoppered test tube and dissolve
in 1 ml of dry pyridinc. Allow the solution to stand at room temperature
for 10 minutes. Add 0.1 ml of trimethylchlorosilane and then 0.1 ml of
hexamethyldisilazane. Under these conditions the reaction was reported
to be complete within a few minutes, although Dalgleish et al., 16 recom-
mended that the reaction should be left overnight to ensure that it had
reached completion. The sample is then ready for analysis by gas
chromatography.
(ii) METHYL ESTERS/TRIMETHYLSILYLETHERS.16 Dissolve about 1 mg
of the acid or of each of a mixture of the acids in 0.1 ml of methanol.
Add a slight excess of ethereal diazomethane (usually 1-2 ml), and
immediately evaporate to dryness under a stream of dry nitrogen. Ex-
posure to diazomcthane should be less than 1 minute. 1~,2~ Add 0.25 ml
of pyridine and to the resultant clear solution add 0.15 ml of hexamethyl-
disilazane and 0.05 ml of trimethylchlorosilane. Seal the tube and set
aside at room temperature overnight. Centrifuge and use the supernatant
directly for injection into the gas liquid chromatography column.
Procedure ]or Keto Acids. ~ (i) TRIMETHYLSILYLOXIME DERIVATIVES.
Place 10 mg of the acids or of each of a mixture of the acids and 10 mg
of hydroxylamine hydrochloride in a ground glass-stoppered 5 ml test
tube and dissolve in 1 ml of dry pyridine. Allow the preparation to stand
for 10 minutes at room temperature; add 0.1 ml of trimethylchlorosilane
and then 0.1 ml of hexamethyldisilazane. The reaction was reported to be
complete within a few minutes at room temperature and a sample of the
resulting mixture was injected directly into the gas liquid chromatog-
raphy column. The conditions for chromatography were the same as
those for the derivatives of the nonketo acids and are described below.
b. Separation oI Trimethylsilyl Derivatives by Gas Chromatography
Apparatus. In both cases where trimethylsilyl derivatives of the
members of the citric acid cycle have been separated by gas chromatog-
raphy, temperature programming has been used? ~,~6 Although Horii
et al., 14 do not state the type of detector used, Dalgleish detected the
derivatives by means of a hydrogen flame detector.
Preparation o] Column and Packing. Either SE-52 (methyl phenyl
siloxane) or F-60 (methyl-p-chlorophenyl siloxane) have been used as
the stationary phases to separate the trimethylsilyl derivatives prepared.
Both of these are nonselective phases. Horning et al., 2° reported difficulty
in obtaining a satisfactory thin film column if an acid-washed support
21p. G. Simmonds, B. C. Pettitt, and A. Zlatkis, Anal. Chem. 39, 163 (1967).
was coated directly with a nonselective phase. Their procedure for

obtaining satisfactory thin film packings involved the use of a silanized
support; the support was deactivated by treatment with hexamethyl-
disilazane or dichlorodimethylsilane. Since Horning et al., ~° stressed the
importance of careful preparation of the column, this detailed procedure
is described. The procedure applies equally well to coating Chromasorb
W which was used by Horii et al., 14 or Gas Chrom P, mesh 80--100,
which was used by Dalgleish et al. 16 The support (50 g) was treated with
400 ml of concentrated hydrochloric acid in a l-liter beaker with oc-
casional stirring for 12 hours. The acid was removed by the use of a
filter stick (coarse porosity). The support was then treated with hydro-
chloric acid in the same manner, with a contact period of 1 hour for each
of three washings. After the final acid treatment, 750 ml of deionized
water was added to the support and the mixture was stirred. The result-
ing suspension was allowed to stand for 2 minutes, and the supernatant
liquid was removed by decantation. This process removed finely divided
particles produced during the washing process. The support was then
washed thoroughly with deionized water by repeating this process. After
the final decantation the support was washed again on a Biichner funnel
with deionized water; the wash should be neutral. The support was then
suspended in acetone or methanol to remove most of the water. After
filtration and preliminary drying at room temperature, the drying process
was completed at 80 ° .
For silanization the dry acid-washed support (25 g) was placed in
100 ml of 5% dichlorodimethylsilane in toluene in a side-arm filter flask.
The pressure was reduced in the flask (by an aspirator) for a period of
a few minutes. The flask was shaken to dislodge bubbles from the surface
of the support, and the pressure was then allowed to return to atmos-
pheric. The treated support was removed by filtration and washed with
100 ml of toluene. It was then washed well with methanol, and after
preliminary air drying the support was dried at 80 °.
The coating procedure was carried out by a filtration process which
leads to a uniform deposition of liquid phase on the surface of the
support. A solution of liquid phase in an appropriate solvent was pre-
pared. The concentration of liquid phase in the solvent should be ap-
proximately 1% where a 1% (w/w) coating is required on Gas Chrom
p.2o If Chromasorb W is used, a concentration of 3% of liquid phase
in solvent is required for a 3% (w/w) coating which was used by Horii
et aI. TM The support (20-25 g) was placed in 100 ml of solution in a side-
arm filter flask. The flask was maintained at a reduced pressure (as-
pirator) for a few minutes, and the flask was shaken to dislodge
bubbles from the surface of the support. The pressure was allowed to
return to atmospheric and the mixture allowed to stand for about 5
minutes. The slurry was placed on a Biichner funnel with a rapid swirling
motion of the flask and the solution was allowed to drain freely through
the bed of support. Reduced pressure was maintained on the filter flask
for about 5 minutes. At the end of this time the filtration process was
complete, and the surface of the filter cake usually appeared to be damp
but not wet. The coated support was spread on a smooth surface for
preliminary drying at room temperature. It was then dried at 80-100 ° .
It is important that preliminary air drying should be carried out before
the support is placed in an oven. Column packings prepared in this way
should flow freely and should have the appearance of a powder.
When a filtration procedure is used for the preparation of thin-film
column packings, the relationship between the concentration of the phase
in solution and the amount of phase on the support must be determined
by experiment. With Gas Chrom P, it has been found that the amount of
phase on the support (w/w) is the same (to within about 0.1%) as the
concentration of phase in solution. If Celite 545 is used, the amount of
phase on the support is approximately two times the concentration of
the phase in solution. The amount of phase on the support may be
determined directly as follows: a 2--3 g quantity of coated support may
be extracted exhaustively in a Soxhlet extractor and the weight of phase
be determined after evaporation of the solvent.
A glass column may be silanized prior to packing by passing a
solution of 5 ~ dichlorodimethylsilane in toluene through it. Glass wool
plugs should be silanized similarly. The column can then be washed with
toluene and finally with methanol, and is ready for filling. It is packed
by attaching one end (plugged previously with a glass wool plug) to a
suction pump, and filling with the aid of a tapered glass funnel. Uniform
packing can be achieved by agitating the column with a mechanical
vibrator while it is being filled. It is plugged on the open end with a
glass wool plug and is then ready for conditioning. Column conditioning
at 300 ° for 12 hours is satisfactory for either F-60 or for SE-52. 2°
Conditions for Chromatography of Trimethylsilyl Derivatives. The
conditions used for separating trimethylsilyl esters or trimethylsilyl
esters/trimethylsilyl ethers or trimethylsilyl oxime derivatives of the
keto acids 1~ are outlined in Fig. 2. Similar conditions of temperature
programming should be suitable for separating the trimethylsilyl ethers/
methyl esters prepared by the method of Dalgleish et al? e
Quantitation
The hydrogen flame is suitable for detecting the derivatives as they
emerge from the column. Its response, however, is not proportional to the
[61] o A s CHROMATOGRAPHY 413
t l I I
T~
I l
10 2O 3O 4O
Time (minutes)
I I I l I
90 II0 130 150 170
Ternperat u re
Fro. 2. Linear temperature programmed sepaxation of trimethylsilyl derivatives

of Krebs cycle and allied acids. Column: size, 6 feet X % inch; packing, 3% SE-52
on Chromasorb W (60-80 mesh); temperature, 90-170° (program rate: 2°/minute).
Flow rate (N2): 35 ml/minute. Components: 1, lactic acid; $, pyruvic acid; 5,
succinic acid; ~, fumaxic acid; 5, malic acid; 6, oxaloacetic acid; 7, a-ketoglutaric
acid; 8, lo.ctoisocitric acid; 9, c/s-aconitic acid; 10, citric acid. Redrawn from Z. Horii,
M. Makita, and Y. Tamura [Chem. & Ind. 34, 1494 (1965)].
mass of substance passing through the flame when there are a variety
of different chemical types present. Hence, for quantitation it is neces-
sary to incorporate into the mixture some standard substance whose
response can be compared with that of a known amount of each com-
pound to be measured. The use of either a fatty acid methyl ester
(Alcoek, unpublished data) or of n-alkanes as advocated by Dalgleish
et al., TM or some other suitable internal standard is imperative.
Identification of Derivatives after C h r o m a t o g r a p h y
1. Characterization of P e a k Position
In order to provide an accurate and reproducible parameter for
characterizing peak position on a chromatograph, Dalgleish et al., 16 in-
cluded consecutive, even-numbered straight-chain hydrocarbons in the
solutions containing the derivatives to he chromatographed. The posi-
tions of the hydrocarbon peaks were used as a reference for determining
the exact position of the peaks from the derivatives studied. Under the
conditions used--programming the temperature at a rate of 2 ° per
minute and using F-60 as the stationary phase--the hydrocarbons were
eluted at intervals that were close to lineal'. Assuming lille'u'ity between
any two adjacent peaks arising from the hydrocarbons which differed by
2 in their number of carbon atoms, the peak position of an unknown
substance was determined as follows: If the distance between the peaks
of hydrocarbons Cn and C.÷2 is x cm, and the distance from hydrocarbon
peak C. to the peak being measured is y cm, the methylene unit value
defined by Dalgleish et al., 16 for the compound is n ~ (2 y / x ) . If the
emergence of the hydrocarbons does not occur at intervals that are near
linear, then the methylene units should be interpolated graphically from
the curve showing the temperature of emergence (or alternatively the
retention time) of the hydrocarbons plotted against hydrocarbon chain
length.
2. I d e n t i t y o] Substance E l u t e d
0nly one group of investigators TM has attempted to confirm the
structure of substances eluted from the gas chromatography column.
Dalgleish et al., ~6 accomplished this by means of coupling a mass
spectrometer which was equipped with a gas chromatographic inlet sys-
tem, thus permitting the direct connection of the gas chromatographic
column to the mass spectrometer to study the structure of many sub-
stances.
Using the mass spectrometer, the kinetics of the rate of formation
of products of the reaction between citric acid and silanizing compounds
was studied; when citric acid and hexamethylsilane alone reacted at 65 °,
the reaction was complete in 35 hours, whereas in the presence of pyridine
and trimethylchlorosilane it was complete within 5 minutes.
Application to Biological Materials
Extraction from biological materials of acids of the citric acid cycle,
and their subsequent determination by gas liquid chromatography, has
only been attempted crudely to date. In no case has the determination
been duplicated by a recognized, reliable method. Ion-exchange chroma-
tography has been used prior to preparation of suitable derivatives for
gas chromatography by Quin et al., 1 in cigarette smoke, Lessard et al. 2
and Kowala et al2 from suint, and Canvin ~2 in plant~ material. The
recovery of the acids studied, added to the starting material, was not
reported by the authors.
Rumsey et al., ~° dried a sample of forage and lyophilized rumen
samples before directly carrying out their esterification procedure; quali-
tative results only were attempted. Mirocha and DeVay 3 used air-dried
fungus culture for the reaction with ethanol and sulfuric acid. The re-
covery of fumaric acid, the only member studied in detail, when added to
the culture was 102-104%. Since in the absence of culture the efficiency
[62] PARTITION COLUMN CHROMATOGRAPHY 415
of the esterification procedure was only 78%, some added catalytic effect
seems to have increased the efficiency.
The procedure described by Dalgleish et al., 16 in which human urine
and rat urine were extracted with ethyl acetate following treatment with
sodium chloride and hydrochloric acid would extract only a portion of
hydrophilic substances such as citric acid, and is therefore unsuitable.
Alcock (unpublished data) added citric acid, succinic acid, and
fumaric acid to 25 ml of human urine, the pH of which had been ad-
justed to 2 with N H2S04. Margaric acid was added in known quantity.
The water was removed by lyophilization. To the dried residue 5 ml of
methanol borontrifluoride (Applied Science Laboratory, State College,
Pennsylvania) was added and the mixture was transferred quantitatively
to a 50 ml centrifuge tube. After centrifugation, the supernatant and two
subsequent washings of the residue (using methanol borontrifluoride)
were allowed to stand overnight. The mixture was diluted with an equal
volume of water and then extracted three times with chloroform. Excess
chloroform was evaporated in a stream of dry nitrogen and the samples
were used for gas chromatography. The recovery of the three acids by
this procedure was consistently between 95 and I00% of that obtained by
direct esterification of the acids.
[62 ] S e p a r a t i o n of C i t r i c A c i d C y c l e a n d R e l a t e d C o m p o u n d s
by Partition Column Chromatography
B y LEo KBSNER and EDWARDMUNTWYLER
Principle
The procedure is essentially that previously described 1 and combines
many of the features of other partition chromatographic systems designed
for the estimation of organic acids? The use of precision pumps, longer
columns, and "indicator titration" provide improved resolution, increased
sensitivity, and continuous, automatic assay. Complex mixtures of acids
ranging from 0.05 to 3 micromoles per acid may be analyzed in less than
5 hours. Simple mixtures may be analyzed in less than an hour by
appropriate changes in gradient, column length, or pump speed.
A gradient elution system composed of chloroform and t-amyl alcohol
is deaerated and pumped through an acidified, hydrated silica gel column
at a uniform pump rate. The effluent is mixed with an excess of indicator
1L. Kesner and E. Muntwylcr, Anal. Chem. 38, 1164 (1966).
H. E. Swim and M. F. Utter, Vol. IV, p. 584.
of the esterification procedure was only 78%, some added catalytic effect
seems to have increased the efficiency.
The procedure described by Dalgleish et al., 16 in which human urine
and rat urine were extracted with ethyl acetate following treatment with
sodium chloride and hydrochloric acid would extract only a portion of
hydrophilic substances such as citric acid, and is therefore unsuitable.
Alcock (unpublished data) added citric acid, succinic acid, and
fumaric acid to 25 ml of human urine, the pH of which had been ad-
justed to 2 with N H2S04. Margaric acid was added in known quantity.
The water was removed by lyophilization. To the dried residue 5 ml of
methanol borontrifluoride (Applied Science Laboratory, State College,
Pennsylvania) was added and the mixture was transferred quantitatively
to a 50 ml centrifuge tube. After centrifugation, the supernatant and two
subsequent washings of the residue (using methanol borontrifluoride)
were allowed to stand overnight. The mixture was diluted with an equal
volume of water and then extracted three times with chloroform. Excess
chloroform was evaporated in a stream of dry nitrogen and the samples
were used for gas chromatography. The recovery of the three acids by
this procedure was consistently between 95 and I00% of that obtained by
direct esterification of the acids.
[62 ] S e p a r a t i o n of C i t r i c A c i d C y c l e a n d R e l a t e d C o m p o u n d s
by Partition Column Chromatography
B y LEo KBSNER and EDWARDMUNTWYLER
Principle
The procedure is essentially that previously described 1 and combines
many of the features of other partition chromatographic systems designed
for the estimation of organic acids? The use of precision pumps, longer
columns, and "indicator titration" provide improved resolution, increased
sensitivity, and continuous, automatic assay. Complex mixtures of acids
ranging from 0.05 to 3 micromoles per acid may be analyzed in less than
5 hours. Simple mixtures may be analyzed in less than an hour by
appropriate changes in gradient, column length, or pump speed.
A gradient elution system composed of chloroform and t-amyl alcohol
is deaerated and pumped through an acidified, hydrated silica gel column
at a uniform pump rate. The effluent is mixed with an excess of indicator
1L. Kesner and E. Muntwylcr, Anal. Chem. 38, 1164 (1966).
H. E. Swim and M. F. Utter, Vol. IV, p. 584.
416 S:~.rA~ATION AND ASSAY METHODS [62]
salt dissolved in a suitable solvent and delivered at a constant rate by a

second pump. As acid appears, an equivalent amount of indicator salt is
converted to its hydrogen form and the acid is neutralized.
l~NaIn -}- xHA --~ xNaA ~ xHIn + (n - x) NaIn
The absorbance of the resulting solution is continuously monitored
by a recording photometer at a wavelength chosen to detect the hydrogen
form of the indicator. Continuous ultraviolet and radioactive detection
may be included when desired. 1
Materials and Methods
Reagents
Silica gel [Mallinckrodt, Acid Silicie A. R., 100 mesh (powd.)],
suitable for chromatographic analysis by the method of Ramsay
and Patterson, is dried to constant weight at 110 ° and stored in
a desiccator in a plastic-lined screw-cap jar.
Chloroform ("Baker Analyzed" reagent) is used directly.
Tertiary amyl alcohol (practical grade tert-pentyl alcohol, 100 °-
103 °, Distillation Products Industries, Division of Eastman
Kodak Co., Rochester, New York) is redistilled in an all-glass
apparatus. The distillate is collected over a 1° temperature range.
Ethanol, 957~ is redistiIled over sodium hydroxide.
Indicator solution. One gram of o-nitrophenol sodium salt (Distil-
lation Products Industries, practical) is dissolved in 2 liters of
freshly boiled ethanol with heat and stirring. The solution is
stored in a brown glass bottle protected from carbon dioxide by
a soda-lime tube.
Apparatus
Acid analyzer. The basic instrument for automatic organic acid
analysis as illustrated in Fig. 1 consists of a gradient producing
device, a glass and Teflon Varigrad, 8 2 pumps, ~ a small magnetic
stirring device for rapid mixing of components, and a recording
photometer. The indicator solution is delivered at a rate of 30 ml
per hour, and the solvent pump for column operation is run at 200
ml per hour under the standard working conditions described. It
is preferable to have pumps whose delivery rate can be varied to
allow for changes in experimental conditions and for application
Buchler Instruments, Fort Lee, New Jersey, Model 4001.
' Milton Roy Co., 1300 East Mermaid Lane, Philadelphia, Pennsylvania: CHMMI-
B57 for solvents and CHMM-1-B29 for indicator.
to different photometric instruments. An instrument containing

most of the components described is now commercially available?
Chromatographic columns. A 9 X 500 m m column 6 with Teflon
joint connectors is convenient. These columns are less expensive
and easier to clean t h a n the ball and socket columns previously
described. 1
Fluid connections. All connections between tubing of Teflon and
glass or metal are made with Swagelok 7 fittings. P u m p outlet to
VARIGRAD
~tOwasleorfraction ~ ~_~
Fro. 1. An automatic organic acid analyzer with module for determining the
ultraviolet absorbing peaks.
¼6 inch i.d. flexible tubing of Teflon s requires a 200-1-2-316

fitting. P u m p inlet to tubing of Teflon utilizes a 200-2-2-316
fitting. Tubing of Teflon to glass connections are at both ends of
the magnetic stirrer mixing vessel, at the connector below the
column and at the connector on the top of the column. These
are made with 400-6-2-316 reducing unions utilizing a ferrule of
Waters Associates, 61 Fountain St., Framingham, Massachusetts.
' Fischer and Porter Co., Warminster, Pennsylvania: column No. 274-738, fitting
274-761, seal 275-305, disk 275-263, O rings 571-158. Fitting ends may be ground to
fit Swagelok connectors or a short piece of 6 mm o.d. glas~ tubing can be fused
on the end.
'Crawford Fitting Co., 884 E. 140 St., Cleveland, Ohio.
' Pennsylvania Fluorocarbon Co., Clifton Heights, Pennsylvania.
418 SEPARATION" A N D ASSAY METHODS [52]
Teflon on the ¼-inch side. A pressure gauge9 is inserted between

the pump and column utilizing two Swagelok 200-2-4-316 fittings.
The tee connection joining the column effluent and the indicator
stream is a 200-3-316 fitting.
Mixing chamber. The mixing chamber is made from a 6-cm length
of 6 mm o.d. tubing. A flattened bulb is blown into the center of
the tube with a diameter sufficient to allow free rotation of a
3~. X 1/~-ineh magnetic stirring bar coated with Teflon. The total
volume of the chamber is about 1 ml. Rapid mixing, at least 800
rpm, is essential for smooth baselines.
Recording photometers. Any stable recording photometer system
may be used which is capable of reading at 350 m~. The Beckman
DB spectrophotometer, TM Technicon Phototube colorimeter, n and
Waters recording colorimeter5 have been found suitable. For
ultraviolet recordings at 280 m~, the Beckman DB and Waters
photometer can be used.
Recorder. A multipoint recorder is convenient for the estimation of
area measurements, but any 0-10 mV single pen recorder whose
input is compatible with the photometer output may be employed.
For the Beckman DB and Waters instruments, a BristoP 2 re-
corder is used.
Timer. A 15-hour timer 13 serves to turn off equipment on runs that
extend after working hours. Several multioutlet boxes are con-
nected through the clock to accommodate all the equipment.
Ultraviolet Absorption. It is often important to determine whether

the acidic substances being measured absorb in the ultraviolet, as many
phenolic and heterocyclic acids absorb strongly in the 260-280 rn~ region.
Ultraviolet absorbing peaks are revealed by passing the effluent from the
column through a suitable detector prior to mixing with the indicator.
A variable resistance connected across the terminals of the ultraviolet
photometer permits simple adjustment of the output voltage so that it
may be balanced with the output from the second photometer, allowing
both to be displayed on the same chart by means of a multipoint recorder.
'Industrial Gauge and Instrument Co., 1407 E. 180 St., New York, New York:
P. 500, 2½ inch-range 0-200 lb., 1A-inch bottom, H-inch flushing connection, ~-inch
process connection, 316 stainless housing complete with M and G type seal.
~0Beckman Instruments, Inc., Fullerton, California.
11Teehnicon Chromatography Corp., Chauncey, New York.
12Bristol Co., Waterbury, Connecticut: Model 4P12HllX571, 4 point multirange
recorder.
12Dimco-Gray Co., 207 E. 6 St., Dayton, Ohio: 15 hour timer with 3 blade parallel
plug.
Continuous Counting o] Radioactive Materials ]rom Column Effluent.

The presence of organic solvents precludes the use of commercially
available flow counting scintillation systems. Organic solvents have
quenching effects and may dissolve the cell housing and scintillating
materials. To avoid these difficulties a fraction of the effluent stream may
be withdrawn by a pump, usually at a maximum rate of 30 ml per hour.
This is applied to a moving strip of ll/2-ineh Whatman No. 4 filter paper
which is transported past the point of application at a rate of 12 inches
per hour. The drive mechanism is part of the Actigraph II, model 1026, TM
4= strip counter. The solvent is rapidly evaporated from the paper by a
heater-blower located directly below the point of application of solvent.
The paper strip is correctly positioned by passing it through a brass guide
fastened to the top of the blower. The heater-blower is constructed from
a hair dryer adapted with special wiring for a 75-volt heating element
whose temperature is controlled by a variable transformer.
Procedure
Solvent System. Although a variety of solvents and gradient devices
have been used with some measure of success, the Varigrad has been
found to be most convenient. It is also important to deaerate the solvents
to prevent column disruption and to obtain exact metering by the pumps.
This can be accomplished by heating the solvent in chamber 1 to a few
degrees below the boiling point of the solvent mixture. A silicone-rubber
insulated heating tape 15 is wrapped around the bottom third of chamber
1 and is held in place by adhesive tape coated with Teflon? 6 The tem-
perature is controlled by a variable transformer.
For complex mixtures of acids such as those found in physiological
fluids, a 5-chamber concave gradient is generated. Chamber 1 is filled
with 200 ml of chloroform. Chambers 2 and 3 contain 7% (v/v) t-amyl
alcohol-chloroform, chamber 4 contains 30% (v/v) t-amyl alcohol-
chloroform, and chamber 5 contains 50% (v/v) t-amyl alcohol-chloro-
form. The volume of solvents used in chambers 2 through 5 is determined
by the density of their solvent mixtures. A volume equal in weight to the
200 ml of chloroform in chamber 1 is placed in these latter chambers.
This gradient was suitable for a 42 cm column of hydrated silica gel
(5.0:9.2 v/w) with an input volume of 200 ml per hour. Less complex
mixtures may be separated at faster rates, simpler gradients and shorter
'* Nuclear Chicago, Des Plaines, Illinois.
1~Gins-Col Apparatus Co., Terre Haute, Indiana: A 70 volt, ½-inch X 6 feet heating
tape.
~Connecticut Hard Rubber Co., New ttaven, Connecticut: Temp-R-Tape t-18,
1A-inch.
420 SEPARATIO~ AND ASSAY METHODS [52]
columns being used. For example, propionie, acetic, and formic acids can
be determined in less than 40 minutes with a 25 em column, a 2 chamber
gradient composed of 200 ml of chloroform and 7 ~ (v/v) t-amyl alcohol-
chloroform, and a pump speed of 400 ml per hour.
Column Preparation. The hydration of the silica gel is a critical factor
in the separation of certain organic acids. Although organic acid separa-
tions using chloroform and t-amyl alcohol are not as sensitive to changes
in hydration as are DNP-amino acids, 17 variations in hydration can
influence relative elution of acids. Thus, when the hydration of the silica
gel is decreased, the positions of the hydroxyl-bearing carboxylic acids
fl-hydroxybutyrie, lactic, malic, and citric acids, are shifted to a greater
extent than the other acids.
To 92 g of oven-dried silica gel in a screw-cap jar is added 50 ml of
0.1 N sulfuric acid. The mixture is stirred with a heavy glass rod until it
is lump free and the powder no longer adheres to the walls. Several small
glass rods are added to aid in dispersing the powder, and the cap is tightly
fitted in place. The jar is then rotated for 15 minutes on a ball mill.
For a 0.9 X 42 cm column, approximately 25 g of the hydrated gel is
used. The gel is mixed with approximately 40 ml of chloroform, and the
slurry is poured into a clean dry column up to the socket. The upper
connector is secured with a clamp, ~8 and chloroform is pumped through
until the level of the silica gel remains constant. The upper connector is
then removed, the excess chloroform above the silica gel is aspirated, and
the remainder of the slurry is poured in. The procedure is repeated until
a packed height of 42 cm is obtained.
Preparation of a column requires about 15 minutes and must be
performed for each run. Silica gel from the previous run is discarded by
removing the bottom connector and pumping chloroform through the
column until the used gel is extruded into a waste container. When a ball
and socket column is employed, the used gel is removed by inverting the
column over a sink and slowly passing a narrow plastic tube with
running water up through the gel. The column is rinsed with distilled
water followed by acetone and then air dried. The flow cell and indicator
mixing chamber are periodically rinsed with 50% ethanol and acetone to
remove material which sometimes deposits on their surfaces.
Sample Addition to Column. To prevent spreading of peaks, the
volume of aqueous solution containing the acids should not be greater
than 1 ml. The sample is first madd acid to Congo red by the addition of
1 or 2 drops of 6 N sulfuric acid. Enough oven-dried silica gel is then
added to make a free-flowing powder; each 0.5 ml of solution requires
1~L. Kesner, E. Muntwyler, G. E. Griffin, and P. Quaranta, Vol. XI [9].
,sThomas, Philadelphia, Pennsylvania: No. 18A screw clamp.
approximately 0.8 g of silica gel. The free-flowing powder is then quanti-

tatively added through a 3 cm layer of chloroform which covers the top
of the silica gel column. The sample is stirred with a glass rod to release
air bubbles and chloroform is added up to the socket. The connector is
placed in position and the pump is activated. Pump performance may be
evaluated by timing the collection of solution in a graduated cylinder as
it leaves the colorimeter. The collection rate is recorded.
Calibration. Calibration is undertaken once optimal conditions for
separation have been established for the substances being investigated.
Stock solutions of acids are prepared in a 50% (v/v) acetone-water
mixture in concentrations of approximately 0.01N. From the stock
solutions a mixture of acids is prepared by combining aliquots of 1 to
10 ml each acid and diluting to a known volume, usually 100 ml. To
standardize each acid in the mixture, a sample equal to the amount trans-
ferred, using the same pipette, is titrated. This minimizes pipetting errors
caused from poor draining which is characteristic of nonaqueous solu-
tions. The stock acids are titrated to a phenolphthalein end point with
0.01 N sodium hydroxide using a stream of nitrogen for stirring. It has
been our experience that calibration on a weight basis is less reliable
than standardization by titration. If an acid is not obtainable in a pm'e
free form, its sodium salt may be used. The salt can be titrated poten-
tiometrically with a platinum silver-silver chloride electrode pair. One
milliliter of a 0.1 N aqueous soIution of the salt is diluted to 25 ml with
glacial acetic acid and the mixture is titrated with 1.0 N perchloric acid
in glacial acetic acid. Potassium hydrogen phthalate serves as the refer-
ence standard for both acid and salt titrations.
Known mixtures of carboxylic acids are analyzed in the concentration
range 0.05-3 microequivalents per acid. The area under the peaks is
obtained by multiplying the net height of a peak in absorbance units by
the width at half the peak height. A calibration constant for each acid is
thus obtained. To determine the area of a curve over a sloping baseline, a
line is drawn which follows the baseline slope under the peak. A general-
ized baseline connecting several low points on the curve should be drawn
across the entire length of the chromatogram. This is because valleys
between certain peaks may not completely descend to the baseline. A
vertical line from the highest point of the peak to the drawn baseline
represents the net height. The line at one half the net height is made
parallel to the chart abscissa, not parallel to the drawn baseline.
In a static system containing uniform concentration of chloroform,
alcohol, and indicator, it is possible to equate changes in 'tbsorbance per
microequivalent of acid regardless of whether the carboxylic grou]) is
contributed by a mono-, di-, ov tricarboxylic acid. Itowever, in :t flowil~g
422 SEPA.I~ATION AND ASSAY METHODS [62]
•.~ ~.o
Z)II~/W .- . ~ ,..4 ~
o .~ ' ~ ;.~ o
~ .= ~ ..-
:)IZlVI m 001 :Di" ""---

~ "~ ~ TM ;~
o
,,.~"~ o ~ .~
•~ ~ ~_,,
~, ~ . ~ ~'~
.,~ ~ ~ ~ -- .~
~ . ~o o ~
l
"" .0
~, ~, .~ ~- ~
[62] PARTITION COLUMN CtIROMATOGRAPHY 423
system, where there is a continuous change in the composition of the

solvent, a uniform calibration constant in terms of absorbance units per
microequivalent of acid is not possible. This is due in part to a change in
the spectral characteristics of the indicator with changes in solvent com-
position. Consequently, calibration constants are not uniform for all
acids and are dependent upon the position of the curve at which they
are eluted. A second effect arising from this phenomenon is a downward
sloping baseline early in the run and an upward sloping baseline near
the end of the run. The latter is also due in part to the leaching of small
amounts of sulfuric acid from the column near the end of the run. How-
ever, the sloping baselines do not introduce serious errors in the calcuta~
tions and are relatively constant as long as the procedure is held constant.
After the elution of the weak organic acids, the absorbances rises
markedly as sulfuric acid in large concentrations now is eluted from the
column. During the course of a run the top of the column becomes trans-
lucent as the hydration of the silica gel is changed. The sulfuric acid
comes off as the translucent area reaches the bottom of the column.
Variations in gradient, silica gel preparation, flow rates, or column
height may influence the calibration factor. Hence it is important to
carry out the calibration after these variables have been standardized
and thereafter adhere strictly to the same procedure. It should be noted
that the standard organic acids prepared in acetone and water deteriorate
on standing. For example, a standard mixture stored at 4 ° for 5 month.~
exhibited a loss of 25% of c/s-aconitic and malic acids; a loss of 15-20%
of citric, fl-hydroxybutyric, and pyruvic acids; and a loss of 5% or less
of fumaric, glutaric, lactic, succinic, and ~-ketoglutaric acids.
Elution Pattern of Organic Acids. Figure 2 is a representation of the
etution pattern of a number of acids which can be detected with the
o-nitrophenol indicator titration system. The position and identity of
some major metabolic acids is displayed directly on the curve whereas
the numbers at the top of the diagram indicate where other acids of
biochemical interest appear.
Oxaloacetic acid is not sufficiently stable to be chromatographed on
columns containing 0.1 N H_~S04 as the stationary phase. In the presence
of 6 N H:S04 oxaloacetic acid is relatively stable and is elutcd, but
under these conditions there is a high blank, and indicator titratio~
cannot be used for the complete run. 0xaloacetic acid shows absorption
in the ultraviolet region permitting ready detection when cluted from
the column, trans-Aconitic acid is always found in samples of cis-aconitic
acid.
When unknown mixtures are chromatographed there is a possibility
that peak effluent volumes may overlap with one of the known acids.
Irregularities in the symmetry of the peak may be sufficient to designate

this. However, when this situation is suspected, the eluate corresponding
to the peak can be collected and its purity confirmed by paper chromatog-
raphy in a variety of developing solvents. In addition, improved resolu-
tion may be obtained by varying the hydration of the silica gel or by
changing the length of the column, the temperature, and the gradient.
Chemical characterization may involve the use of earbonyl reaction
reagents such as 2,4-dinitrophenylhydrazine or oxidizing agents such as
permanganate or ceric sulfate. Since only specific structures are attacked
by these reagents, 2 analysis of the products of such a reaction may
provide information useful in identifying the structure of the original
compound.
Analysis o] Biological Fluids and Tissues. In determining the acid
content of actively metabolizing tissue, two major problems are en-
countered. Alterations due to metabolic transformations have been
shown to occur with extreme rapidity after removal from the body. In
addition, the chemical stability of certain acids during extraction, storage,
and separation must be considered once the metabolic processes have
been terminated. The contribution of acids from extracellular fluids
trapped in tissues can be a considerable source of error under some cir-
cumstances. Certain metabolically inert acids rapidly appear in the
urine and plasma after they are eaten or administered. Thus trans-
aconitic acid as well as several unidentified acids appear in large amom,~s
in the urine obtained h'om rats on the standard checker diet in contrast
to urine from rats fed a synthetic casein diet. When D-malic acid is
administered to rats, it promptly appears in the urine whereas the
L-isomer is metabolized and is not excreted.
No preliminary extraction or concentration of urine or plasma is
required prior to analysis; 0.2 to 0.5 ml of acidified urine is usually
required for a complete analysis. Because of the small amount of urine
needed for a determination, it is possible to follow the pattern of acid
excretion in individual rats by analyzing each sample of urine voided. 19
Protein containing fluids such as plasma and homogenates are analyzed
in a similar manner. A 0.5 or 1.0 ml sample is acidified with 6 N sulfuric
acid. Dry silica gel is added and the mixture is stirred until free flowing.
The entire mixture is added to the top of the column. No prior protein
precipitation is required.
Whole tissues such as liver, muscle and kidney are treated in an
analogous manner except that acidification and mixing is done in a
ceramic mortar to ensure disruption of cells and uniform distribution of
the intracellular material. The tissue should be instantly frozen, weighed,
'*L. Kesner, ]. Biol. Chem. 240, 1722 (1965).
[63] ION-EXCHANGE CHROMATOGRAPHY 425
and then ground to ensure against metabolic alteration of acid con-

stituents.
Sulfuric acid is used in preference to other acids such as trichloro-
acetic or perchloric acid where it is necessary to terminate a biochemical
reaction. Most of the commonly used protein precipitating reagents
appear as peaks on the chromatogram and will swamp a particular area
of analysis. The pH of the sample is adjusted to below 2 to keep the
acids in a nonionized form. Due to the increased sensitivity of the
method, the requirement for preliminary extraction and concentration is
unnecessary in most instances. Hence better yield, fewer artifacts, and
smaller losses from volatilization or polymerization of certain sub-
stances are obtained.
Sources of Error. Limitations in reproducibility are the result of a
number of factors, of which the calculation of the peak area may be of
considerable magnitude. The height-width method for determining area
is the most convenient procedure when utilizing a nonlinear absorbance
photometer. Errors may also arise from lack of photometric stability or
potentiometer reproducibility and deviations in the pump rates of the
two or three pumps used. When biological specimens are to be evaluated,
it is of utmost importance that the investigator be aware of potential
losses during the sample preparation.
[63] I o n - E x c h a n g e C h r o m a t o g r a p h y of C i t r i c A c i d
Cycle Components and Related Compounds
B y R. W. VON KORFF
Ion-exchange chromatography of citric acid cycle components and

related compounds was first applied to biochemical studies by Busch
et al., 1 who used Dowex 1 in the formate cycle and a formic acid gradient
to elute organic acids. The variants ~,a of this procedure include use of
ammonium formate-formic acid 4 and acetate columns with an acetic
acid gradient? ,6
These procedures are used widely to separate appreciable amounts of
H. Busch, R. B. Hurlbert, and V. R. Potter, J. Biol. Chem. 196, 717 (1952).

A. W. Norman and H. F. De Luca, Bioehem. J. 91, 124 (1964).
' G. R. Bartlett, J. Biol. Chem. 234, 459 (1959).
' J. L. Gamble, J. Biol. Chem. 240, 2668 (1965).
BIt. Busch, Cancer Res. 13, 789 (1953).
• See Vol. III [70].
and then ground to ensure against metabolic alteration of acid con-

stituents.
Sulfuric acid is used in preference to other acids such as trichloro-
acetic or perchloric acid where it is necessary to terminate a biochemical
reaction. Most of the commonly used protein precipitating reagents
appear as peaks on the chromatogram and will swamp a particular area
of analysis. The pH of the sample is adjusted to below 2 to keep the
acids in a nonionized form. Due to the increased sensitivity of the
method, the requirement for preliminary extraction and concentration is
unnecessary in most instances. Hence better yield, fewer artifacts, and
smaller losses from volatilization or polymerization of certain sub-
stances are obtained.
Sources of Error. Limitations in reproducibility are the result of a
number of factors, of which the calculation of the peak area may be of
considerable magnitude. The height-width method for determining area
is the most convenient procedure when utilizing a nonlinear absorbance
photometer. Errors may also arise from lack of photometric stability or
potentiometer reproducibility and deviations in the pump rates of the
two or three pumps used. When biological specimens are to be evaluated,
it is of utmost importance that the investigator be aware of potential
losses during the sample preparation.
[63] I o n - E x c h a n g e C h r o m a t o g r a p h y of C i t r i c A c i d
Cycle Components and Related Compounds
B y R. W. VON KORFF
Ion-exchange chromatography of citric acid cycle components and

related compounds was first applied to biochemical studies by Busch
et al., 1 who used Dowex 1 in the formate cycle and a formic acid gradient
to elute organic acids. The variants ~,a of this procedure include use of
ammonium formate-formic acid 4 and acetate columns with an acetic
acid gradient? ,6
These procedures are used widely to separate appreciable amounts of
H. Busch, R. B. Hurlbert, and V. R. Potter, J. Biol. Chem. 196, 717 (1952).

A. W. Norman and H. F. De Luca, Bioehem. J. 91, 124 (1964).
' G. R. Bartlett, J. Biol. Chem. 234, 459 (1959).
' J. L. Gamble, J. Biol. Chem. 240, 2668 (1965).
BIt. Busch, Cancer Res. 13, 789 (1953).
• See Vol. III [70].
organic acids (5-200 micromoles).1 The removal of large amounts of

formic or acetic acids, or their ammonium salts, is time-consuming, and
it is not well adapted for either the separation of nanomole quantities of
citric acid cycle components or the simultaneous monitoring of radio-
activity where isotopic acids are employed.
Bartlett et al./used Dowex 1 (CI-) with HC1 as the eluting agent to
chromatograph carbohydrate intermediates and related cofactors of
erythrocytes. They observed that HC1 facilitates recovery of compounds
by evaporation, rechromatography after neutralization and dilution, and
sampling for 14C radioactivity assay on "infinitely thin" planchets.
The use of Dowex 1 (C1-) columns with an HC1 gradient offers
several advantages:
1. Washed analytical grade resin in the (Cl:) cycle is readily avail-
able.
2. The organic acids, in general, are most stable in acid solution, and
the eluates may be stored frozen with little decomposition.
3. The column eluate may be passed through a scintillation flow cell
for simultaneous monitoring of radioactivity.
4. Fractions containing volatile acids, e.g., acetate, lactate, or pyru-
vate, may be neutralized to pH 8--9, without introduction of
excessive amounts of salt, and concentrated to dryness.
However, quantities of organic acids greatly in excess of 5 micromoles
of individual components may alter the pH and lead to peak distortion
and displacement or both. For example, succinate and malate, separated
readily in amounts up to 5 mieromoles, merge as the amounts increase;
the succinate region increases in size, while malate decreases as the acids
apparently interact.
Complete separation of acids eluting in closely adjacent regions is
also a function of the relative amounts of the two compounds. While two
acids may elute in separate peaks in a ratio of 5A to 1B (or 1A to 1B)
resolution may be poor at a ratio of 1A to 5B. The procedure may be
modified for particular situations but is most generally useful in separat-
ing trace amounts of radioactive materials.
Chromatographic Procedure. Dowex l-X8 (C1-), 200--400 mesh (ana-
lytical grade) 8 is washed with water to remove impurities and acid.
After washings reach pH 4.5, the resin suspension in water is neutralized
to pH 6.8, and stored as a slurry. After about one month the resin should
be rewashed and neutralized.
G. R. Bartlett, E. Savage, L. Hughes, and A. R. Marlow, J. Applied Physiol. 6,
51 (1953).
Dowex I-X10 does not appear to offer any advantages over the X8 resin.
A colunm (1 X 17 cm) is formed by pouring water-suspended resin

into a tube (1 X 30 cm) having a sintered disk, a 12/2 ball of a (S~)
joint at the base and an 18/9 socket at the top. Flow can be controlled by
a Teflon needle valve (Manostat Corporation, New York City) having
a 12/2 socket (Sj) at one end and a drip tip at the other. About 2 mm
of water should remain above the resin.
Add 1-2 ml of organic acid sample, 9 pH 6.8-7.2, to the resin bed using
a long-tip pipette, and drain it into the resin. Wash the sample container
with about 1 ml of water, drain it into the resin. Close the stopcock until
the sample is eluted. First, neutralize perchloric acid filtrates 1° to pH 6.8
with 4 N KOH, and keep at 0 ° until the KC10~ has settled; then add
the fluid to the column and wash the KC1Q once with cold water.
For elution of the acids, fill the column with water and attach a 250-
ml Erlenmeyer flask with a 35/20 socket (S~) and a 12 cm side arm
ending in a right-angle with an 18/9 ball (Sj) ; fill the flask with water,
add a stirring bar, and mount above a magnetic stirrer. At the upper
end of the flask attach a 35/20 ball (Sj) through which a 5 mm tube
extends to within 1 inch of the bottom. If radioactivity is monitored with
a scintillation flow cell, connect this tube to a micropump (Buchler Instru-
ments, Inc., Fort Lee, New Jersey) to supply a constant rate of flow.
Remove the stopcock from the lower end of the column, attach a 12/2
socket (S~), in turn attached to Teflon tubing passing to a scintillation
flow cell assembly. To begin elution, connect the pump to a source of
0.05 N HC1. After 50 fractions of 2 ml have been collected, connect the
pump to a source of 0.10 N HC1. The approximate positions of a number
of organic and amino acids chromatographed with this system are listed
in the table. Oxaloacetic acid cannot be recovered from Dowex 1 as it
undergoes decomposition on the basic nitrogenous resin.
If the sample is nonradioactive, or if fractions are to be counted
individually, gravity flow from a separatory funnel extending to within
1 inch of the bottom of the mixing flask may be used in place of the
micropump assembly. Fractions may be collected directly into tubes
using the elution schedule described above.
Where certain acids elute together (overlap), chemical reduction
permits a rapid second separation using the same chromatographic sys-
tem. For example, citrate and pyruvate overlap, while suecinate and
acetoacetate, and fumarate and a-ketoglutarate elute as single peaks. In
Samples at high salt concentration should be diluted to reduce the salts to less
than 0.15M. At higher initial salt concentrations, acids may be partially eluted in
abnormal postions in the ehromatogram.
loWhen 14C0, is present, the perchloric acid solution should first be treated with
pieces of dry ice to displace the radioactive C01.
PROFILE OF ORGANICACID ELUTIONFROMDOWEX 1-(XS) (C1-) COLUMNS

USING AN H C I GRADIENT
Approximate volume
at peak
Acid (ml)
Alanine and other monoamino 8

monocarboxylic acids
COs, aspartate, glutamate, 46
B-hydroxybutyrate, acetate
Lactate 52
Butyrate" 56
~-Methyl-fl-hydroxyglutarate 62
Suceinate, acetoacetate 66
a-Hydroxyglutarate 72
Glyoxylate 80
Malate 86
Isocitrate 108
Citrate 130
Pyruvate, malonate 150
Fumarate, a-ketoglutarate 204
a Unsubstituted acids of greater chain length than C-4 do not elute from the Dowex 1
columns. These may be separated on silicic acid columns.
each case the combined fraction containing the compounds to be sepa-

rated is neutralized to p H 6-7 and treated with 1 to 2 mg of sodium
borohydride to reduce the keto acids? 1 After acidification to destroy
excess borohydride and neutralization to p H 7, the sample is placed on a
Dowex 1 column and rerun as described previously. The reduced samples
containing citrate and lactate, succinate and fl-hydroxybutyrate, and
fumarate and a-hydroxyglutarate are separated easily by chromatog-
raphy on Dowex 1 with an HCI gradient.
To date, a separation of acetate, fl-hydroxybutyrate, glutamate, and
aspartate has not been effected with the chloride system. Lactate follows
these compounds and m a y overlap partially. I t m a y be separated com-
pletely, however, by using 0.01 N HC1 to form the initial gradient. In
this case, subsequent peaks, caused by a gradual acidity gradient, m a y
be broader and appear later. After elution of lactate, change the HC1
from 0.01 N to 0.05 N. The Bessman TM Gradient Elution Device (National
Instruments Laboratories, Inc., Rockville, Maryland) is useful for fur-
ther experimentation with different gradients.
Separation o] Acetate plus fl-Hydroxybutyrate and Glutamate plus
11In the case of acetoa~etate, this should be done as soon as possible after chromatog-
raphy to avoid possible losses.
uS. P. Bessman, Anal. Biochem. 18, 256 (1967).
Aspartate. The peak containing these acids is neutralized to pH 7 and

passed through a 1 X 3 cm column of Dowex 50-X8, 200-400 mesh (ana-
lytical grade). Acetate and fl-hydroxybutyrate are not held on the column.
Wash the column with 5-6 times the volume of sample, usually not less
than 40 ml. The eluate is then neutralized; if radioactivity is to be deter-
mined, aliquots are taken for counting to determine the total activity
present. Concentrate the remainder of the sample to dryness on a rotat-
ing evaporator. The acetate and fl-hydroxybutyrate may be separated on
a silicic acid column using a modification of the procedure of Bulen et
al.13-15
Wash silicic acid (Mallinckrodt Chemical Works), 100 mesh (labeled
"suitable for chromatographic analyses by the method of Ramsey and
Peterson"), to remove fines, as described by Bulen, et aI. Dry the washed
material thoroughly at 105 ° and prepare the stationary phase as de-
scribed by Kinnory et al. TM Mix silicic acid, 8.8 g, in a mortar with 5.4
ml of 0.05 N H~SQ until the powder is finely divided. Then wash chloro-
form in a separatory funnel with a small amount of 0.05 N H~SO, and
pass the chloroform layer through dry filter paper to remove water drop-
lets. Bring the silicic acid to a slurry with chloroform in a beaker and
pour it into a column (1.4 X 35 cm) that has a small glass-wool plug, a
stopcock at the lower end, and a 24/40 joint at the top. Acidify the
neutralized sample, not over 0.6 ml in volume, with 0.05 ml of 3 N
H~S04. Very small amounts of radioactive acetate and fl-hydroxybuty-
rate may be determined by adding 5-10 micromoles of nonradioactive
carrier acids to aid in locating the peaks. The sample is mixed well with
1.0 g silicic acid and added as a chloroform slurry to the top of the silieic
acid column, drained previously until only 1-2 mm of chloroform remains
above the silicic acid. After the sample has settled, add to the column a
5-cm layer of chloroform and connect to a reservoir containing 250 ml of
acid-washed chloroform and a magnetic mixing bar. A separatory funnel
containing 1-butanol-chloroform, 40:60 (v/v), to which 1 ml of 0.05N
H2S04 has been added per 100 ml of mixed solvent, is connected to the
reservoir, and in turn mounted on a fraction collector and above a
magnetic mixer. Fractions (3 ml) are collected into tubes containing 1.0
ml of a neutralized methyl red indicator solution (a 1:20 dilution with
water of a 0.04% methyl red solution in ethanol). The diluted methyl red
should be neutralized to an orange color and tested for the highest sensi-
tivity to traces of acidity. Acids eluting from the column are detected
~*W. A. Bulen, J. E. Varner, and R. C. Burrell, Anal. Chem. 24, 187 (1952).
~'See Vol. III [64].
~R. W. Von Korff, J. Biol. Chem. 240, 1351 (1965).
~aD. S. Kinnory, Y. Takeda, and D. M. Greenberg, J. Biol. Chem. 212, 379 (1955).
readily, as 0.1 mieroequivalent of acidity per tube produces a red color.

The acids may be determined by-titration_with 2 X 10-8 N NaOH using
phenol red indicator (0.04~) and a Vortex mixer to extract all acid into
the aqueous layer during titration. To determine radioactive acetate and
fl-hydroxybutyrate, combine the tubes for a given peak, and, after extrac-
tion of the acid into the aqueous phase (previously made slightly alka-
line), count an aliquot to-determine the radioactivity present in the
fraction. The total activity in each acid may be calculated from the
percentage of activity found in each acid after chromatography multiplied
by the total counts in the organic acid fraction prior to chromatography.
Elute the glutamate and aspartate retained on the Dowex 50 column
during separation of the acetate and fl-hydroxybutyrate with six 2 ml
portions of 1 N HC1; concentrate and dissolve the residue in 1 ml of
water. When radioactive acids are present, determine total activity on
a small aliquot (0.05-0.10 ml). In earlier studies,15 aspartate and gluta-
mate were separated using a procedure of Moore and Stein 17 substituting
a 50-cm column for the 100-cm column used by Moore and Stein. The
elution volumes (aspartate approximately 40 ml and glutamate 70 ml)
are about one-half of those reported here for the 100 cm column. The
distribution of radioactivity was determined by passing the eluate through
a scintillation flow counter connected to a recorder. The fractional areas
under each peak give the percentage of activity in each acid.
In more recent work, thin-layer chromatography has been used as
recommended by Sch~ifer.TM The solvent system is EtOH-NH,OH, 70:30
(v/v), and slow developing plates TM should be used. Radioactive acids
may be counted in a thin-layer chromatogram scanner, or a control strip
using nonradioactive acids may be sprayed with ninhydrin to locate the
amino acids. In this system glutamate has a higher Rr value than
aspartate, whereas in the Moore and Stein system the acids are eluted
in the reverse order.
The techniques described in this report are useful in studying the
dynamic aspects of metabolism in mitochondrial systems. ~5,~°-24
~7S. Moore and W. H. Stein, J. Biol. Chem. 192, 663 (1951)

G. Sehiffer, personal communication.
Rapid developing plates do not yield separation of the two acids. We have found
the Brinkman F2s precoated plates to be satisfactory for this purpose.
M. S. Olson and R. W. Yon Korff, J. Biol. Chem. 242, 325 (1967).
21M. S. Olson and R. W. Von Korff, J. Biol. Chem. 242, 333 (1967).
"~C. Bauer and R. W. Yon Korff, Biochim. Biophys. Acta 131, 280 (1967).
**G. Seh~fer, P. Balde, and W. Lamprecht, Nature 214, 20 (1967).
**R. W. Von Korff, Nature 214, 23 (1967).
[64] T H I N - L A Y E R CHROMATOGRAPHY 431
[64] Thin-Layer Chromatography of Citric

Acid Cycle Compounds
By WILLIAM F. MYERS and Ku~-YE.'," HUANG
Principle. The citric acid cycle intermediates and some related amino
acids are separated by two-dimensional cellulose thin-layer chroma-
tography. After separation the compounds are visualized by spraying
with an acid-base indicator or ninhydrin. 1
Materials and Reagents
Thin-layer chromatography jars, suitable for 20 cm X 20 cm plates
Glass plates, 20 cm X 20 cm
Whatman crystalline cellulose powder, grade CC41 (W. R. Balston,
Ltd.)
Diethyl ether-formic acid (90%)-water (7:2:1).z The ether should
be anhydrous, and both ether and formic acid should be reagent
grade.
Phenol-water-formic acid (90%) (75:25:1). 3 The phenol and
formic acid should be reagent grade. If any discoloration of the
phenol is observed it should be redistilled2 Avoid phenol contain-
ing H3P02 as a preservative since the latter may interfere with
visualization of spots when an acid-base indicator is used. If
liquid phenol (90%) is used, the ratios are 83: 17:1.
Bromcresol green indicator. Dissolve 0.04 g of bromcresol green in
100 ml ethanol (96%). Add 0.1 N NaOH until a blue coloration
just appears. Other indicators which may be used include
bromphenol blue, chlorophenol red, or bromcresol purple. In each
case they should be adjusted slightly on the alkaline side of their
indicator range.
Ninhydrin reagent. Dissolve 0.25 g of ninhydrin in 100 ml of
acetone. Other ninhydrin formulations may be substituted.
Procedure. Cellulose plates of 250 t~ thickness are prepared by the
standard method recommended by Stahl? Since fumarie acid runs very
close to the solvent front in the first dimension, it may be advisable to
prewash the plates in the ether-formic acid-water solvent before spot-
'W. F. Myers and K. Y. Huang, Anal. Biochem. 17, 210, (1966).
2j. K. Palmer, Conn. Agr. Exptl. Sta. Bull. 589, 12 (1955).
sj. B. Stark, A. E. Goodban, and It. S. Owens, Anal. Chem. 23, 413 (1951).
~J. 0. Draper and A. L. Pollard, Science 109, 448 (1949).
E. Stahl, "Thin-Layer Chromatography." Academic Press, New York, 1965.
ting. However, if high-purity cellulose is used and the plates are made
freshly, this step may be omitted. The plates are loaded with 2.5 ~l of
a mixture of known compounds, each 10 mM, prepared in distilled water.
An appropriate volume of the carbon-14-1abeled biological extract may
then be spotted directly over the former. Spotting volumes should be
minimized by prior concentration of the sample to avoid a "doughnut"
effect.
The plates are subjected to two-dimensional development with
solvent 1 (ethyl ether-formic acid-water) and with solvent 2 (phenol-
water-formic acid). Each solvent front is developed 15 cm, approxi-
mately 1 hour being required for development in solvent 1, and 2 hours
for soh'ent 2. The plates are dried in a stream of air. Complete drying
requires an additional 1 and 3 hours, respectively, although this time
may be shortened if vacuum drying facilities are available. Since some
of the keto acids are labile, particularly oxaloacetic acid, it is not
advisable to employ heat to shorten drying time. The leading edge of the
solvent in the first dimension preferably is removed to minimize solvent
flow distortion in the second dimension.
For detection of the carboxylic acids the plates are sprayed with
bromcresol green indicator. If it is desired to visualize the related amino
acids, alanine, aspartic acid, glutamie acid, and glutamine, this may be
done easily by covering the amino acids before spraying with bromcresol
SEPARATION OF INTERMEDIATES OF THE CITRIC ACID
CYCLE AND RELATED COMPOUNDS
RI × 100
Compound Solvent 1~ Solvent 2 b
a-Ketoglutarate 70 59
Succinate 81 71
Fumarate 96 66
Malate 54 54
Oxaloacetato 82 54
Citrate 38 42
c/s-Aconitate 62 51
Isocitrate 38 39
Glutamine 14 61
Glutamate 18 50
Alanine 39 62
Aspartate 16 38
Pyruvate 88 73
° Ether: formic acid: water (7/2/1).

b Phenol: water: formic acid (75/25/1).
[64] THIN-LAYER CHROMATOGRAPHY 433
green, then the earboxylie acids are covered and the amino acids are
developed by spraying with ninhydrin. Alternatively, glutamic acid and
aspartic acid may be visualized with the acid-base indicator.
The RI values of the compounds tested are listed for each solvent in
the table and a diagram of the results of two dimensional chromatography
is presented in Fig. 1.
The separation of the eight intermediates of the citric acid cycle is
good with the exception of the citratc-isocitrate spots. If the initial spot
Q FurnarGte
/ 0 Pyruvate
O×aloacetate
0 ~JSuccinate
~J
or"
Off-Ketoglutarate
C/s-Aconi~0
tale Malate
LU
I
C3
L)
to
n-
O
Isocitrote~ Citrate0 hlanine
U-
i
n,"
LU
-r-
UJ
Glutamate
Aspartate0 ~ ~ Glutamine
i
...."
Origin
2. PHENOL-WATER-FORMI
&CID(75:25
C I~
Fro. 1. Thin-layer chromatogram of intermediates of citric acid cycle and related
compounds. The intermediates and pyruvic acid are detected by acid-base indicator,
and amino acids by ninhydrin reagent. The broken line divides carboxylic acids from
amino acids except alanine.
is kept small, separation of the latter two compounds is possible. There
is a partial overlapping between succinate and pyruvate, but separation
can be obtained by using solvent 1 in two dimensions.
Biological samples may be deproteinized prior to chromatography by
trichloroacetic acid or perchloric acid. The former may be removed by
evaporation and the latter removed by adding an equivalent amount of
KOH or KHCOs. Excessive amounts of salts, particularly Na +, may
cause tailing. Huang 6 experienced no difficulty in this regard, using a
diluent containing 0.1 M KC1, 15 mM NaC1, and 50 mM potassium phos-
e K. Y. Huang, J. Bacteriol. 93, 853 (1967).
434 SEPARATmN AND ASSAY METHODS [55]
phate, pH 7.0. The sample may, of course, be desalted, using ion

exchange techniques.
Since the compounds studied are normally present in only trace
amounts in most biological extracts, it is often necessary to use sub-
strates labeled with isotopes. In these cases the spots are identified with
the help of mixtures of unlabeled known compounds. Techniques for the
radioassay of the plates have been developed well. ~ This chromatographic
technique, including the usage of compounds labeled with ~4C, has been
adopted successfully in metabolic studies, s
7F. Snyder and N. Stephens, Anal. Chem. 4, 128 (1962).
[65] Assays of Intermediates of the Citric Acid Cycle and

Related Compounds by Fluorometric Enzyme Methods ~
By JOHNR. WILLIAMSON2 and BARBARAE. CORKEY
Introduction 435
Preparation of Samples 437
Tissues: Acid Extraction 437
Tissues: Alkaline Extraction 439
Mitochondria 439
Modifications of Extraction Procedures for Assays of CoA and Its Deriva-
tives 439
General Assay Techniques 440
Calculations and Expression of Results . 443
Spectrophotometric Standardization of Solutions 443
Expression of Results 444
Determination of the End Point of a Reaction in the Presence of Drift 444
Content of Citric Acid Cycle Intermediates in Various Tissues 445
Citrate 446
A. Determination with Aconitase and Isocitrate Dehydrogenase 446
B. Determination with Citrate Lyase and Malate Dehydrogenas~ 450
Isocitrate--Determination with Isocitrate Dehydrogenase 453
~-Ketoglutarate--Determination with Glutamate Dehydrogenase 455
Succinate--Determination with Succinate Thiokinase, Pyruvate Kinase and
Lactate Dehydrogenase . 458
Fumaratc--Determination with Fumarase and Malate Dehydrogcnasc~ 463
Malate---Determination with Malate Dehydrogenase 466
Oxaloacetate--Determination with Malate Dehydrogenase 468
Glutamate--Determination with Glutamate Dehydrogenase . 471
Aspartate--Determination with Glutamate-Oxaloacetate Transaminase amt
Malate Dehydrogenase 473
1Supported by grants from the U.S. Public Health Service (GM 12202-04) and the
American Heart Association.
Established Investigator of the American Heart Association.
phate, pH 7.0. The sample may, of course, be desalted, using ion

exchange techniques.
Since the compounds studied are normally present in only trace
amounts in most biological extracts, it is often necessary to use sub-
strates labeled with isotopes. In these cases the spots are identified with
the help of mixtures of unlabeled known compounds. Techniques for the
radioassay of the plates have been developed well. ~ This chromatographic
technique, including the usage of compounds labeled with ~4C, has been
adopted successfully in metabolic studies, s
7F. Snyder and N. Stephens, Anal. Chem. 4, 128 (1962).
[65] Assays of Intermediates of the Citric Acid Cycle and

Related Compounds by Fluorometric Enzyme Methods ~
By JOHNR. WILLIAMSON2 and BARBARAE. CORKEY
Introduction 435
Preparation of Samples 437
Tissues: Acid Extraction 437
Tissues: Alkaline Extraction 439
Mitochondria 439
Modifications of Extraction Procedures for Assays of CoA and Its Deriva-
tives 439
General Assay Techniques 440
Calculations and Expression of Results . 443
Spectrophotometric Standardization of Solutions 443
Expression of Results 444
Determination of the End Point of a Reaction in the Presence of Drift 444
Content of Citric Acid Cycle Intermediates in Various Tissues 445
Citrate 446
A. Determination with Aconitase and Isocitrate Dehydrogenase 446
B. Determination with Citrate Lyase and Malate Dehydrogenas~ 450
Isocitrate--Determination with Isocitrate Dehydrogenase 453
~-Ketoglutarate--Determination with Glutamate Dehydrogenase 455
Succinate--Determination with Succinate Thiokinase, Pyruvate Kinase and
Lactate Dehydrogenase . 458
Fumaratc--Determination with Fumarase and Malate Dehydrogcnasc~ 463
Malate---Determination with Malate Dehydrogenase 466
Oxaloacetate--Determination with Malate Dehydrogenase 468
Glutamate--Determination with Glutamate Dehydrogenase . 471
Aspartate--Determination with Glutamate-Oxaloacetate Transaminase amt
Malate Dehydrogenase 473
1Supported by grants from the U.S. Public Health Service (GM 12202-04) and the
American Heart Association.
Established Investigator of the American Heart Association.
[65] FLUOROMETRICASSAYS USING ENZYMATIC METHODS 435
D-3-Hydroxybutyrate--Determination with 3-Hydroxybutyrate Dehydrogenase 476

Acetoacetate--Determination with 3-Hydroxybutyrate Dehydrogenase 478
Pyridine Nucleotides . . . 481
A. Nicotinamide-Adenine Dinucleotide--Determination with Alcohol
Dehydrogenase . 481
B. Nicotinamide-Adenine Dinucleotide Phosphate-Determination with
Glucose-6-phosphate Dehydrogenase 483
C. Reduced Nicotinamide-Adenine l~'ucleotides--Determination with
Lactate Dehydrogenase and Glutamate Dehydrogenase 485
Adenine N'ucleotides . . . . . . . 488
A. Adenosine 5'-triphosphate--Determination with Hexokinase and Glu-
cose-6-phosphate Dehydrogenase . . 4&~
B. Adenosine 5'-triphosphate--Determination with Phosphoglycerate Ki-
nase and Glyceraldehyde-3-phosphate Dehydrogenase . . . 491
C. Adenosine 5'-diphosphate and Adenosine 5'-monophosphate--Deter-
ruination with Pyruvate Kinase, Myokinase, and Lactate Dehydrogenase 494
CoA, Acetyl-CoA, and Long-Chain Fatty Acyl-CoA 497
A. Determination with a-Ketoglutarate Oxidase and Phosphotransacetylase 497
B. Acetyl-CoA--Determination with Citrate Synthase and Malate Dehy-
drogenase 501
L-(--)-Carnitine and Long-Chain Fatty Acylcarnitine Derivatives--Determina'-
tion with Acetylcarnitine Transferase, Succinate Thiokinase, Pyruvate
Kinase, and Lactate Dehydrogenase 505
Acetylcarnitine--Determination with Acetylcarnitine Transferase, Citrate Syn-
thase, and Malate Dehydrogenase 509
Introduction
T h e characteristic absorption of the reduced but not the oxidized

forms of pyridine nucleotides (including N - a c e t y l pyridine nucleotide)
has been used for a number of years for the enzymatic measurement of
metabolic intermediates. Methods for the spectrophotometric measure-
m e n t of most of the intermediates of the citric acid cycle have been
described by B e r g m e y e r 2 About a 100-fold gain in sensitivity is achieved
b y measuring the fluorescence change rather t h a n the absorption changes
of N A D H or N A D P H in the enzyme reactions. 4
The reduced forms of N A D + and N A D P + absorb light at 340 m~ and
emit a fluorescent band at a longer wavelength which has a p e a k at
about 465 m~. The fluorescence emission occurs as photons are lost from
the excited state of the molecule, but transitions to the ground state
without emission of light are possible. This causes the fluorescence
q u a n t u m yield (light quanta emitted/light quanta absorbed) to be a
~H. U. Bergmeyer, (ed.), "Methods of Enzymatic Analysis," 2rid ed. Academic
Press, New York, 1965.
O. H. Lowry, J. V. Passonneau, F. X. Hasselberger, and D. W. SchuIz. J. Biol.
Chem. ~39, 18 (1964).
436 S~.PARATmN AND ASSAY METHODS [55]
variable, depending on such factors as the solvent, pH, and temperature.

A change in the quantum yield forms the basis of the quenching artifacts
that are observed in some of the assays.
Several commercial filter fluorometers are available with the required
sensitivity, and most of these have both recording and temperature
equilibration facilities. In the author's laboratory, either an Eppendorf
fluorometer (modified by the incorporation of a Keithley 151 micro-
voltmeter and a bucking voltage between the fluorometer and input to a
1 mA recorderS), or a metabolite fluorometer (designed and constructed at
the Johnson Research Foundation 6) is used. These instruments are capa-
ble of giving a full-scale deflection of the recorder with 0.25/~M NADH,
with a noise level less than 27~. At such high sensitivities the full progress
of each enzymatic reaction is recorded. This enables suitable corrections
to be made for unavoidable drifts and for fluorescence artifacts intro-
duced by the addition of enzymes. The enzyme concentration used in the
assays is adjusted, if possible, so that the reaction is complete in 1-3
minutes, particularly when the instrument is used at high sensitivities.
At lower sensitivities and when the reactions require more than l0
minutes for completion, it is more convenient not to make a complete
recording of each assay. In such cases a series of reactions are run
simultaneously in different cuvettes and individual readings are taken
at the beginning and end of the reactions in a manner similar to that
used by Lowry e t al. 4
The following special precautions must be taken when the fluorometer
is used at high sensitivities. These account for the majority of difficulties
and inaccuracies commonly encountered with fluorometrie enzyme
methods.
1. All solutions should be dust and particle free. Buffers and stock
solutions should be filtered through fritted glass filters, and glassware
must be scrupulously clean. Optical surfaces are best cleaned with lens
tissue. The use of paper tissues for wiping pipettes should be avoided.
The maximum useful sensitivity attained by any fluorometer is deter-
mined by the optical clarity of the solution in the cuvette.
2. The cuvettes should be temperature equilibrated, preferably by
having the cuvette chamber water-jacketed.
3. Particular care should be taken to avoid contamination of solutions
with enzymes, or cross-contamination of different enzymes in the same
assay.
4. Each enzyme solution should be used at the highest possible
dilution compatible with a rapid reaction, and fresh dilutions should be
made each day.
~R. W. Estabrook and P. K. Maitra, Anal. Biochem. 3, 369 (1962).
D. Mayer and J. R. Williamson, in preparation.
[65] FLUOROMETRICA S S A Y S USING ENZYMATIC METHODS 437
5. Solutions of pyridine nucleotides are best prepared each day and

stored on ice. NAD + and NADP ÷ are most stable in a slightly acid
solution, and may be diluted with distilled water. NADH and NADPH
should be dissolved and diluted in alkaline buffer, e.g., 0.1M tri-
ethanolamine buffer, pH 8.0-9.0.
6. Standard substrate solutions should be prepared daily if unstable
in solution, or diluted each day from frozen stock solution. All standard
solutions should be neutralized, and assayed spectrophotometrically on
the day of use.
7. Enzyme and substrate additions are best made on the flattened tip
of a small glass rod, which is then used for mixing the contents of the
cuvette and is rinsed and stored in a small flask of distilled water.
8. Micropipettes should be calibrated carefully.
9. Relatively wide variations in background fluorescence of cuvettes
may be encountered. Cuvettes should be of low fluorescence glass or
quartz to minimize the blank fluorescence reading.
Preparation of Samples
Metabolic intermediates other than reduced pyridine nueleotides,
total CoA, fatty acyl-CoA, and fatty acylearnitine compounds are meas-
ured in neutralized perchloric acid extracts of tissues. Perchloric acid is
generally more convenient to use than trichloroacetic acid for the
extraction, because it may be removed by precipitation as the potassium
salt.
Tissues: Acid Extraction

It is important to freeze samples of animal tissues as quickly as
possible because the tissue levels of many intermediates change rapidly
either postmortem or during the anoxic interval upon removal of the
tissue from the animal. ',7 The best means of freezing the tissue is to
press it between plates of aluminum tongs which have been cooled in
liquid N2:8 this procedure has the advantage of increasing the surface
area of the tissue at the instant of freezing. It is undesirable to drop a
lump of tissue into liquid N2 because the insulating layer of trapped gas
which forms at the tissue surface delays the freezing process. If it is not
practical to use the tongs, then the preferred method is to allow the tissue
to fall into liquid freon ("Freon-12" dichlorodifluoromethane: Virginia
Chemicals, Inc., West Norfolk, Virginia), which is maintained at a low
temperature, e.g., --140 °, by cooling with liquid N2.
' J . R. Williamson, J. Biol. Chem. 241, 5026 (1966).

' A. Wollenberger, O. Ristau, and G. Schofla, Arch. Ges. Physiol. 270, 399 (1960).
The frozen tissue is powdered in a stainless steel or Teflon percussion

mortar previously cooled in dry ice or liquid N2. An aliquot of the
powdered tissue (0.5-1 g) is placed in a 10 ml homogenizing tube stored
in powdered dry ice. Immediately before and after addition of the
powder, the tubes are weighed to the nearest milligram. The weighing
should be as rapid as possible to minimize errors caused by the conden-
sation of moisture on the tubes.
Approximately 3.5 volumes of 8% (v/v) HC104 in 40% (v/v) ethanol
are added to the cold powder, mixed quickly with a spatula or glass rod,
and homogenized for 2 minutes at --10 °, a Teflon pestle being used. The
contents of the homogenizing tube are decanted into a /5 ml glass
centrifuge tube and centrifuged in the cold at 25,000 g for 10 minutes. The
supernatant is decanted into a graduated conical centrifuge tube, and any
precipitate remaining in the homogenizing tube is transferred to the
pellet in the centrifuge tube by washing with an additional 2.5 volumes
of 6% (v/v) HCI04. The pellet is reextracted with perchloric acid wash
by mixing it with a Teflon rod, into a smooth paste. After centrifugation,
the supernatants are combined, the volume is recorded, and the solution
is adjusted to pH 5.5-6.0 by the slow addition of 3 M K2C0~ containing
0.5 M triethanolamine base. During neutralization, the contents of the
tube are mixed continuously to avoid areas of local alkalinity in the solu-
tion. The tube is then centrifuged to remove the precipitated KC10,, and
the supernatant is stored at --20 °. The perchloric acid-insoluble material
may be stored frozen and used for the extraction of fatty acyl-CoA and
fatty acylcarnitine.
In order to obtain complete extraction of metabolites from tissues
relatively high in lipid, we have found it essential to use the double
extraction method outlined above with 8% HCIO~ in 40~b ethanol for
the first extraction, and 6% HCI0~ for the second extraction. Incorpora-
tion of ethanol in the first extraction has the additional advantage of
making it possible to obtain a homogeneous mixture of tissue and extrac-
tion medium at temperatures below zero degrees, thus ensuring that the
enzymes remain inactive prior to contact with perchloric acid. Removal
of the deproteinized pellet prior to neutralization is necessary to cir-
cumvent the possible reactivation of certain enzymes that are not de-
stroyed by the acid treatment, e.g., aldolase and adenylate kinase2 It is
recommended that the percentage recovery of each intermediate be
ascertained after the addition of a known amount of metabolite to the
frozen powder. In order to ensure complete extraction of adenine and
pyridine nucleotides and CoA derivatives, it is useful to compare the
totals of each of these types of compounds in a series of samples prepared
from aerobic and anaerobic tissues. 4,T
[65] FLUOROMETRIC ASSAYS USING ENZYMATIC METHODS 439
Tissues: Alkaline Extraction

The following method is recommended for extraction of the powdered
tissue for reduced pyridine nucleotides. An aliquot (0.2 g) of cold
powdered tissue is transferred to preweighed 10 ml homogenizing tubes
which are kept cold in liquid N~. A volume of 0.25 N K 0 H in 50% (v/v)
ethanol equal to ten times the weight of the powder is added to the tube
and mixed by brief homogenization. The concentration or the volume of
the added KOH may be varied for convenience, but it is essential to
keep the final pH of the mixture between 10.5 and 11.0 in order to
obtain maximal extraction of both NADH and NADPH. The tube i~
heated at 55 ° for 1 minute with constant agitation and cooled in ice; the
contents are adjusted to pH 8.5 by the slow addition of 1 M triethanol-
amine-HC1 (pH 5.5). Vigorous mixing is necessary to avoid local acidity.
The mixture is decanted into a glass centrifuge tube and centrifuged in
the cold for 10 minutes at 25,000 g to obtain a clear supernatant.
Tubes containing tissue samples for the alkaline extraction should
not be cooled in dry ice, since acidification of the powder by the COs
vapor causes rapid destruction of the reduced pyridine nucleotides.
Mitochondria
To make perchlorie acid extractions of mitochondria, the mito-
chondrial sample containing 2-4 mg of protein per milliliter is added
rapidly to an equal volume of 12~b (v/v) HC10,, mixed and centrifuged
at 25,000 g for 5 minutes. An aliquot of the supernatant is neutralized to
pH 6.0 with 3 N K2CO3 containing 0.5 M triethanolamine base, and the
precipitated KC104 is removed by centrifugation. To make alkaline ex-
tracts, 0.5 ml of mitoehondria (2--6 mg of protein per milliliter) is added
to 0.25 ml of N KOH in 100% ethanol, mixed, heated for 1 minute at 37 °,
neutralized to pH 8.5, and centrifuged at 25,000 g for 5 minutes.
Modifications of Extraction Procedures for Assays of CoA
and its Derivatives
Acid-Soluble CoA and Acetyl-CoA. The perchlorie acid extraction
procedure described in the section above on acid extraction of tissues is
modified (a) by minimizing the time interval from addition of per-
chloric acid to the tissue to assaying the sample for CoA and acetyl-CoA,
and (b) by the addition of dithiothreitol (10 ~I/ml, 0.1 M) to the acid
extract immediately after neutralization.
Total CoA. Add 1.0 ml of 0.25N KOH in 50% (v/v) ethanol per
100 mg weight of powdered tissue at liquid N~ temperature. Homogenize
briefly and add 20 /~1 of 1 M dithiothreitol per milliliter of extract. Heat
for 10 minutes at 55 ° with stirring, cool in ice and neutralize to pH
5.0 ± 0.5 with 0.5 M triethanolamine-HCl (TRA) in 6 ~ perchloric acid.

Centrifuge in a refrigerated centrifuge for 10 minutes at 25,000 g and
decant supernatant into a cold test tube. Assay for CoA immediately.
Total CoA is extracted from mitoehondria] suspensions (2-6 mg of
protein per milliliter) by adding 2 volumes of mitochondria to 1 volume
of N K 0 t t in ethanol containing 20 mM dithiothreitol, mixing vigorously,
followed by a 5-minute incubation at 55 °. The mixture is neutralized to
pH 5.0 ± 0.5 and centrifuged as described above.
Problems of poor recovery can result from a suboptimal pH during
extraction. The final pH after addition of KOH should be 11.0--11.5. A
lower pH results in incomplete hydrolysis of CoA derivatives, and a
higher value causes degradation of CoA. Assay for total CoA may be
made with the same extraction solution as that used for measurement
of NADH and NADPH by incorporating the above modifications.
Long-Chain Fatty Acyl-CoA Derivatives. The denatured protein
precipitate formed during the perchlorie acid extraction is used for the
assay of long-chain fatty acyl-CoA derivatives after hydrolysis to free
CoA. The precipitate is washed once with 2 volumes of 0.6% HC10, and
once with 2 volumes of distilled water. Two milliliters of 10 mM dithio-
threitol per gram of tissue is added to the washed precipitate and mixed
into a smooth paste with a Teflon rod. The mixture is brought to pH 11.5
by the addition of N KOH and incubated for 10 minutes at 55 °. After
neutralization to pH 5.0 with 6 ~ HC104 containing 0.5M triethanol-
amine-HC1, the mixture is centrifuged in the cold at 25,000 q for 10
minutes, and the supernatant is decanted into cold tubes. The extract is
assayed for free CoA immediately. The washed precipitate may be
stored at --20 ° for several weeks prior to extraction.
Long-Chain Fatty Acyl-L-(--)-carnitine Derivatives. These are pre-
cipitated along with denatured protein during extraction of the tissue
with perchloric acid. The precipitate is washed as described above and is
mixed with 2 ml of distilled water per gram of tissue. The pH is adjusted
to 12.5 with KOH, and the mixture is incubated for 2 hours at 70 ° in
glass-stoppered test tubes. After hydrolysis, the mixture is neutralized to
pH 7.0 with 12~ (v/v) ttC10, and centrifuged at 25,000 g for l0
minutes. The supernatant is used for the assay of v.. (_)_carnitine.
General Assay Techniques
A suitable fluorometer should have a noise level of 1-2% when 1
millimicromole of NADH gives a full-scale deflection of the recorder and
a baseline drift of not more than 5~o of full scale over a 10 minute period.
It is recommended that a calibrated zero shift be incorporated between
the output of the fluorometer and input to the amplifier or recorder. This
offset voltage should be capable of bucking out the background fluores-

cence of a 1:I dilution of extract when the fluorometer sensitivity is
adjusted to give full-scale deflection of the recorder with 0.5 millimicro-
moles of NADH. The calibration on the zero shift allows a scale expan-
sion of the recorder, so that a 5 inch chart-width is satisfactory for
recording the beginning and end of the assay. The sample chamber should
be maintained at a constant temperature and must have the capacity to
hold several cuvettes.
Enzymes, substrates, cofactors, and standards should be prepared in
small tubes, placed for convenience in an aluminum block resting on
crushed ice. Before attempting to assay intermediates in samples, each
reaction should be tested with known standards in order to determine the
maximum dilution of the enzymes required fur the reaction to reach
completion within a few minutes. If the enzyme has a strong native
fluorescence or quenching effect, a compromise is made between the
size of the enzyme blank and speed of the reaction. Addition of standard
to the cuvette containing the full complement of enzymes and cofactors
also serves to calibrate the fluorometer so that the required sensitivity
adjustments can be made. The standard should be added to the same
cuvette several times consecutively in order to check the range over
which the fluorescence change is linear. The choice of sensitivity is deter-
mined by the amount of intermediate present in the tissue extract, the
volume of extract available, inhibitor effects of the sample on the
reaction, and whether the particular enzyme reaction is characterized
by a drift. A sensitivity of 1-2 millimicromoles change of reduced pyri-
dine nucleotide per chart-width is convenient for most assays.
It is essential that labile intermediates, such as CoA, oxaloacetate,
pyruvate, a-ketoglutarate, and pyridine nucleotides, be assayed as soon
after the preparation of the extracts as possible, e.g., for oxaloacetate no
more than 1 hour should elapse between the beginning of extraction and
assay.
All extracts contain material with a fluorescence band which overlaps
that of reduced pyridine nucleotides. This background fluorescence is
greater in plant preparations and in tissues containing blood than in
blood-free preparations, but is relatively small in mitochondrial extracts.
Consequently, a sample dilution in the assay cuvette of less than 1 in 10
tends to cause assay problems due to (1) the large bucking voltage
required to compensate for the high native fluorescence, (2) a decrease
in the signal to noise ratio, and (3) the increased tendency toward drifts
and quenching artifacts. In this context, "quenching" is the term used to
describe a fluorescence change during a reaction which is smaller than
expected: e.g., when the sample dilution in the cuvette is about 1-5, it is
442 SEPAnATION AND ASSAY METHODS [65]
often observed that a smaller reaction is given with internal than with
external standards. For this reason, internal standards are preferable and
standard solutions of intermediates should be added to each cuvette
after thc initial reaction has reached completion. This applics also to
enzyme blanks, since differences are occasionally observed between
enzyme blanks in the presence and absence of sample. Goldberg et al2
have recommended the use of Florisil to decrease the native fluorescence
of the sample. When this treatment is used, it is necessary to establish
that metabolites are not lost from the sample.
Standard curves have been made with all the assays described in this
chapter, and most of them were linear over the range from zero to 5
concentration of intermediate in the cuvette. The exceptions are discussed
separately under the heading for the particular assay. However, it is
advisable to test whether different volumes of sample produce a linear
fluorescence response in each of the assays. It is unusual to observe
linearity over a wide range of sample volumes; particularly when highly
fluorescent tissue extracts are being assayed. Generally, it is sufficient to
adjust the sensitivity of the fluorometer so that reasonable changes are
observed within the linear range of the assay. When marked nonlinearity
is observed, a sample volume containing 1-2 millimicromoles of metabo-
life is chosen, and a standard curve is prepared in the presence of this
volume of sample. The assay is then performed with the predetermined
volume of sample such that the range of values in a series of unknown
samples falls within the linear range of the standard curve. Linearity
studies also serve to determine the correct enzyme blank when there is a
discrepancy between the size of the internal and external blanks, as in
the a-ketoglutarate assay. The correct enzyme blank is then given by the
point of intersection of the linear portion of the curve with the ordinate.
Special procedures and precautions required for each assay are de-
scribed in detail in later sections under separate assay headings, but it is
hoped that the above general principles will be useful, particularly to
investigators who are not familiar with fluorometry and enzyme assay
techniques.
Commercial Sources o] Materials. All reagents used should be of the
highest possible purity, and dilutions should be made with double glass-
distilled water. Use of chromic acid solutions for cleaning glassware
should be strictly avoided. Enzymes, cofactors, and substrates needed
for the assays are obtained from the commercial suppliers listed below.
Care should be taken to order enzymes that have the greatest specific
activity and the lowest listed contamination.
N. D. Goldberg, J. V. Passonneau, and O. It. Lowry, J. Biol. Chem. 241, 3997
(1966).
Sigma Chemical Co., 3500 DeKalb St., St. Louis, Missouri.

Calbiochem, P.O. Box 54282, Los Angeles, California 90054.
Boehringer-Mannheim Corporation, 20 Vesey St., New York, New York 10007.
Worthington Biochemical Corporation, Freehold, New Jersey.
Nutritional Biochemicals Corporation, 26201 Miles Road, Cleveland, Ohio.
Mann Research Laboratories, Inc., 136 Liberty St., New York, New York.
P-L Biochemicals, Inc., 1037 West McKinley Ave., Milwaukee, Wisconsin 53205.
In the text, enzymes are referred to by their common usage names,
but in order to avoid confusion, the names and number key recommended
by the International Union of Biochemistry, 1964, is also given for each
enzyme.
Calculations and Expression of Results
Spectrophotometric Standardization of Solutions

The fluorescence change of N A D H or N A D P H in the enzyme reac-
tions does not provide an absolute measure of the concentration of
metabolite in the unknown solution. The fluorescence change is therefore
compared with the change observed upon addition of 5-20 ~I of a
standard solution of the particular metabolite to the cuvette. The con-
centration of metabolite in this solution is determined spectrophoto-
metrically by measuring the change in optical density of reduced pyridinc
nucleotide at 340 m ~ in the enzyme test. Standard solutions are usually
made up to a concentration of 0.1 raM, and 0.5 ml is used for the spectro-
photometric analysis. Cuvettes containing the standard solution of
metabolite to be assayed are prepared in duplicate together with a blank
cuvette in which distilledwater replaces the metabolite standard. In the
extrapolation from fluorometric to spectrophotometric tests, care has to
be taken that the cuvette contains a suitable excess of substrates and
enzymes needed for the enzyme tests.
The concentration of metabolite in the standard solution used for
the assay is calculated from the following formula:
Cone (mM) -- 6.2---2

V1 X ~1 X [(RF - R~) - h blank]
where
V1 is the total final volume of solution in the cuvette
V2 is the volume of standard added to the cuvette
RF is the final optical density at 340 m/~
R1 is the initial optical density at 340 m~
A blank is the optical density change which results from the addition
of enzyme to a cuvette which contains all ingredients except stand-
ard, which is replaced by distilled water
444 SEPARATION AND ASSAY METHODS [6S]
All readings of optical density are made against distilled water. The
millimolar extinction coefficient of NADH and NADPH at 340 m~ is
taken as 6.22 for a light path of 1 era.
Expression of Results
Metabolite levels in tissues are generally expressed in millimicromoles
or micromoles per gram, wet weight or dry weight. The dry weight basis
is preferable, particularly with perfused organs or incubated tissue slices,
since the water content of samples can be a variable factor. For tissues
with a high fat content, expression of results in terms of the fat-free dry
weight or the N~ content is suitable. Metabolite levels in mitochondria
are usually expressed per milligram of protein.
For the determination of the water content, an aliquot of powdered
tissue is placed in a preweighed weighing bottle, which is then reweighed,
dried overnight at 105 °, and weighed again after the bottle has been
allowed to cool in a desiccator. When the wet weight of the powder used
for extraction and the percentage water content are known, it is possible
to calculate the dry weight of the tissue aliquot.
The tissue content of each metabolite in millimicromoles per gram,
dry weight (A) is calculated from the formula:
A= (V,+Vb)(V~+Vd) XB
Vo×W
where
Ira = total volume of HCI04 added during the extraction
Vb = amount of water in the sample of tissue powder
Vo = volume of aliquot used for neutralization
Va = volume of K~COs added to neutralize the above aliquot Vo
W= dry weight of tissue sample in grams
B = concentration of metabolite in extract (m#moles/ml)
When a known quantity of metabolite is added to an extract to deter-
mine the recovery, the amount added in millimicromoles per gram, dry
weight, is calculated by dividing the total number of millimicromoles
added by W.
Determination of the End Point of a Reaction in the Presence of Drift
If the reaction in any assay system terminates with a drift (e.g.,
acetoacetate and succinate determinations), two methods may be used
to calculate the end point. The first method entails extrapolation of the
linear part of the reaction curve back to the time at which enzyme was
added (to), and measuring the number of divisions of fluorescence change
from the beginning of the reaction to the point of intersection of the
drift extrapolation with to. Although this method is often satisfactory,

errors can arise if the extrapolation is long because of a slow reaction.
An alternative method, commonly used in our laboratory, is to t a k e the
end point as the point of inflection between the reaction curve and the
linear p a r t of the reaction tail (see Fig. 12). This entails little error, if
the drift rate is constant, since the same method is used to calculate the
end point of the reaction in the unknown sample and in the internal
standard. I f the drift rate is not constant, the first method is preferable.
Content of Citric Acid Cycle I n t e r m e d i a t e s in Various Tissues

For convenience of reference, the content of citric acid cycle inter-
mediates and related compounds in a number of tissues of the r a t are
presented in Tables I and II. The absolute level of certain intermediates
TABLE I
CONTENTS OF CITRIC AcID CYCLE INTERMEDIATES
IN V A R I O U S TISSUES OF T H E RAT ~
Heart~ Brain Liver Liver~ Kidney

Metabolite (perfused) (in rico) (in rico) (per/used) (in rico)
Citrate 1030 1470 900 1800 1100

Isocitrste 120 72 75 110 125
a-Ketoglu~rate 160 570 640 1200 1100
Succinate w 3000 1600 1100
Fumarate w 330 -- 130
MMate 450 2000 1380 425 570
Oxaloacetate 5-26 18 15 16 40
Acetyl-CoA 15 -- 165 167
CoA 330 -- 300 362 --
Values given as millimicromoles per gram, dry weight.
b Heart from fed rat perfused with 5-10 mM glucose and insulin.
"Liver from 24 hours starved rat perfused with 10 mM alanine.
as reported by different laboratories shows some variation, but generally,

when fiuorometric methods have been used, the agreement is good. The
values given in the tables are from our laboratory, 1°-14 d a t a reported by
Goldberg et al., ~ together with some unpublished data obtained by us and
by Drs. N. N a g a t a and H. Rassmussen.
2oj. R. Williamson, J. Biol. Chem. 240, 2308 (1965).

n B. Chance, D. Jamieson, and J. R. Wilhamson, Proc. 8rd Intern. Con]. Hyperbaric
Medicine, Natl. Acad. Sci., Washington, p. 15 (1966).
~2j. R. Williamson, Biochem. d. 1Ol, II C (1966).
~J. R. Williamson, R. A. Krcisberg, and P. W. Felts, Proc. Natl. Acad. Sci. U.S. 56,
247 (1966).
~4j. R. Williamson, E. T. Browning, R. Scholz, R. A. Kreisberg, and I. B. Fritz,
Diabetes 17, 194 (1968).
TABLE I I
METABOLITE CONTENTS IN VARIOUS TISSUES OF THE RAT a
Heart b Brain Liver Liver" Kidney

Metabolite (perfused) (in vivo) (in vivo) (perfused) (in vivo)
ATP 21.7 13.3 10.5 8.6 9.7

ADP 2.49 1.49 2.46 2.37 1.67
AMP 0.35 0.23 0.40 0.76 0.35
NAD + 3.95 1.55 3.22 3.26 2.58
NADH 0.24 0.20 0.38 0.20 0.20
NADP + 0.20 0.02 0.39 0.63 0.22
NADPH + 0.30 0.13 1.20 1.76 0.50
Glutamate 29.3 35.0 12.2 21.4 6.3
Aspartate 9.2 8.5 2.8 2.1 --
Carnitine 2.30 -- 0.80 0.64 --
Aeetylcarnitine 0.15 -- 0.28 0.28 --
Values are given as micromoles per gram, dry weight,

b Heart from fed rat perfused with 5-10 mM glucose and insulin.
c Liver from 24 hours starved rat perfused with 10 mM alanine.
Citrate
A. D e t e r m i n a t i o n w i t h A c o n i t a s e '5 a n d I s o c i t r a t e D e h y d r o g e n a s e ~e
Principle
Isoci~rate d e h y d r o g e n a s e ( I C D H ) c a t a l y z e s t h e o x i d a t i v e d e c a r -
b o x y l a t i o n of threo-Ds-isocitrate b y N A D P ÷ a c c o r d i n g to E q . (1).
M n ++
threo-D,-Isocitrate -{- N A D P + ICDI~
a - k e t o g l u t a r a t e -{- C02 -{- N A D P H +H + (1)
T h e e q u i l i b r i u m of t h i s r e a c t i o n lies f a r to t h e right. V a l u e s b e t w e e n
1.1 a n d 7.7 m M for t h e e q u i l i b r i u m c o n s t a n t of t h e o v e r a l l r e a c t i o n h a v e
been r e p o r t e d Y ,~s M a n g a n o u s or m a g n e s i u m ions a r e r e q u i r e d for
activity.
A c o n i t a s e c a t a l y z e s t h e c o n v e r s i o n of c i t r a t e to i s o c i t r a t e :
Fe++
Citrate ~- • (cis-aconitate) ~ - i s o c i t r a t e (2)
aconitase
l~Citrate (isocitrate) hydro-lyase, EC 4.2.1.3.

threo-I)s-Isocitrate :NADP oxidoreductase (decarboxylating), EC 1.1.1.42.
"S. Ochoa, J. Biol. Chem. 174, 133 (1948).
'BE. Racker, Biochim. Bix~phys. Acta 4, 211 (1950).
[6S] FLUOROMETRIC ASSAYS USING ENZYMATIC METtiODS 447
At equilibrium, 91% citrate, 3~o cis-aconitate, and 6% isocitrate are

present at pH 7.4. TM Ferrous ions activate this enzyme. :°
When aconitase is coupled with excess isocitrate dehydrogenase, all
the citrate is converted to a-ketoglutarate according to Eq. (3).
ICDH
Citrate -4- NADP+ a-ketoglutarate + C02 + NADPH + H + (3)
aconitase
The disappearance of citrate is measured by the fluorescence increase due

to N A D P H formation.
Assay Reagents
Buffer: 0.1 M triethanolamine(TRA), pH 7.4. Adjust pH with NaOH
(0.1 M K * ions inhibit the reaction). Store at 2-4 °.
NADP ÷ (sodium salt), 10 mg/ml. NADP ÷ is most stable at slightly
acid pH. Solution may be stored frozen for several weeks with
only slight loss.
MnS04, 10 mM. This solution is best stored in the frozen state.
Citrate standard, 0.1 mM. A stock solution of 10 mM sodium citrate
may be prepared and stored frozen.
Enzymes
a. Aconitase, approximately 1 U/mg. Dilute 1:5 after activation.
Aconitase is not commercially available (this volume [6]). In
the authors' laboratory this enzyme is prepared by the method of
Morrison, 21 with the following modifications: (1) use of tri-
carballylic acid (propane tricarboxylic acid) instead of citric acid
to stabilize the enzyme ;9 (2) taking the enzyme purification only
as far as the second ethanol fractionation; (3) discarding protein
precipitating at ethanol concentrations below 40% in the first
ethanol fractionation, and using that precipitating between 40
and 50% ethanol for the succeeding steps. The enzyme loses
activity with age and requires activation before use (see below).
Aconitase prepared by the above method contains sufficient iso-
citrate dehydrogenase so that a separate addition of more enzyme
is unnecessary.
b. Isocitrate dehydrogenase 1 mg/ml (1.8 U/mg). Dilute com-
mercial enzyme (10 mg/ml) 1:10 or 1:20 with distilled water.
19H. A. Krebs, Biochem. J. 54, 78 (1953).

S. R. Dickman and A. A. Cloutier, J. Biol. Chem. 188, 379 (1951).
~lj. F. Morrison, Biochem. J. 56, 99 (1954).
Activation o] Aconitase
The method of aconitase activation is based on that described by
Morrison, 21,2~ except that a high concentration of Fe ÷* ions (5 raM) and
different incubation conditions for activation are used. This modified
method is more reproducible and causes a greater increase in specific
activity than Morrison's original method or Siebert's 28 modification.
Method. Prepare activating solution (5 mM ferrous ammonium
sulfate, 20 mM cysteine) as follows: Weigh 98.3 mg of ferrous ammonium
sulfate hexahydrate and 136 mg of cysteine hydrochloride monohydrate
into a 50 ml narrow-necked flask; add 45 ml of distilled water and mix
by bubbling N2 through the solution. Continue bubbling N2 for 5-10
minutes to remove oxygen. Slowly adjust the pH to 7.4 with 1 N NaOH
and make up to 50 ml. Add one volume of activating solution to one
volume of aconitase solution (0.1 rag) in a small tube. Flush out air and
maintain an inert atmosphere above the liquid surface with a stream of
N2 gas. Incubate for 5 minutes at 30% Place on ice and dilute with ice
cold water if necessary. (See also this volume [6].)
Discussion. The preceding activation procedure yields a 2- to 400-fold
increase in enzyme activity, depending upon the age of the enzyme; e.g.,
the activity of freshly prepared enzyme is increased 2-fold; of 3-month-
old enzyme, 20-fold; and of 2-year-old enzyme, 400-fold. It has been
observed that, with the activation procedure described by Morrison,
activated enzyme retained activity for only 3 or 4 hours, whereas with
the above procedure, 8 5 ~ activity was retained for 5 hours and 60%
activity for 18 hours. The requirement for the Fe** ion increased with
the age of the enzyme; e.g., a freshly prepared aconitase solution was
maximally activated with 0.5 mM ferrous ammonium sulfate; a 3-month-
old solution with 2 mM ferrous ammonium sulfate; and a one-year-old
solution with 5 mM ferrous ammonium sulfate. The use of glutathione or
dithiothreitol to replace cysteine as thiol reagents in the activating pro-
cedure was found to give less effective activation.
Assay Procedure
Sensitivity. Adjust the sensitivity of the fluorometer so that addition
of 1 millimicromole of N A D H (10 ~1 of 0.1 mM solution) to 2.0 ml of
buffer in a 1 cm ~ cuvette gives a deflection of 70--90~ of the full scale
on the recorder.
External Standard and Enzyme Blank. Pipette into euvette: 2.0 ml
nj. F. Morrison, Biochem. J. 58, 685 (1954).

~8G. Siebert, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), p. 318.
Academic Press, New York, 1963.
[65] FLUOROMETRIC
ASSAYS USING ENZYMATIC METHODS 449
of buffer, 0.1M TRA, pH 7.4; 10 ~l of NADP ÷, l0 mg/ml; 10 ~1 of

MnSO4, 10 mM. Mix thoroughly and place in fiuorometer. Read fluores-
cence level on the recorder. When temperature equilibration is complete
(1-2 minutes), add 5 /zl of aconitase. An increase in fluorescence occurs
(external blank), and a new baseline is established. Add 10 #l of citrate
standard to cuvette. Within 1-3 minutes the increase in fluorescence
ends as citrate is quantitatively converted to a-ketoglutarate (external
standard). The standard and enzyme may be added a second time and
should cause the same change in fluorescence as that originally recorded.
Citrate Measurements in Unknown Samples. Sample aliquots contain-
ing 0.5-12 millimicromoles are used. The required volume of sample must
be determined by trial and error. The buffer volume is decreased so that
Reset
Reset
~ ¢ . ,
--/- -'| Reset
• ,-:, T: /
-I
Aconilose
I ...... j ,Ira/;mole Citrote
Fluorescence Increasel
Fro. I. Determination of citrate with aconitase. A 0.05 ml sample of neutralized

perchloric acid extract from rat liver was used for assay (8 mg fresh wt).
buffer plus sample is equal to 2.0 ml. Cuvettes are prepared as for the
external standard, and when the reaction is complete, a second addition
of enzyme is made (internal blank), followed by addition of 10 gl of
citrate standard (internal standard) (Fig. 1).
Discussion. The method described measures total citrate, c/s-aeonitate,
and isocitrate. Isocitrate can he measured in the same cuvette by adding
isocitrate dehydrogenase prior to aconitase. However, owing to the low
concentration of isocitrate in tissue samples under most conditions, it is
more accurate to measure it separately at a higher sensitivity using a
Iarger volume of sample (see section on isocitrate).
Difficulty in assaying citrate is most frequently caused by inactive
aconitase or high cation concentration.
INACTIVITY OF ACONITASE. This may be due to denaturation which
increases with age of the enzyme. Improper activation also yields an
inactive enzyme. The activating solution should be clear amber (purple

or brown color is caused by oxidation of Fe ÷÷ to Fe s÷, and cloudiness is
due to cystine formation). Maximal activation is pH dependent and is
not achieved above pH 7.5.
CATION CONCENTRATION.The presence of a high concentration of K ÷
ions inhibits the reaction; e.g., buffer neutralized with KOH instead of
N a 0 H completely prevents measurement of small amounts of citrate.
Low concentrations of Mn ~ ions are necessary for isocitrate dehydro-
genase activity. However, the range of Mn *+ concentration for maximum
activity is rather small and an increase from 0.05 mM to 0.25 mM causes
inhibition. Concentrations of Mg ÷÷ ions above 5 mM inhibit the overall
reaction.
FLUOROCITRATE. Aconitase is competitively inhibited by fluoroci-
trate,~4,25 thus preventing the use of this assay procedure in its presence.
Determination of citrate by citrate lyase (as described in the following
section) is an alternative, highly satisfactory method.
Standardization
The concentration of citrate in the standard solution is determined
spectrophotometrically by adding the following reagents to a 1 cm 2
cuvette, in duplicate, with distilled water replacing the citrate standard
solution for the blank: 1.87 ml of buffer, 0.10 ml of NADP ÷, 0.02 ml of
MnS0,, 0.50 ml of citrate standard.
After mixing the sample, record the initial optical density at 340 m#
(R1). Add 0.01 ml of aconitase and take readings until the reaction
reaches completion (R2). The change in optical density produced by
addition of 0.01 ml of aconitase to the blank is subtracted from the dif-
ference R2 -- R1.
B. Determination with Citrate LyasC 6 and Malate Dehydrogenase '7
Principle
Citrate lyase (CL) (citritase or citrase) catalyzes the cleavage of
citrate to oxaloacetate and acetate according to Eq. (1).
Mg++
Citrate. " oxaloacetate + acetate (1)
CL
Oxaloacetate + N A D H + H + , L-malate + NAD + (2)
MDH
2, R. A. Peters, Discussions Faraday Soc. 20, 189 (1955).
'~ R. A. Peters, Johns Hopkins Hosp. Bull. 97, 21 (1955).
'* Citrate oxaloacetate-lyase, E C 4.1.3.6.
"~L-Malate:NAD oxidoreductase, E C 1.1.1.37.
[65] FLUOROMETRIC
The molar equilibrium constant of the citrate lyase reaction is 1.56

in favor of citrate synthesisf-s The reaction is coupled to malate dehydro-
genase [Eq. {2)] for tile quantitative removal of oxaloacetate, thereby
pulling the overall reaction [Eq. (3) ] from left to right.
Citrate 4- NADH W H + Mg++ , L-mal~te ~- acetate ~ NAD + (3)

MDH, CL
The accompanying decrease in fluorescence of :NADH can either be
followed in a fluorometer or measured spectrophotometrically by the
change in optical density at 340 m/~.29
Assay Reagents
Buffer, 0.05M triethanolamine-HC1 (TRA), 10 mM MgSO~, 5 mM
EDTA (ethylenediaminetctraacetic acid), pH 7.4. Adjust the pH
with KOH and store at 2-4 °.
NADH (2 mg/ml). Purified NADH available from P-L Biochemicals,
Inc. (P-L 6500) is recommended. Dissolve NADH in 0.1 M TRA,
pH 8.2.
Citrate standard (0.2 mM). A stock solution of 10 mM sodium
citrate may be prepared and stored frozen. Dilute stock solution
1:50 with distilled water each day.
Enzymes
a. Malate dehydrogenase (this volume [18-21]), 1 mg/ml (720 U/
mg). Make up 0.2 ml by diluting commercial enzyme (10 mg/ml)
l:10 with distilled water.
b. Citrate lyase (this volume [28]), 20 mg/ml (I0 U/mg). Dis-
solve 10 mg of citrate lyase from Boehringer-Mannhein Corpora-
tion in 0.5 ml of 10 mM TRA, pH 7.6, containing 6% (NH4)~SO~
and 0.04% ZnC12, as recommended by the manufacturers.
Assay Procedure
of 2 millimieromoles of :NADH (10 ~l of 0.2 mM solution) to 2.0 ml of
buffer in a 1 cm ~ cuvette, gives a deflection of 70-90% of the full scale on
the recorder.
External Standard and Enzyme Blank. Pipette into cuvette: 2.0 ml of
buffer (0.05 M TRA, 0.01 M MgS04, 0.005 M EDTA, pH 7.4); 10 ~l of
NADH, 2 mg/ml; 5 ~! of MDH, 1 mg/m!.
After mixing the contents of the cuvette, record the fluorescence
" H . 1=[. Daron, and I. C. Gunsalus, in "Biochemists' Handbook" (C. Long, ed.), p.
471. Van Nostrand, Princeton, New Jersey, 1961.
~*H. Moellering and W. Gruber, Anal. Biochim. 17, 369 (1966).
level. When temperature equilibration is complete (1-2 minutes), add 10

~1 of citrate lyase. A small increase in fluorescence is caused by addition
of the enzyme (external blank). When a new baseline is established, add
10 ~l of citrate standard to the cuvette. Within 2-5 minutes the fluores-
cence level will have reached a new baseline (external standard). The
standard and/or enzyme may be added a second time and should cause
the same deflection (number of divisions) as originally recorded. The
slight drift occasionally caused by addition of enzyme will remain
constant: it is proportional to the volume of enzyme added.
Citrate Measurements on Unknown Samples. Sample aliquots con-
taining 0.5-10.0 millimicromoles of citrate are used. The required volume
of sample must be determined by trial and error. The buffer volume is
decreased so that buffer plus sample is equal to 2.0 ml. Cuvettes are
prepared as for the external standard, and the reaction is started by
Citrate Lyase Reset
irc-~--~/ , 1 2rain I "~"~
-'luorescenceIncrease~ 1.87mFmoleCitrate
s
FIO.2. Determinationof citrat~withcitrate|yase.A 0~ m] ~mpleof neutralized
perch|oricacidextractfrom rat liver mitochondriawasusedfor assay.
addition of citrate lyase. When the reaction is complete, a second addition
of citrate lyase is made (internal blank) followed by addition of citrate
standard (internal standard) (Fig. 2).
Discussion. Citrate lyase requires cations for activation. Mg**~ Zn++,
Fe**, Co *+, Mn++~NH4 ÷ serve this purpose. 2~,so The enzyme shows marked
product inhibition, sl-s4 The Michaelis constant for Mg +* is 3 raM, and
for citrate is 0.18 raM22 In the presence of large volumes of extract the
reaction may take longer than 5 minutes to reach completion. This
problem can be minimized by increasing the enzyme concentration and/or
decreasing the sample volume.
uS. Dagley, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), 2nd ed.,
p. 313. Academic Press, New York, 1965.
s~T. J. Bowen and L. J. Rogers, Biochim. Biophys. Acta 77, 685 (1963).
aS. Dagley and E. A. Dawes, Biochim. Biophys. Acta 17, 177 (1955),
~S. S. Tare and S. Datta, Bioehem. J. 94~ 470 (1965).
R. W. Wheat and S. J. Ajl, J. Biol. Chem. 217, 909 (1955).
[65] FLUOROMETRIC
Standardization
The concentration of the citrate in the standard solution is determined
spectrophotometrieally by measuring the decrease in optical density at
340 mt~ accompanying the disappearance of NADH in the following re-
action mixture: buffer, 2.17 ml; NADH, 0.05 ml; MDH, 0.01 ml; citrate
standard or distilled water, 0.25 ml.
The contents of the cuvettes are mixed, then the initial optical density
at 340 m/~ is read against water (R1) ; 0.02 ml of citrate lyase is added,
readings being taken at 1 minute intervals until reaction is complete (R2).
The optical density change obtained upon addition of enzyme to a blank
cuvette, in which water replaces the citrate standard solution, is sub-
tracted from the difference R 1 - R~.
Isocitrate
D e t e r m i n a t i o n with Isocitrate D e h y d r o g e n a s e ~6
Principle
Isocitrate dehydrogenase (ICDH) catalyzes the oxidative decar-
boxylation of isocitrate by NADP ÷ according to Eq. (1).
Mn++
threo-I).-Isocitrate -{- NADP+ ICDI~
a-ketoglutarate -b CO~ + NADPH + H + (1)
The equilibrium of this reaction lies far to the right, and quantitative
conversion of isocitrate is possible at optimal pH and Mn +* concentration.
The increase in fluorescence due to the formation of NADPH can be
followed in a fluorometer.
Assay Reagents
Buffer. 0.I M triethanolamine-HCt (TRA), pH 7.4. Adjust the pH
with NaOH and store at 2-4 °.
NADP÷, 5 mg/ml. Solutions made up in distilled water are stable for
several weeks when frozen.
MnSO4, 10 mM. The solution may be stored frozen several week~
without appreciable oxidation of Mn ++ ions.
Isocitrate standard, 50 t~/. A stock solution of 5 mM L-isocitrate may
be prepared and stored frozen. Dilute stock solution 1:100 each day
with distilled water.
Enzyme: Isocitrate dehydrogenase 1 mg/ml (1.8 U/rag). Make up
0.2 ml by diluting commercial enzyme (10 mg/ml) l:10 with di~-
tilled water.
Assay Procedure
Sensitivity. Adjust sensitivity of fluorometer so that 0.5 millimicro-
moles of N A D H (10/~l of 50 ~ / ) added to 2.0 ml of buffer in a 1 cm 2
cuvette gives a deflection of 70-90~ of the full scale on the recorder.
External Standard and Enzyme Blank. Pipette into the euvette:
2.0 ml of buffer (0.1 M TRA, pH 7.4) ; 10 ~l of NADP ÷, 5 mg/ml; 10/~l
of MnS04, 10 raM.
After mixing, place the cuvette in the fluorometer and record the
fluorescence level. When temperature equilibration is complete (1-2
minutes), add 5/~l of isocitrate dehydrogenase. A very small increase in
fluorescence is caused by addition of the enzyme (external blank). Add 10
~1 of isocitrate standard to the cuvette. Within 1-3 minutes the increase
in fluorescence ends as isocitrate is quantitatively converted to a-keto-
glutarate. The difference between the initial and final base lines as
recorded on the chart is proportional to the concentration of isocitrate
Isocltrate l,,..,~-,~----"~
Dehydrocjenase.~
' /I
FIO. 3. Determination of isocitrate with isocitrate dehydrogcnase. A 0.2 ml sample

of neutralized perchloric acid extract from rat liver was used for assay (34 mg
fresh wt).
in the standard solution. When sa,mple is absent this is called the external
standard. The standard and/or enzyme may be added a second time and
should cause the same deflection as recorded originally.
Isocitrate Measurements on Unknown Samples. Sample aliquots con-
taining 0.1-1.0 millimicromole of isocitrate are used. The required volume
of sample must be determined by trial and error. However, because of
the low levels of isocitrate in most tissues, a maximal volume, i.e., 0.5-1.0
ml, may be required. The volume of buffer plus sample is kept constant
at 2.0 ml. When only a small volume of sample is available, it may be
desirable to adapt the assay for use with a smaller cuvette. Cuvettes are
prepared as for the external standard and the reaction started by addi-
tion of isocitrate dehydrogenase (Fig. 3). When the reaction is complete,
a second addition of isocitrate dehydrogenase is made (internal blank)
followed by addition of isocitrate standard (internal standard). Because
of the large sample volume usually required for this assay, fluorescence
[65] FLUOROMETRIC
quenching of N A D P H causes smaller internal standards than external

standards. The fluorescence change produced by addition of standard
isocitrate solution in the presence of sample, therefore, provides a better
basis for comparing the fluorescence change due to isocitrate in the
sample than the change produced by the addition of isocitrate as an
external standard.
Discussion. A very slow reaction may be caused either by the presence
of a high concentration of K ÷, e.g., by neutralization of tile TRA buffm"
with KOH instead of NaOH, or by excess or insufficient Mn ÷+. A back drift
is caused by reoxidation of N A D P H formed during the reaction and is
generally due to contamination with N A D P H oxidase. This may be
alleviated by further dilution of the enzyme. A drift toward an increase
of fluorescence frequently accompanies the high fluorescence of the
extract. In view of the very large sample volume usually required for
this assay and the very high sensitivity at which it is generally carried
out, it may be necessary to extrapolate an end point from the drift, as
described by Bergmeyer. 3s
Standardization
The concentration of the isocitrate standard solution is determined
spectrophotometrically in 1 cm 2 cuvettes using the following reaction
mixture: buffer, 1.37 ml; NADP ÷, 0.1 ml; MnSO~, 0.02 ml; standard or
distilled water, 1.0 ml.
The contents of the cuvettes are mixed, and the initial optical
density at 340 m~ is read against water (R~); 0.01 ml of isoeitrate
dehydrogenase is added, readings being taken at 1 minute intervals until
the reaction ends (R~). The change of optical density in the blank is
subtracted from the difference R ~ - R~.
a-Ketoglutarate
Determination with Glutamate Dehydrogenase 36
Principle
Glutamate dehydrogenase (GDH) catalyzes the reaction [Eq. (1)].
a-Ketoglutarate 4- NADH ~- NH, + GDH' L-glutamate 4- NAD + W H20
(1)
The equilibrium for this reaction lies far to the right (Keq = 1.8 X 10-1~)~7
~tt. U. Bergmeyer (ed.), in "Methods of Enzymatic Analysis," 2nd ed., p. 38.
L-Glutamate:NAD oxidoreductase (deaminating), EC 1.4.12.
~ H. J. Strecker, in "Biochemists' Handbook" (C. Long, ed.), p. 333. Van Nostrand,
Princeton, New Jersey, 1961.
thus permitting quantitative determination of a-ketoglutarate in the

presence of excess NH4 ÷ and NADH. The concomitant conversion of
NADH to NAD ÷ can be followed fluorometrically or spectrophoto-
metrically.
Assay Reagents
Buffer: M/15 KH2PO~, pH 7.0, or 50 mM triethanolamine-HC1
(TRA), 10 mM MgS04, 5 mM EDTA, pH 7.0. Neutralize with
K 0 H and store at 2-4 °. The two buffers are equally effective for
the assay with most extracts. However, a faster reaction is some-
times achieved by the use of the phosphate buffer. In the presence
of tissue extracts, precipitation of phosphate salts in the cuvette is
occasionally observed after addition of the enzyme.
NADH, 1 mg/ml. 'Dissolve 1 mg NADH in 1.0 ml of 0.1 M TRA,
pH 8.2.
a-Ketoglutarate standard, 0.1 raM. A stock solution of 10 mM potas-
sium a-ketoglutarate (pH 6.0 ± 0.5) may be prepared and stored
frozen for several weeks with only a small decrease in concentra-
tion. Dilute stock solution l:100 with distilled water each day.
Ammonium sulfate, 5 M
Enzyme: glutamate dehydrogenase, 20 mg/ml (3 U/rag).
Assay Procedure
Sensitivity. Ad]ust~ the sensitivity of the fluorometer so that the addi-
tion of 1 millimicromole of I~ADH (10 t~l of 0.1 raM) to 2.0 ml of buffer
in a 1 cm ~ euvette, gives a deflection of 70--90% of the full scale on the
recorder.
buffer; 10/~l of NADH, 1 mg/ml; 5 ~l (NH4)2SO~, 5.0M.
Mix the contents, place the euvette in the fluorometer, and record the
fluorescence level. When temperature equilibration is complete (1-2 min-
utes), add 5-10 #l of glutamate dehydrogenase. An increase in fluores-
cence will be recorded (external blank), and a new baseline is established.
Add 10 ~1 of a-ketoglutarate standard to cuvette. Within 2-5 minutes the
fluorescence will decrease to a new baseline (external standard). The
standard may be added a second time and should cause the same deflec-
tion as originally recorded. Because of a change in the enhancement of
fluorescence of enzyme-bound NADH, the glutamate dehydrogenase
blank will vary depending on the NADH level, thus leading to a con-
siderably smaller blank after the second addition of glutamate deby-
drogenase (see next section).
Measurements o] a-Ketoglutarate in Unknown Samples. Aliquots of
[65] FLUOROMETRIC ASSAYS USING ENZYMATIC METtIODS 457
sample containing 0.2-4.0 millimicromoles of a-ketoglutarate are used.

The required volume of sample must be determined by trial and error.
The volume of buffer is adjusted so that buffer plus sample volume is
equal to 2.0 ml. Cuvettes are prepared as for the external standard, and
the reaction is started by the addition of glutamate dehydrogenase (Fig.
4). When the reaction is complete, a second addition of glutamate dehy-
drogenase is made (internal blank) followed by addition of a-kcto-
glutarate standard (internal standard). Internal and external standard~
Glutamate
Dehydrogenase 1
--Stir . . . . . .
. . . . . . . . : . . . . I
* 2min---~
Glutamate
Dehydrocjenase - - -
Fluorescence Decrease I aO.93mp.mole

- ketocjlutarote
FIn. 4. Determination of a-ketoglutarate with glutamate dehydrogenase. A 0.2 ml

sample of neutralized perchloric acid extract from rat liver was used for assay (34
mg fresh wt).
should be in good agreement. However, large differences will be noted

between internal and external blanks. In order to determine the correct
blank, 10 ~l of standard a-ketoglutarate solution may be added to a
cuvette containing buffer, NADH, and N H G the reaction started by
addition of en~.yme, and 10 ~l of standard a-ketoglutarate solution added
a second time. The difference between the two values obtained will equal
the en~,yme blank. Alternatively, different volumes of sample may be
assayed and the fluorescence change in divisions plotted against the
sample volume. The enzyme blank is determined by extrapolating the
line drawn through the points to the ordinate.
Discussion. Problems with the assay arise chiefly from insufficient or
inactive enzyme. To determine the location of the difficulty the following
procedure may be followed.
1. Place a cuvette containing 2.0 ml of buffer in the fluorometer.
2. Add l0 ~l of NADH solution. This gives a large increase of fluores-
cence equivalent to approximately 12 millimicromoles of substrate.
3. Add N H G this addition should produce no appreciable fluorescence
change. A large drif~ at this point is caused by precipitation of phosphate
salts and indicates the need for a different buffer.
4. Add glutamate dehydrogenase. This addition gives an increase of

fluorescence due to the fluorescence enhancement of enzyme-bound
NADH, but there should be no subsequent drift. If addition of a-keto-
glutarate gives an excessively long reaction (greater than 5 minutes),
this is usually indicative of poor enzyme, insufficient NH4÷ or the
presence of inhibitors.
5. Repeat the above procedure in the presence of sample. If the
reaction is good with external standard, but poor with sample, check for
the presence of contaminating enzymes in the GDH. Try enzyme from
a new bottle or from a different commercial supplier. Inhibition of the
reaction by the extract may be caused by high concentrations of gluta-
mate and aspartate in the extract, and can frequently be alleviated by
decreasing the sample volume or increasing the amount of enzyme.
Standardization
The concentration of a-ketoglutarate in the standard solution is
determined spectrophotometrically by measuring the decrease in optical
density at 340 m/~ accompanying the disappearance of NADH from the
following reaction mixture: buffer, 1.92 ml; NADH, 0.05 ml; NH4÷,
0.01 ml; standard or distilled water, 0.50 ml.
Mix the sample thoroughly and record the initial optical density at
340 m~ (R1) ; add 0.02 ml of glutamate dehydrogenase and take readings
until the reaction is completed (R~). The change in optical density in a
blank cuvette is subtracted from the optical density difference R 1 - R2.
Alternative Method
a-Ketoglutarate may also be measured by coupling glutamate-
oxaloacetate transaminase with malate dehydrogenase. The assay system
is similar to that described later for the aspartate assay, with the excep-
tion that excess aspartate is used in place of a-ketoglutarate.
Succinate
Determination with Succinate Thiokinase, 3s Pyruvate Kinase, s9

and Lactate Dehydrogcnase ~°
Principle
Succinate thiokinase (STK) from Escherichia coli catalyzes the
phosphorolytic cleavage of succinyl-CoA by ADP according to Eq. (1).
~Succinate:CoA ligase (ADP), EC 6.2.1.5.
*PATP.'pyruvate phosphotransferase, EC 2.7.1.40.
L-Lactate : NAD oxldoreductase, EC 1.1.I.27.
[65] FLUOROMETRIC ASSAYS USINO ENZYMATIC METHODS 459
Mg ++
Succinyl-CoA + P~ q- ADP ~ " succinate q- ATP q- CoA (I)
STK
Succinic thiokinase from bacterial and plant sources is specific for

ADP '1,42 whereas enzyme prepared from mammalian tissues uses GDP
or IDP# ~ The reaction shown in Eq. (1) is freely reversible and can be
shifted from right to left by removing one of the end products, as
indicated by Eqs. (2) and (3).
Mg ++
ADP q- phosphoenolpyruvate PK ' pyruvate q- ATP (2)
Pyruvate q- NADH q- H + , L-lactate q- NAD + (3)

LDH
When suecinate thiokinase (STK) is coupled with pyruvate kinase

(PK) and lactate dehydrogenase (LDH), succinate is quantitatively
measured by the disappearance of NADH.
Mg++
Succinate q- CoA q- phosphoenolpyruvate q- NADH q- H +
STK, PK, LDH
L-lactate q- succinyl-CoA q- P~ q- NAD + (4)
This reaction is followed by measuring either the decrease in fluores-

cence of NADH in a fluorometer or the decrease in optical density at 340
m~ in a spectrophotometer.
Assay Reagents
Buffer: 0.05M triethanolamine base (TRA), 10 mM MgS04, 5 mM

EDTA, pH 7.4. Adjust pH with HC1 and store at 2-4 °.
NADH, 2 mg/ml. Dissolve 2 mg NADH in 1.0 ml of alkaline buffer,
e.g., 0.1 M TRA, pH 8.2.
The following solutions are prepared freshly each week by dissolving

the substance in distilled water. They are stored frozen:
Phosphoenolpyruvate, Na ÷ salt (PEP), 25 mg/ml

Adenosine triphosphate, Na ÷ salt, 10 mM
41S. Kaufman and S. G. A. Alivisatos, J. Biol. Chem. 216, 141 (1955).

R. A. Smith, I. R. Frank, and I. C. Gunsalus, Federation Proc. 1~},251 (1957).
~ D. R. Sanadi, D. M. Gibson, P. Ayengar, and M. Jacob, J. Biol. Chem. 218, 505
(1965).
CoA, lithium salt, 5 mM

Succinate standard, 0.2 raM. Prepare a stock solution of 10 mM
succinic acid (neutralized with 0.1 N KOH) and store frozen.
Dilute stock solution 1:50 with distilled water each day.
Enzymes
a. Lactate dehydrogenase 0.8 mg/ml (125 U/rag). Dilute beef
heart lactate dehydrogenase (~40 mg/ml) 1:50 with distilled
water.
b. Pyruvate kinase (PK), 2 mg/ml (125 U/rag). Dilute commercial
enzyme (10 mg/ml) 1:5 with distilled water.
c. Succinate thiokinase, 0.6 mg/ml (35 U/rag). Succinate thio-
kinase is not commercially available. It can be prepared from
E. coli according ~ the method of Bridger et al., ~ or from pig
heart, by the method of Cha et al. 4~ The mammalian enzyme has
the disadvantage of requiring GTP. Commercial preparations of
GTP may be contaminated with GDP, which prevents the use
of an optimal GTP concentration in the assay, since a large excess
of NADH must be added to react with the GDP in the added
GTP. Since the K,~ of succinate thiokinase from Rhodopseudo-
monas spheroides for succinate is of the order of 1 raM, ~e a large
amount of enzyme is needed for the measuring of succinate con-
centrations between 1 and 10/~/'.
Assay Procedure
of 2 millimicromoles of NADH (10 #l of a 0.2 mM solution), to 2.0 ml of
buffer in a 1 cm~ cuvette, gives a deflection of 70-90% of the full scale
on the recorder.
External Standard and Enzyme Blank. Pipette into euvette: 2.0 ml
of buffer (50 mM TRA, 10 mM MgS04, 5 mM EDTA, pH 7.4) ; 10 ~l of
NADH, 2 mg/m]; 10 ~l of phosphoenolpyruvate, 25 mg/ml; 10 ~l of
ATP, 10 mM; 10 ~l of CoA, 5 mM; 5 ~l of lactate dehydrogenase, 0.8
mg/ml; 10 ~l of pyruvate kinase, 2 mg/m].
Mix the contents, place the cuvette in the fluorometer, and record
the fluorescence level. When temperature equilibration is complete (1-2
minutes), add 10 #l of succinate thiokinase. A small decrease in fluores-
cence is caused by addition of the enzyme (external blank), and a new
W. A. Bridger, 1t. F. Ramaley, and P. D. Boyer, this volume [14].

'sS. Cha, C.-J. M. Cha, and R. E. Parks, Jr., J. Biol. Chem. ~,42, 2577 (1967); this
volume [13].
WB. F. Burnham, Acta Chem. Scand. 17, S 123 (1963).
baseline is established. Add 10 ~1 of succinate standard (0.2 mM) to the

cuvette. Within 2-4 minutes the decrease in fluorescence will end (ex-
ternal standard). The standard and/or enzyme may be added a second
time and should cause the same deflection as originally recorded.
Succinate Measurements in Unknown Samples. Sample aliquots con-
taining 0.5-6.0 millimicromoles of succinate are used. The required vol-
ume of sample must be determined by trial and error. The buffer volume
is decreased so that buffer plus sample volume is equal to 2.0 ml. Cu-
vettes are prepared as for the external standard, and the reaction is
started by the addition of succinate thiokinase (Fig. 5). When the
reaction is complete, a second addition of succinate thiokinase is made
(internal blank) followed by addition of suceinate standard (internal
standard). The reaction rate is also decreased by the accumulation of
1.89m/~moles
Succinic Succinote
Thiokinase
End Point
J
Standard
Succinate
_ =Stir ~ ,
Reset'
j~ ~ I min ,,P- Fluorescence

Increase - -
Reset I I
Fro. 5. Determination of succinate with succinate thiokinase. A 0.05 ml sample of
neutralized perchloric acid extract from rat liver was used for assay (8 mg fresh wt).
The end point is taken as the point in the reaction curve at which the drift
becomes constant.
NAD ÷ due to the presence of large amounts of ADP, GDP, or pyruvate

in the samples or the added reagents.
Discussion. Problems with the assay arise from many sources. Local-
ization of specific difficulties may be facilitated by proceeding as follows:
1. Place a cuvette containing 2.0 ml of buffer in the fluorometer. Add
10 ~l NADH, which gives a large fluorescence increase equivalent to 24
millimicromoles succinate. Add 5 ~1 of lactate dehydrogenase. Any
decrease of fluorescence is caused by contamination of the solutions with
pyruvate (care should be taken in this assay to avoid the possibility of
introducing pyruvate inadvertently into the cuvette when making addi-
tions). If a drift of the baseline occurs after addition of lactate dehydro-
genase, this is usually overcome by further dilution of the lactate dehydro-
genase. When the reaction with LDH has reached completion, add 10 ~l
of 0.2 mM pyruvate. The reaction should require at least 20 seconds and
not more than 1 minute for completion.
2. Add 10 ~l of phosphoenolpyruvate. If a large decrease of fluores-
cence results, this is caused by contamination of the phosphoenolpyruvate
solution with pyruvate. Add 10 ~l of pyruvate kinase, which should
produce a negligible fluorescence change. Add 10 #l of 0.2 mM ADP. The
reaction should be complete in 2 minutes. If not, add more pyruvate
kinase until a rapid reaction is obtained. A further addition of pyruvate
kinase should produce a similar fluorescence change to the first addition,
if there is no ADP contamination.
3. Add 10 ~l of ATP. A large decrease in fluorescence is caused by the
presence of ADP in the ATP solution; in this case fresh ATP should
be prepared. Add successively 10 ~l of CoA, 10 ~l of succinate thiokinase,
and 10 #l of suecinate standard (0.2 raM). A slow reaction at this point
is caused by insufficient CoA, ATP, or succinate thiokinase. Excessive
drifting after completion of the reaction is caused by the breakdown of
suceinyl-CoA, or contamination of succinate thiokinase with NADH
oxidase. If a purer suecinate thiokinase preparation is not available, it
is necessary to extrapolate the end point from the drift and use the lowest
concentration of succinate thiokinase compatible with a reasonable rate
of reaction.
4. The above procedure is repeated in the presence of sample.
Troublesome drifts may be caused by contamination of one of the added
enzymes. By adding the enzyme and cofactors in the order given above,
the source of the contamination can be ascertained. Drifts at the end
of the enzyme reactions can often be minimized by decreasing the sample
volume, without undue loss of accuracy in the assay.
Standardization
The concentration of succinate in the standard solution is determined

spectrophotometrically by measuring the decrease in optical density at
340 m~ accompanying the disappearance of NADH in the following
reaction mixture: buffer, 2.02 ml; NADH, 0.05 ml; phosphoenolpyruvate,
0.05 ml; ATP, 0.05 ml; CoA, 0.05 ml; lactate dehydrogenase, 0.01 ml;
pyruvate kinase, 0.01 ml; standard succinate or H20, 0.25 ml.
After thorough mixing, the initial optical density at 340 m~ (R~) is
read against water, 0.01 ml suecinate thiokinase is then added, and read-
ings are taken at 1 minute intervals until reaction is completed (R2).
The change in optical density of the blank upon addition of succinate
thioki"~e is subtracted from the difference R1--R2.
Fumarate
D e t e r m i n a t i o n w i t h F u m a r a s e 4T a n d M a l a t e Dehydrogenase '-'7
Principle
Fumarase catalyzes the reversible hydration of fumarate to form
malate according to Eq. (1).
Fumarate + H,.,O ~ • L-malate (1)
Fumarase
L-Malate + NAD + ~= , oxaloacetate + NADH + H + (2)
MDH
Malate dehydrogenase (MDH) catalyzes the oxidation of malate by
NAD ÷ to oxaloacetate and NADH (Eq. 2). The equilibrium of this
reaction lies far to the left, so that quantitative oxidation of malate is
possible only if oxaloacetate is removed from the reaction medium. An
alkaline reaction medium is used to decrease the H ÷ concentration, and
oxaloacetate is removed by converting it to the hydrazone derivative.
Quantitative measurement of fumarate is then possible according to Eq.
(3).
pH 8.5
Fumarate -t- H20 -t- NAD + -~ hydrazine
Fumarase -t- MDI~
oxaloacetate-hydrazone + NADH + H,O + (3)
The accompanying increase in fluorescence of NADH can be observed

in a fluorometer or measured spectrophotometrically by following the
increase in optical density at 340 m~.
Assay Reagents
Buffer: 0.1 M Tris base, 0.4M hydrazine hydrate, 10 mbl MgS04, 5
mM EDTA, pH 8.5. Although Mg ÷* and EDTA are not essential for
the reactions, a faster rate was not obtained by including them in
the buffer. This buffer is also used for the assay of glutamate and
3-hydroxybutyrate.
NAD ÷, 80 mg/ml
Fumarate standard, 0.2 raM. A stock solution of 10 mM sodium
fumarate is prepared and stored frozen. The stock solution is
diluted 1:50 with distilled water.
Enzymes (use undiluted)
a. Malate dehydrogenase, 10 mg/ml (720 U/rag)
b. Fumarase, 2 mg/ml (350 U/mg)
*' L-Malate hydro-lyase,EC 4.2.1.2.
Fumarate Assay
of 2 millimicromoles of NADH (10 gl of 0.2 mM solution) to 2.0 ml of
buffer in a I cm 2 cuvette, gives a deflection of 70-90~ of the full scale on
the recorder.
buffer (0.1 M Tris, 0.4 M hydrazine, 10 mM MgS04, 5 mM EDTA, pH
8.5) ; 10 #l of NAD +, 80 mg/ml; 5 gl of malate dehydrogenase, 10 mg/ml.
Mix thoroughly, and place the cuvette in the fluorometer. When
temperature equilibration is complete (1-2 mifiutes), add 10 #l of
fumarase. A small increase in fluorescence is caused by addition of the
enzyme (external blank). Add 10 gl of fumarate standard to the cuvette.
Within 6-12 minutes the reaction should reach completion (external
standard). The standard and/or enzyme may be added a second time and
should cause the same deflection as the first addition.
Fumarase I I |
1 Reset J ~ - - I
| I [ ~--F Fluo es,cence In,creoset' '

3.4rap.moles
F'umarate
F~a. 6. Determination of fumarate with fumarase. A 0.2 ml sample of neutralized
perchloric acid extract from perfused rat liver was used for assay (34 mg fresh wt).
Malate dehydrogenase was added to the cuvette prior to the first addition of
fumarase.
Fumarate Measurements on Unknown Samples. Sample aliquots con-

taining 1-5 millimicromoles of fumarate are used. The volume of buffer
is decreased so that buffer plus sample is equal to 2.0 ml. Cuvettes are
prepared as for the external standard. After addition of malate dehydro-
genase, the initial r.eaction is allowed to reach completion, and the fluores-
cence baseline is recorded. Fumarase (10 ~l) is then added. When the
reaction is complete, a second addition of enzyme is made (internal
blank) followed by addition of 10 ~l of fumarate standard (internal
standard) (Fig. 6). Although it is theoretically possible to measure
malate and fumarate in the same cuvette, the relative slowness of both
reactions, and the 4- to 6-fold greater concentration of malate in tissue
extracts makes the combined assay impractical.
Discussion. Problems with the assay, such as a slow reaction or a
large baseline drift, can arise from inactive enzymes, decomposition of
the hydrazine in the buffer, and the presence of inhibitors in the extract.
The following procedure is useful to determine the nature of the diffi-

culty:
I. Place the cuvette containing 2.0 ml of buffer into fiuorometer, and
add 10 t~l of NAD +. An interaction between NAD ÷ and hydrazine may
necessitate waiting several minutes until a constant baseline is obtained.
Add 5 ul of malate dehydrogenase. The reaction should reach completion
in 3-5 minutes. If this reaction is slow, or does not have a clear end point,
deterioration of the hydrazine or insufficient enzyme is indicated.
2. Add 10 #l of fumarase. A small deflection will occur, but there
should be little or no drift. Add 10 gl of fumaratc standard. The reaction
should end in less than 10 minutes. A slow reaction indicates the need
for more enzyme.
Repeat procedure in the presence of the unknown sample. If the re-
actions are faster in the absence than in the presence of sample, inhibition
of one of the added enzymes by the extract is indicated. This problem
can be alleviated by increasing the enzyme concentration or decreasing
the sample volume. If the reaction is followed by a drift after addi-
tion of malate dehydrogenase, which is abolished upon subsequent addi-
tion of fumarase, contamination of the malate dehydrogenase by fume-
ruse is indicated.
Fumarase is inhibited competitively by succinate, citrate, and gly-
cine, and noncompetitively by C1-.4s The optimum pH for enzyme activ-
ity is shifted toward more alkaline pH by sulfate, arsenite, or citrate ions
with an increase in reaction rate, and toward a more acid pH by phos-
phate or arsenate. 49
Standardization
The concentration of the fumarate standard solution is determined
spectrophotometrically by measuring the increase in optical density at
340 mt~ accompanying the appearance of N A D H in the following reaction
mixture: buffer, 2.17 ml; NAD ÷, 0.05 ml; malate dehydrogenase, 0.01 ml;
standard or distilled water, 0.25 ml.
Mix thoroughly, read the initial optical density at 340 n ~ (R1)
against water, add 20 gl of fumarase, and take readings at 2 minute
intervals until the reaction is complete (R2). The change in optical
density obtained upon addition of 20 ~l of fumarase to a blank cuvette
is subtracted from the difference R2--R1.
A lte.rna tire Method
Fumarate may also be measured by coupling fumarase to malic en-
zyme [L-Malate: NADP oxidoreductase (decarboxylating~, EC 1.1.1.40].
'sV. Massey, Biochcm J. 55, 172 (1953).
'~ V. Masse, y, Biocbem. J. 53, 67 (1953).
The increase of N A D P H is measured fluorometrically or spectrophoto-

metrically.
Malate
Determination with Malate Dehydrogenase 27
Principle
Malate debydrogenase (MDH) catalyzes the oxidation of L-malate
to oxaloacetate in the presence of NAD ÷ according to Eq. (1).
L-Malate 4- NAD + . ' oxaloacetate + N A D H -4- H + (1)
MDH
The equilibrium of the reaction lies far to the left, but it can be
shifted in favor of N A D H formation by removal of thc end products. An
alkaline reaction medium is used to decrease the H + concentration, and
oxaloacetate is trapped as the hydrazone derivative. Quantitative meas-
urement of malate is thus possible. The accompanying increase in fluores-
cence of N A D H can be followed in a fluorometer or be measured spectro-
photometrically by the increase in optical density at 340 m~.
Assay Reagents
Buffer: 0.4M hydrazine hydrate, 0.5211 glycine, pH 9.5. Adjust pH
with 5 N KOH. Prepare daily.
N A D +, 80 mg/ml
Malate standard, 0.2 raM. A stock solution of 10 mM malate, pH
6.5 ± 0.5, may be prepared and stored frozen. Dilute stock solution
1:50 with distilled water for use each day.
Enzyme: malato dehydrogenase, 10 mg/ml (720 U/rag)
Assay Procedure
Sensitivity. Adjust the sensitivity of the fluorometer so that the
addition of 2 millimicromoles of N A D H (10 t~l of 0.2 mM solution) to
2.0 ml buffer in a 1 cmz cuvette, gives a deflection of 70-90?5 of the full
scale on the recorder.
buffer (0.4 M hydrazine, 0.5 M glycine, pH 9.5); and 10 #1 of NAD ÷,
80 mg/ml.
Mix thoroughly and place in fluorometer. Record the fluorescence
level. When temperature equilibration is complete (1-2 minutes) add 10
t~l of MDH. A small increase in fluorescence is caused by addition of the
enzyme (external blank). Add 10 gl of malate standard to the cuvette.
Within 3-5 minutes the increase in fluorescence will end (external stand-
ard). The standard, the enzyme, or both may be added a second time and
should cause the same deflection as was originally recorded.
Malate Measurements on Unknown Samples. Sample aliquots con-
taining 1-5 millimicromoles of malate are used. The required volume of
sample must be determined by trial and error. The buffer volume is de-
creased so that the volume of buffer plus sample is equal to 2.0 ml.
Cuvettes are prepared as for the external standard, and the reaction is
started by the addition of malate dehydrogenase. When the reaction is
complete, a second addition of enzyme is made (internal blank) followed
by addition of malate standard (internal standard) (Fig. 7).
Malote Dehydrocjenase
--~ 2min t~-
Stir - I - ~
I 0.92 mN.mole Molote

Molote
Dehydrocjenose FluorescenceIncreose'I
Fro. 7. D e t e r m i n a t i o n of malate with malate dehydrogenase. A 0.2 ml sample of
neutralized perchloric acid extract from rat liver mitochondria was used for assay
(34 mg fresh wt).
Discussion. Problems with the assay, such as a slow rate of reaction,

are caused by inactive enzyme, decomposition of the hydrazine or NAD ÷
in the buffer mixture, or inhibition of the enzyme by tissue extracts. The
procedure outlined below is useful to determine the nature of the problem
in the assay:
1. Place a cuvette containing 2.0 ml of buffer in the fluorometer, and
add 10 td NAD ÷. An interaction between NAD ÷ and hydrazine may
necessitate waiting a few minutes until a constant baseline is obtained.
A persistent drift indicates that either the hydrazine-glycine buffer or the
NAD ÷ solution should be replaced. Add 10 #l malate dehydrogenase. This
may produce a small change of fluorescence, but should not result in a
drift. Add 10 td of malate standard. The reaction should end in 3-5
minutes. If it takes longer than 5 minutes to reach completion, add more
enzyme. A high rate of drift at the end of the reaction indicates deteriora-
tion of the hydrazine buffer.
2. Repeat the above procedure in the presence of the sample. If a good
reaction is obtained with external standard, but not in the presence of
the sample, a contaminated enzyme or inhibition of the enzyme by the

tissue extract is probable. The latter problem can be alleviated by in-
creasing the enzyme concentration, or by decreasing the sample volume.
The Km of malate dehydrogenase for malate is 55/a~/. 5° The low Km
for malate, together with the low fluorescence artifact produced by
malate dehydrogenase make possible a rapid, reliable assay.
Standardization
The concentration of the malate standard solution is determined
spectrophotometrically by measuring the increase in optical density at
340 m/z accompanying the appearance of NADH in the following reaction
mixture: buffer, 2.19 ml; NAD ÷, 0.05 ml; standard or distilled water,
0.25 ml.
After thorough mixing of the cuvette, the initial optical density at
340 m~ is read against water (Rx), and 0.01 ml of malate dehydrogenase
is added. Readings are taken at 1 minute intervals until the reaction is
complete (R2). The change in optical density of the blank upon addition
of malate dehydrogenase is subtracted from the difference R~--R1.
Oxaloacetate
Determination with Malate Dehydrogenase 2T

Prlnc@le
Malate dehydrogenase catalyzes the reduction of oxaloacetate to
L-malate in the presence of NADH (Eq. 1).
Oxaloacetate q- NADH W H + ~ L-malate Jr NAD + (1)
The equilibrium of this reaction lies far to the right and quantitative
conversion of oxaloacetate to malate is achieved in the presence of a
slight excess of NADH. The decrease of NADH fluorescence or the
optical density change at 340 m~ may be used to follow the reaction.
Assay Reagents
Buffer: 50 mM triethanolamine-HC1 (TRA), 10 mM MgS04, 5 mM
EDTA. The buffer is adjusted to pH 7.4 with KOH and stored at
2--4°. (Although Mg ~ and EDTA are not needed for the reaction,
the above buffer is a convenient one to use since it is suitable for
several other assays.)
~J. R. Stern, in "Biochemists' Handbook" (C. Long, ed.), p. 329. Van Nostrand,
NADH, 0.5 mg/ml. Dissolve 0.5 mg NADH in 1.0 ml of alkaline

buffer, e.g., 0.1 M TRA, pH 8.2.
Oxaloacetate standard, 50 ~ / . Prepare a small volume of 5 mM
oxaloacetic acid solution, and adjust pH to 5.0-6.0. Dilute an
aliquot of this solution 1"100 with distilled water.
Malate dehydrogenase, 0.5 mg/ml (720 U/mg). Dilute commercial
malate dehydrogenase (10 mg/ml) 1:20 with distilled water.
Assay Procedure
Sensitivity. Adjust the sensitivity of the fluorometer so that 0.5 milli-
micromoles of NADH (10 #l of 50 ~d~/) added to 2.0 ml of buffer in a
1 em 2 cuvette gives a deflection of 70-90% of full-scale deflection on the
recorder.
External Standard and Enzyme Blank. Pipette into the cuvette:
2.0 ml of buffer (50 mM TRA, 10 mM MgCl~, 5 mM EDTA, pH 7.4);
5 ~l of NADH, 0.5 mg/ml.
Mix and place the cuvette in the fluorometer. When temperature
equilibration is complete and a constant baseline is reached (3-4 min-
utes), add 5 ~l of malate dehydrogenase. A small increase of fluorescence
results upon addition of the enzyme (external blank). Add 10 ~l of
oxaloacetate standard to the cuvette. The reaction will end within 1-2
minutes, and a new baseline is then established {external standard). The
the same deflection as originally recorded.
Oxaloacetate Measurements on Unknown Samples. Sample aliquots
containing 0.1-1.0 millimicromole of oxaloacetate are used. The required
volume of sample must be determined by trial and error. However, be-
cause of the low levels of oxaloacetate in tissue samples, a maximal
sample volume, i.e., 0.5-1.0 ml, is usually required. Alternatively, it may
be desirable to adapt the assay for use with a smaller cuvette, which
requires a total volume of 0.8 ml. With the smaller cuvette, a sample
volume of 0.5 ml may be used, together with 0.3 ml of 0.1 M TRA buffer,
pH 7.4, 2 #l of NADH, and 5 ~l of malate dehydrogenase. Otherwise, the
volume of buffer plus sample should be kept constant at 2.0 ml. Cuvettes
are prepared as for the external standard, and the reaction is started by
the addition of 5 ~l of malate dehydrogenase (Fig. 8). When the reaction
is complete, a second addition of 5 ~1 of malate dehydrogenase is made
(internal blank) followed by the addition of oxaloacetate standard
(internal standard). Because of the large sample volume usually required
for this assay, quenching of the NADH fluorescence by the sample causes
smaller internal than external standards.
470 SEPAR&TION A.ND ASSAY METHODS [55]
Discussion. The extreme lability of oxaloacetate requires special

precautions: (a) It must be borne in mind that the standard solution
may deteriorate during the course of the assay, thus necessitating
standardization both before and after the assay. (b) Samples must be
analyzed as soon as possible after extraction. (c) Oxaloacetate present
either in samples or in the standards must not be left in the cuvette
longer than is necessary to attain temperature equilibration. The ex-
tremely high sensitivity, together with the large volume required for this
assay, results in a high noise level and also produces mixing and drift
artifacts. However, the rapidity of the reaction makes the assay reliable,
with a possible error of 10-15%.
0.SmFmole
Oxaloacetate
Malate
Dehydrocjenase
'~-2rnin--~ ~ ~-~'~
Molote
Dehydrogenese Stir
FluorescenceIncreaseI
F1o. 8. Determination of oxaloacetate with malate dehydrogenase. A 0.4 ml
sample of neutralized perchloric acid extract from rat liver was used for assay (68
mg fresh wt).
Standardization
The concentration of the oxaloacetate in the standard solution is
determined spectrophotometrically by measuring the decrease in optical
density at 340 m~ accompanying the disappearance of NADH in the
following reaction mixture: buffer, 1.29 ml; NADH, 0.20 ml; standard or
distilled water, 1.00 ml.
After thorough mixing of the contents of the cuvette, the initial
optical density at 340 m~ is read against water (R1), and 10 ~l of malate
dehydrogenase is added. Readings are taken at 1 minute intervals until
the reaction ceases (R2). The change in optical density of the blank upon
addition of malate dehydrogenase is subtracted from the difference
R1--R2.
Glutamate
D e t e r m i n a t i o n w i t h G l u t a m a t e D e h y d r o g e n a s e ~G
Principle
Glutamate dehydrogenase catalyzes the oxidative deamination of
L-glutamate to a-ketoglutarate and NH~ + according to Eq. (1).
L-Glutamate + NAD + + H20 ~ a-ketoglutarute + NADH + NH, + (1)
The equilibrium of the reaction lies far to the left, thus necessitating
the removal of end products. By using an alkaline reaction medium to
decrease the H ÷ concentration, and hydrazine to remove a-ketoglutarate
as the hydrazone derivative, quantitative measurement of glutamate is
possible. The accompanying increase in fluorescence of NADH can be
followed in a fluorometer or measured spcctrophotometrically by the
increase in optical density at 340 m~.
Assay Reagents
Buffer: 0.1 M Tris base, 0.4M hydrazine hydrate, 10 mM MgCI~, 5
mM EDTA, pH 8.5
NAD +, 80 mg/ml
Glutamate standard, 0.5 raM. A stock solution of 10 mM L-glutamic
acid is prepared, neutralized, and stored frozen. The stock solution
is diluted 1:20 with distilled water.
Glutamate dehydrogenase in glycerin solution, 10 mg/ml (3 U/mg)
Assay Procedure
of 5 millimicromoles of N A D H (10 td of 0.5 mM solution) to 2.0 ml of
buffer in a 1 cm ~ cuvette, gives a deflection of 70-90% of the full scale
on the recorder.
External Standard and Enzyme Blank. Pipette into cuvette: 2.0 ml
buffer (0.1 M Tris, 0.4 M hydrazine, 10 mM MgCI2, 5 mM EDTA, pH
8.5); 10 ~1 of NAD ÷, 80 mg/ml.
After mixing the contents, place the cuvette into the fluorometer, and
record the fluorescence level. When temperature equilibration is complete
(1-2 minutes), add 20 ul of glutamate dehydrogenase. An increase in
fluorescence will be recorded (external blank). Add 10 ul of glutamate
standard to the cuvette. The reaction will end (external standard) within
8-15 minutes and a new baseline is then established. The standard may be
added a second time, and should cause the same deflection as originally
recorded. Because of the fluorescence enhancement of enzyme-bound
472 SEPARATION AND ASSAY M E T H O D S [65]
NADH, addition of glutamate dehydrogenase after a reaction causes

an increase in fluorescence which is dependent upon the level of N A D H in
the reaction mixture. Thus, the external blank will usually be smaller
than the internal blank. If the discrepancy is large, the correct enzyme
blank may be determined as described in the following paragraph.
Glutamate Measurements on Unknown Samples. Sample aliquots
containing 2-10 millimicromoles of glutamate are used. The required
volume of sample must be determined by trial and error. The buffer vol-
ume is decreased so that the volume of buffer plus sample is equal to 2.0
ml. Cuvettes are prepared as for the external standard, and the reaction
is started by the addition of glutamate dehydrogenase (Fig. 9). When
Glutamate ~ _ s e t - -
~-Dehydr°qenose/t~e I
Glutamate 5.6mF.moles Glutan ~te

Dehydrocjenase
Fluorescence Increasel
Fzo. 9. Determination of glutamate with glutamate dehydrogenase. A 0.05 ml
sample of neutralized perchloric acid extract from perfused rat liver was used for
assay (8 mg fresh wt).
the reaction is complete, a second addition of glutamate dehydrogenase is
made (internal enzyme blank) followed by addition of 10 pl glutamate
standard (internal standard). Internal and external standards should be
in good agreement. The correct enzyme blank is determined by adding
10 ~I of standard glutamate solution to a euvette containing buffer and
NAD ÷. The reaction is started by addition of enzyme, and 10 ~l of stand-
ard glutamate solution is then added a second time. The difference be-
tween the size of the two reactions will be equal to the correct enzyme
blank. Alternatively, different volumes of sample may be assayed, and
the increase of fluorescence plotted against the sample volume as the
abscissa. The blank is given by the intersection of the line drawn
through the points with the ordinate.
Discussion. Problems with the assay resulting from a slow reaction
can arise because of insufficient or inactive enzyme, deterioration of the
hydrazine in the buffer, or inhibition of the enzyme by the extract. The
Tris--Mg÷÷-EDTA buffer may be stored as a stock solution, but hydra-
zine should be added on the day of use, and the pH adjusted to 8.5.
[65] FLUOROMETRICASSAYS USING ENZYMATIC METItODS 475
A slow reaction may also be caused by the presence of NH, ÷ ions,

which favor the back reaction, or by high concentrations of glutamine or
aspartate, which inhibit the enzyme2 * Glutamate dchydrogenase sus-
pended in ammonium sulfate is unsuitable for this assay.
Standardization. The concentration of the glutamate standard solution
is determined spectrophotometrically by measuring the increase in optical
density at 340 ms in the reaction mixture prepared as follows: buffer,
2.32 ml; NAD*, 0.05 ml; 0.5 mM glutamate standard, 0.10 ml; or distilled
water, 0.1 ml.
Mix thoroughly, read the initial optical density at 340 m~ against
water, add 0.02 ml of glutamate dehydrogenase, and take readings at 1
minute intervals until the reaction reaches completion (R2). The change
in optical density caused by addition of 0.02 ml of glutamate dehydro-
genase to the blank is subtracted from the "difference R2--R~.
Aspartate
Determination with Glutamate-Oxaloacetate Transaminase 52

and Malate Dehydrogenase 27
Principle
Glutamate-oxaloacetate transaminase (GOT) catalyzes the trans-
amination of L-aspartate and a-ketoglutarate to oxaloacetate and
L-glutamate, according to Eq. 1.
L-Aspartate ~ a-ketoglutarate ~ = • oxaloacetate -I- ~-glutamate (1)
GOT
If oxaloacetate is reduced to malate with malate dehydrogenase
(MDH) and NADH, according to Eq. (2), the disappearance of NADtt
in the combined assay system is stoichiometrically proportional to the
concentration of L-aspartate (Eq. 3).
0xaloacetate -t- NADH ~ H + • L-malate ~ NAD + (2)
MDH
L-Aspartate + a-ketoglutarate -k NADH + H + MDH'
GOT
~glutamate -t- L-malate "l- NAD + (3)
Assay Reagents
Buffer: 50 mM triethanolamine-HC1 (TRA), 10 mM MgS04, 5 mM
EDTA, pH 7.4. Adjust the pH of the buffer with KOH and store
at 2-4 ° .
61j. A. Olson and C. B. Anfmsen, J. Biol. Chem. 202, 841 f1953).
~L-Aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1.
a-Ketoglutarate, 0.1 M; adjust the pH to 6.5 +__0.5 with KOH and

store frozen for no more than 2 weeks. Since ~-ketoglutarate
deteriorates rapidly in solution, it is best made up daily.
NADH 2 mg/ml. Dissolve 2.0 mg NADH in 1.0 ml of alkaline buffer,
0.1 M TRA, pH 8.2.
L-Aspartate standard, 0.5 raM. A stock solution of 10 mM L-aspartic
acid may be prepared (pH adjusted to 6.5 ± 0.5) and stored frozen.
Dilute stock solution 1:20 with distilled water.
Enzymes:
Malate dehydrogenase 1 mg/ml (720 U/rag).
Dilute commercial malate dehydrogenase (10 mg/ml) 1:10 with
distilled water.
Glutamate-oxaloacetate transaminase 10 mg/ml (180 U/rag). Use
undiluted. A small quantity should be transferred to a new vial
for use each day, to avoid the possibility of contaminating the
stock enzyme solution.
Assay Procedure
Sensitivity. Adjust the sensitivity of the fluorometer so that the addi-
tion of 5.0 millimicromoles of N A D H (10 ~l of 0.5 mM) to 2.0 ml of buf-
fer in a 1 cm ~ cuvette gives a deflection of 70-90% of the full scale on the
recorder.
External Standard and Enzyme Blank. Pipette into cuvette: 2.0 m]
of buffer (50 mM TRA, l0 mM MgS04, 5 mM EDTA, pH 7.4) ; 20 ~l of
a-ketoglutarate, 0.1 M; 20 ol of NADH, 2 mg/ml; 5 ~l of MDH, 1 mg/
ml.
Mix and place the cuvette in the fluorometer. When temperature
equilibration is complete (3-4 minutes), add 10 .~l of glutamate-oxalo-
acetate transaminase. A small increase in fluorescence results from addi-
tion of the enzyme (external blank). Add l0/~l of aspartate standard to
cuvette. The decrease in fluorescence will reach completion within 2-4
minutes (external standard), and a new baseline is obtained. The stand-
ard and/or enzyme may be added a second time and should cause the
same deflection as originally recorded.
Aspartate Measurements on Unknown Samples. Sample aliquots con-
taining 1-20 millimicromoles of aspartate are used. The required volume
of sample must be determined by trial and error. The volume of buffer
plus sample is kept constant at 2.0 ml. Cuvettes are prepared as for the
external standard and the reaction is started by addition of l0 ~1 of
glutamate-oxaloacetate transaminase (Fig. 10). When the reaction is
complete, a second addition of 10 /~l of glutamate-oxaloacetate trans-
aminase is made (internal blank) followed by addition of 10 ~l of
aspartate standard (internal standard).
reaction is obtained in the absence, but not in the presence of sample,

contamination of the glutamate-oxaloacetate transaminase is indicated.
Standardization
The concentration of L-aspartate in the standard solution is deter-
mined spectrophotometrically by adding the following reagents to a 1
cm s cuvette, in duplicate, with distilled water replacing the aspartate
standard solution for the blank: 2.20 ml of buffer; 0.10 ml of a-keto-
glutarate (0.1 M ) ; 0.05 ml of NADH (2 mg/ml) ; 0.01 ml of MDH (1
mg/ml) ; 0.10 ml of L-aspartate standard.
Mix, then read the initial optical density at 340 r ~ (R1). Add 0.02 ml
of glutamate-oxaloacetate transaminase and take readings until the re-
action reaches completion (R~). The change in optical density produced
by addition of 0.02 ml glutamate-oxaloacetate transaminase to the blank
cuvette is subtracted from the difference RI--R2.
D-3-Hydroxybutyrate
Determination with 3-Hydroxybutyrate Dehydrogenase53
Pr~wiph~
3-Hydroxybutyrate dehydrogenase catalyzes the oxidation of v-3-hy-
droxybutyrate by NAD ÷ to acetoacetate and NADH according to Eq.
(1).
v~3-Hydmxybutyrate + NAD + ~ acetoacetate + NADH ~ H + (1)
The equilibrium for this reaction lies to the left, necessitating removal
of the end products in order to drive the reaction to the right. This is
achieved by using a hydrazine buffer to remove acetoacetate as the
hydrazone derivative, an alkaline pH to decrease the H ÷ concentration,
and a large excess of NAD ÷. The conversion of NAD ÷ to NADH can be
followed fluorometrically~ or spectrophotometrically2~
Assay Reagents
Buffer: 0.1 M Tris[(hydroxymethyl)aminomethane] base (Tris),
0.4M hydrazine hydrate, 10 mM MgSO,, 5 mM EDTA, pH 8.5.
The hydrazine in the buffer tends to decompose with time. It should
be added to the stock Tris-Mg÷+-EDTA buffer on the day of use,
and the pH adjusted to 8.5.
NAD ÷, 80 mg/ml
n D-3-Hydroxybutyrate: NAD oxidoreducta~, EC 1.1.1.30.
UD. H. Williamson and J. Mellanby, in "Methods of Enzymatic Analysis" (H. U.
Bergmeyer, ed.), p. 459. Academic Press, New York, 1965.
[65] FLUOROMETRIC ASSAYS USING ENZYMATIC M E T H O D S 477
D-3-Hydroxybutyrate standard, 0.5 mM. A stock solution of 10 mM

D-3-hydroxybutyrate (pH 6.5 +__0.5) may be prepared and stored
frozen. Dilute the stock solution with distilled water 1:20.
3-Hydroxybutyrate dehydrogenase, 5 mg/ml (3 U/mg). The com-
mercial enzyme is used undiluted.
Assay Procedure
of 5 millimicromoles of N A D H (10 t~l of 0.5 raM) to 2.0 ml of buffer in a
1 cm 2 cuvette, gives a deflection of 70-90~ of the full scale on the
recorder.
buffer (0.1 M Tris, 0.4M hydrazine, 10 mM MgS04, 5 mM EDTA, pH
8.5), 10 t~l NAD ÷ (80 mg/ml).
After mixing the contents, place the cuvette in the fluorometer, and
record the fluorescence level. When temperature equilibration is complete
(1-2 minutes), add 20 ~l of 3-hydroxybutyrate dehydrogenase. A small
increase of fluorescence will occur upon addition of the enzyme (external
blank). Add 10 t~l of 3-hydroxybutyrate standard to the cuvette. The
increase in fluorescence will end within 8-15 minutes (external standard),
and a new baseline is formed. The standard and/or enzyme may be added
a second time and should cause the same deflection as originally recorded.
A slow reaction, ending in a pronounced drift, is indicative of insufficient
enzyme or decomposition of the hydrazine in the buffer. If the reaction is
not appreciably facilitated by doubling the amount of enzyme added, the
buffer should be made up fresh using new hydrazine hydrate.
3-Hydroxybutyrate Measurements on Unknown Samples. Sample
aliquots containing 1 to l0 m/~moles of 3-hydroxybutyrate are used. The
required volume of sample must be determined by trial and error. The
buffer volume is decreased so that buffer plus sample volume is equal to
2.0 ml. Cuvettes are prepared as for the external standard, and the
reaction is started by the addition of 20 t~l of 3-hydroxybutyrate dehy-
drogenase (Fig. 11). When the reaction is complete, a second addition of
enzyme is made (internal blank) followed by addition of 10 t~1 of
3-hydroxybutyrate standard (internal standard).
Discussion. Problems with the assay, characterized by a slow reaction
or excessive drifting, may arise owing to inactive or contaminated en-
zyme. Although the enzyme is generally stable for several months, oc-
casional lots have been encountered in this laboratory that either lack
activity or lose it after a short period of time. Increasing the enzyme
concentration may increase the speed of the assay. However, it is also
likely to create further problems by increasing the drift. An imprecise
end point may be caused by enzyme impurities in the 3-hydroxybutyrate

dehydrogenase reacting with substrate in the sample (e.g., malate or
glutamate). This problem may be alleviated either by adding the enzyme
contaminant (e.g., malate dehydrogenase) to the cuvette containing
sample prior to the addition of 3-hydroxybutyrate dehydrogenase, or by
decreasing the sample aliquot. Laurate, AMP, oxaloacetate, and D-fruc-
tose have been shown to have an inhibitory effect on the enzyme24,55
.B-Hydroxybutyrote
Dehydrogenose
.B-Hydl oxybutyrote 4.Sm/~rnoles

Dehydrocjenase ,B- Hydroxybutyrate
Fluorescence Increosel
Fro. 11. Determination of 3-hydroxybutyrate with 3-hydroxybutyrate dehydro-

genase. A 0.1 ml sample of neutralized perchloric acid extract from rat liver was used
for assay (17 mg fresh wt).
Standardization
The concentration of 3-hydroxybutyrate in the standard solution is
determined spectrophotometrically by adding the following reagents to
1 em 2 cuvettes, in duplicate, with distilled water replacing the 3-hy-
droxybutyrate standard solution for the blank cuvette: buffer, 2.33 ml;
NAD+, 0.05 ml; 3-hydroxybutyrate standard, 0.10 ml.
After mixing, record the initial optical density at 340 m~ (R1). Add
0.02 ml of 0.5 m M 3-hydroxybutyrate dehydrogenase, and take readings
until the reaction reaches completion (R~). The change in optical density
produced by addition of 0.02 ml of 3-hydroxybutyrate dehydrogenase to
the blank cuvette is subtracted from the difference R2 -- R1.
Acetoacetate
Determination with 3-Hydroxybutyrate Dehydrogenase2 3
Principle
3-Hydroxybutyrate dehydrogenase catalyzes the reduction of aceto-
acetate to 3-hydroxybutyrate according to Eq. (l).
~ M. N. Berry, Biochim. Biophys. Acta 92~ 156 (1964).
[65] FLUOROMETRICASSAYS USING ENZYMATIC METHODS 479
Acetoacetate + NADH + H ÷ ~ D-3-hydroxybutyrate + NAD + (1)

The equilibrium for this reaction at pH 7.056 lies far to the right,
thus making possible the quantitative measurement of acetoacetate by
following the oxidation of N A D H either fluoromctrically ~ or spectro-
photometrically. 5s
Assay Reagents
Buffer: 0.05M triethanolamine base (TRA), 10 mM MgC12, 5 mM
EDTA, pH 7.0. Adjust the pH with HC1 and store at 2-4 °.
N A D H (2 mg/ml). Dissolve 2 mg N A D H in 1.0 ml alkaline buffer,
e.g., 0.1 M TRA, pH 8.2.
Acetoacetate standard (0.5 raM). A stock solution of 10 mM aceto-
acetate pH 6.5 ± 0.5 may be prepared and stored frozen for several
weeks. Dilute stock solution with distilled water 1:20 for use each
day. A stock solution of acetoacetate of approximately 1 M was
prepared according to Krebs and Eggleston. ~9
Enzyme: 3-hydroxybutyrate dehydrogenase, 5 mg/ml (3 U/rag). The
commercial enzyme is used undiluted.
Assay Procedure
of 5 millimicromoles of NADH (10 ~I of 0.5 raM) to 2.0 ml of buffer in a
1 cm 2 cuvette, gives a deflection of 70-90% of the full scale on the
recorder.
External Standard and EnzFme Blank. Pipette into a 1 cm~ cuvette:
2.0 ml buffer (50 mM TRA, 10 mM MgC12, 5 mM EDTA, pH 7.0) ; 10
#l N A D H (2 mg/ml).
Mix, place the cuvette in the fluorometer, and record the fluorescence
level. When temperature equilibration is complete (1-2 minutes), add
20 t~l of 3-hydroxybutyrate dehydrogenase. A small increase of fluores-
cence will occur upon addition of the enzyme (external blank). Add 10 #l
of acetoacetate standard to the cuvette. Within 4--8 minutes the fluores-
cence level will decrease to a new baseline (external standard). The
the same deflection as originally recorded.
Acetoacetate Measurements in Unknown Samples. Sample aliquots
containing 1-10 millimicromoles of acetoacetate are used. The required
~H. U. Bergmeyer and E. Berndt, Enzymol. Biol. Clin. 8, 6.5 (1965).
5, D. A. B. Young and A. E. Renold, Clin. Chim. Acta 13, 791 (1966).
~J. Mellanby and D. It. Williamson, in "Methods of Enzymatic Analysis" (H. U.
Bergmeyer, ed.), p. 454. Academic Press, New York, 1965.
H. A. Krebs and L. V. Eggleston, Biochem. J. 39, 408 (1945).
volume of sample must be determined by trial and error. The buffer

volume is decreased so that buffer plus sample volume is equal to 2.0 ml.
started by the addition of 20 /Ll of 3-hydroxybutyratc dehydrogenase.
When the reaction has reached completion, a second addition of enzyme
is made (internal blank) followed by addition of l0 ~l of acetoace~ate
standard (internal standard) (Fig. 12).
Ftuorescence Increasel
/'J-Hydroxybutyrate
~-Hydroxybutymte
Dehydroqenase
FIn. 12. Determination of acetoacetate with 3-hydroxybutyrate dehydrogenase.
A 02 ml sample of neutralized perchloric acid extract from perfused rat liver was
used for assay (34 mg fresh wt).

or excessive drifting, may arise because of inactive or contaminated
enzyme. Although the enzyme is generally stable for several months,
occasional lots have been encountered in this laboratory which either
lack activity or lose it after a short period of time. The biggest problem
with this assay is the occurrence of a baseline drift after addition of the
enzyme to the buffer in the presence of NADH. Since the drift occurs in
the absence of sample, it may be caused by an N A D H oxidase as an
enzyme contaminant in the 3-hydroxybutyrate dehydrogenase. This may
make the addition of large quantities of 3-hydroxybutyrate dehydro-
genase impractical. With a good enzyme preparation, the NADH con-
cent~ration can be raised to increase the speed of reaction without appreci-
ably increasing the drift rate. The end point of the reaction is determined
as noted in Fig. 12.
Standardization
The concentration of acetoacetate in the standard solution is deter-
mined spectrophotometrically by adding the following reagents to 1 cm 2
cuvettes, in duplicate, with distilled water replacing the acetoacetate
standard solution for the blank: buffer, 2.33 ml; NADH, 0.05 ml; aceto-
acetate standard, 0.10 ml.
Mix, and record the initial optical density at 340 m~ (R1). Add 0.02
ml of 3-hydroxybutyrate dehydrogenase and take readings until the
reaction has reached completion (R2). The change in optical density
produced by the addition of 0.02 ml of the enzyme to the blank cuvette
is subtracted from the difference R1 -- R2.
Pyridine Nucleotidese°
A. Nicotinamide-Adenine Dinucleotide--Determination with
Alcohol Dehydrogenaseel
Principle
Alcohol dehydrogenase from yeast catalyzes the reduction of NAD +
by ethanol according to Eq. (1).
Ethanol -t- NAD + ~ acetaldehyde -t- NADH -t- H + (1)
The equilibrium for this reaction lies far to the left. It is shifted in
favor of NADtt formation by the use of an alkaline assay medium and
a hydrazine buffer.
Assay Reagents
Buffer: 0.1 M Tris[(hydroxymethyl)aminomethane] base (Tris),
0.4 M hydrazine hydrate, pH 8.5. This buffer is not stable and
should be prepared daily. A stock solution of Tris-MgS0~-EDTA
may be prepared and stored at 2-4 °. Hydrazine hydrate is added
to the buffer immediately prior to use, and the pH is adjusted
to 8.5.
Ethanol, 1D0%.
Enzyme: yeast alcohol dehydrogenase, 3 mg/ml (180 U/mg). Dilute
commercial alcohol dehydrogenase (30 mg/ml) 1:10 with distilled
water.
NAD ÷ standard solution (0.1 raM). Add 1.43 ml of distilled water to
1 mg of NAD*, and dilute an aliquot 1:10.
Assay Procedure
of 1 millimicromole of NADH (10 ~l of 0.1 mM solution) to 2.0 ml of
buffer in a 1 em2 cuvette, gives a deflection of 70-90% of the full scale
on the recorder.
~oR. W. Estabrook, J. R. Williamson, R. Frenkel, and P. K. Maitra, Vol. X, p. 474.
oLAleohol:NAD oxidoreductase~ E C 1.1.1.1.

of buffer (0.1 M Tris, 0.4 M hydrazine, pH 8.5) ; 10/~l of ethanol.
Mix, place the cuvette in the fluorometer and read the fluorescence
~l of alcohol dehydrogenase. A very small increase in fluorescence will be
recorded (external blank). Add 10 #1 of NAD ÷ standard to the cuvette.
Within 1-3 minutes the increase in fluorescence will end (external stand-
ard). The standard and/or enzyme may be added a second time and
should cause the same number of divisions of deflection as originally
recorded. If the enzyme blank is more than 5 ~ of the fluorescence change
upon addition of standard NAD + solution, dilute the enzyme a further 1:2.
AIcohol
Dehydrogenose
S l i r , /Reset
Stir [ - - ~ ~ ~J:
-~1 2rain M-
AlcoholDehydrogenose 30mFrnolesNAD°
FluorescenceIncreose
Fig. 13. Determination of NAD* with alcohol dehydrogenase. A 0.05 ml sample of
neutraliy,ed perchloric acid extract from perfused rat liver was used for assay (8
mg fresh wt).
NAD* Measurements on Unknown Samples. Sample aliquots con-

taining 0.2-6 millimicromoles of NAD ÷ are used. The required volume of
sample must be determined by trial and error. The buffer volume is
decreased so that the volume of buffer plus sample is equal to 2.0 ml.
Cuvettes are prepared as for the external standard and the reaction is
started by the addition of alcohol dehydrogenase. When the reaction has
reached completion a second addition of enzyme is made (internal blank)
followed by the addition of 10 ~l of NAD * standard solution (internal
standard) (Fig. 13).
Discussion. If the reaction takes longer than 3 minutes to reach
completion, and successive additions of NAD ÷ give a progressively smaller
fluorescence change, an inactive enzyme or deterioration of the hydrazine
buffer is indicated. A drift toward reoxidation of N A D H indicates the
presence of N A D H oxidase in the enzyme. This is usually corrected by
further dilution of the enzyme. Although the enzyme is generally quite

stable when refrigerated, occasional lots have been observed to lose
activity on storage. The stock enzyme solution should be kept airtight or
be stored in the lyophilized state and fresh dilutions made on the day of
use.
Sta nda ~'dization

The concentration of NAD + in the standard solution is determined
spectrophotometrically in 1 cm 2 cuvettes using the following reaction
mixture: buffer, 1.98 ml; ethanol, 0.01 ml; NAD + standard, 0.50 ml.
Mix, and read the initial optical density at 340 m~ (R1). Add 0.01 ml
of alcohol dehydrogenase and take readings until the reaction has reached
completion (R..). The optical density change upon addition of 0.01 ml of
alcohol dehydrogenase to a blank cuvette in which distilled water replaces
the NAD + solution is subtracted from the difference R2 -- R1.
B. Nicotinamide-Adenine Dinucleotide Phosphate-Determination with

Glucose-6-phosphate Dehydrogenase ~2
Principle
Glucose-6-phosphate dehydrogenase catalyzes the reduction of NADP*
by glucose-6-phosphate according to Eq. (1).
D-Glucose-6-phosphate % NADP+
6-phosphogluconate + N A D P H + H + (1)
The equilibrium for this reaction lies far to the right, thus permitting
quantitative measurement of NADP + by following the increase in fluores-
cence or absorption of NADPH.
Assay Reagents
Buffer: 0.05M triethanolamine-HC1 (TRA), 10 mM MgC12, 5 mM
EDTA, pH 7.4. Adjust the pH of this solution with KOH, and
store at 2-4 ° .
Glucose-6-phosphate, 0.1 M
NADP + standard, 50 v-M. Add 1.2 ml of distilled water to 1 mg of
NADP +, and dilute an aliquot a further 1:20.
Enzyme: glucose-6-phosphate dehydrogenase, 0.2 mg/ml (140 I U /
rag). Dilute the commercial enzyme (1 mg/ml) 1:5 with distilled
water.
u D-Glucose-6-phosphate:NADP oxidoreductase, F_A21.I.1.49.

484 SZPARATION ANn ASSAY METHODS [55]
Assay Procedure
of 0.5 millimicromole of NADH (5 ~l of 0.1 mM solution) to 2.0 ml of
buffer in a 1 cm ~ cuvette, gives a deflection of 70-90% of the full scale
on the recorder.
of' buffer (50 mM TRA, 10 mM MgCl2, 5 mM EDTA, pH 7.4); 10 ~l
of glucose {}-phosphate (0.1 M).
Mix, place the cuvette in the fluorometer, and record the fluorescence
~l of glucose-6-phosphate dehydrogenase. A very small increase in fluores-
cence will result (external blank). Add 10 #l of NADP ÷ standard to the
~eset -~1 2min
A
/_ Stir
Glucose-6-Phosphate 0.66 m/~moleNADP"

Dehydro~lenase
FluorescenceIncreaset
Fie. 14. Determination of NADP ÷ with glucose-6-phosphate dehydrogenase. A 0.5
ml sample of neutralized perchloric acid extract from rat liver mitochondria was used
for assay.
cuvette. Within 1-3 minutes the increase in fluorescence will end (external
standard). The standard and/or enzyme may be added a second time,
and should cause the same number of divisions deflection as originally
recorded.
NADP* Measurements on Unknown Samples. Sample aliquots con-
taining 0.1-1.0 m~mole are used. The required volume of sample must be
determined by trial and error. The volume of buffer is decreased so that
the volume of buffer plus sample is equal to 2.0 mh Cuvettes are prepared
as for the external standard, and the reaction is started by the addition
of glucose-6-phosphate dehydrogenase (Fig. 14). When the reaction is
complete, a second addition of enzyme is made (internal blank) followed
by the addition of 10 ~1 of NADP ÷ standard (internal standard). Large
volumes of highly fluorescent tissue extracts cause quenching of the
NADPH fluorescence resulting in lower values for the internal as corn-
[65] FLUOROMETRIC
pared with the external standards. Consequently the internal standard

should be used to calibrate the assay.
or a drift toward reoxidation of N A D P H after the reaction has ended,
indicate contamination of the enzyme with glutathione reductase or
N A D P H oxidase. The enzyme should be diluted until the backdrift is
abolished. If this is ineffective, use a different batch of enzyme.
Standardization
The concentration of :NADP ÷ in the standard solution is determined
spectrophotometrically in 1 cm ~ cuvettes using the following reaction
mixture: buffer, 1.48 ml; G-6-P, 0.1 ml; NADP ÷ standard, 1.00 ml.
Mix, and read the initial optical density at 340 m~ (R1). Add 0.01
ml of glucose-6-phosphate dehydrogenase until the reaction has reached
completion (R2). The optical density change upon addition of 0.01 ml of
glucose-6-phosphate dehydrogenase to a blank cuvette in which distilled
water replaces the NADP + solution is subtracted from the difference
R~ -- R1.
C. Reduced Nicotinamide-Adenine Nucleotides--Determination with

Lactate D e h y d r o g e n a s e 4° and G l u t a m a t e D e h y d r o g e n a s e 36
Principle
Lactate dehydrogenase catalyzes the conversion of pyruvate to L-
(+)-lactate in the presence of N A D H according to Eq. (1).
Pyruvate + N A D H -F H + ~ L-(+)dactate + NAD + (1)

The equilibrium for this reaction lies far to the right, so that in tile
presence of excess pyruvate, N A D H is quantitatively oxidized to NAD*.
N A D P H can replace N A D H in this reaction but reacts much more slowly
(at about one hundredth the rate obtained with NADH).63
Glutamate dehydrogenase catalyzes the reductive amination of a-keto-
glutarate to glutamate in the presence of ammonium ions and reduced
nicotinamide-adenine dinucleotides, according to Eq. (2).
a-Ketoglutarate + NH~+ + N A D P H (or NADH) --~

L-glutamate + NADP + (or NAD +) + H20 (2)
The equilibrium for this reaction lies far to the right, so that in the
presence of excess ammonium ions and a-ketoglutarate, N A D P H is
quantitatively converted to NADP *.
e~A. Meister, J. Biol. Chem. 184, 117 (1950),

Assay Reagents
Buffer: 0.1 M triethanolamine-HC1 (TRA), pH 7.4
Pyruvate, 0.3 M
a-Ketoglutarate, 0.3M. The pyruvate and a-ketoglutarate stock
solutions are neutralized to pH 6.0 with 1 M NaHC03, and may be
stored frozen for several weeks.
Ammonium sulfate, 3.0M. A substrate solution is prepared freshly
by mixing 0.1 ml of each of the above solutions of pyruvate,
~-ketoglutarate, and ammonium sulfate.
N A D H standard, 0.1 raM. Dissolve 0.5 mg of NADH in 0.64 ml of
0.1 M TRA, pH 8.2. Make a 1:10 dilution with the same buffer.
Solutions of N A D H and N A D P H should be prepared freshly prior
to use.
N A D P H standard (0.1 mM). Dissolve 1 mg of N A D P H in 1.1 ml of
0.1 M TRA buffer, pH 8.2. Make a 1:10 dilution with the same
buffer.
Enzymes
a. Lactate dehydrogenase, 0.2 mg/ml (125 U/mg). Beef heart
lactate dehydrogenase (40 mg/ml) is diluted 1:200 with distilled
water.
b. Glutamate dehydrogenase, 4 mg/ml (3 U/mg). Dilute glutamate
dehydrogenase (20 mg/ml) 1:5 with distilled water.
Assay Procedure
addition of 1.0 millimicromole of N A D H (10 #l of 0.1 mM solution) to
2.0 ml of buffer in a 1 cm 2 cuvette gives a deflection of 70-90% of the
full scale on the recorder.
buffer (0.1 M TRA, pH 7.4) ; 10 #l substrate solution.
Mix, place the cuvette in the fluorometer and record the fluorescence
level. When a constant baseline is reached, add 10 t~l of N A D H standard
(NADH external standard). Add 5 t~l of lactate dehydrogenase. When
the reaction is complete make a second addition of 5 i~1 of lactate
dehydrogenase. The decrease in fluorescence upon addition of the enzyme
(after correction for the lactate dehydrogenase enzyme blank) should
equal the N A D H external standard. The procedure is repeated by adding
10 ~l of NADPH, to a fresh cuvette, followed by two successive additions
of 5 tfl of GDH. The reaction upon addition of lactate dehydrogenase is
almost instantaneous, whereas that upon addition of glutamate dehydro-
genase should bc complete within 1-3 minutes.
Reduced Nicotinamide-Adenine Dinucleotide Measurements on Un-

known Samples. Sample aliquots containing 0.2-6 millimicromoles of
NADH and NADPH are used. The required volume of sample must be
determined by trial and error, and the volume of buffer plus sample is
maintained at 2.0 ml. Cuvettes are prepared as for the external standard,
and the reaction is started by addition of 5 #1 of lactate dehydrogenase.
The reaction ends with a slight drift, which is caused by the slow rate of
reaction of NADPH with lactate dehydrogenasc. Add 5 u] of glutamate
dehydrogenase, followed by a second addition of 5 ~l glutamate dehydro-
genase (internal glutamate dehydrogenase blank), and 5 ul of lactate
dehydrogenase (internal lactate dehydrogenase blank) (Fig. 15).
Lactate
Dehydrogenase - ~ 2rnin
4
--.l.--]---i I I I I ~ |Dehydrogenase
_s,i I i--l-lll-l-str r-- 1
I I--LIU oluta~ote
I ~ i II Dehydrocjenose
_End Pointl~ I J
JResets
Glutamate
Dehydrogenase
FluorescenceIncrease1'
FIG. 15. Determination of NADH and NADPH by lactate dehydrogenase and
glutamate dehydrogenase. A 0.2 ml sample of neutralized perchloric acid extract from
perfused rat liver was used for assay (34 mg fresh wt).
Internal standards are prepared as follows: place a cuvette containing

sample, buffer, and substrates in the fluorometer and add 10 ~l of NADH.
Record the change in fluorescence, and proceed with the assay as de-
scribed above. The internal NADH standard is equal to the difference
between the fluorescence change upon addition of lactate dehydrogenase
to a cuvette containing sample plus NADH standard, and an identical
euvette containing sample but no added NADH.
Discussion. Problems with the assay, characterized by a slow reac-
tion or excessive drifting, may arise as a result of inactive or con-
taminated enzymes or inadequate substrata concentration. Locating the
precise difficulty may be facilitated by proceeding as follows:
1. Place a cuvette containing 2.0 ml of buffer into the fluoromcter.
Add l0 #l of standard NADH solution and record the increase in fluores-

cence. Add 10 #l of 0.1 M pyruvate which should give a negligible change
in fluorescence. Add 5 /zl of lactate dehydrogenase, whereupon the reac-
tion should reach completion after 30-60 seconds. Adjust the concentra-
tion of lactate dehydrogenase to conform to these limits.
2. Repeat the above procedure with NADPH, using a-ketoglutarate
and NH4 ÷ as substrates and add 5 /A of glutamate dehydrogenase. If
the reaction continues for more than 2 minutes, increase the concentra-
tion of glutamate dehydrogenase or prepare fresh a solution of a-ketoglu-
tarate.
3. Because of the lability of reduced pyridine nucleotides, it is
essential to have the assay working optimally before the tissue extrac-
tion is begun. The time interval between the start of extraction and
proceeding with the assay should be as short as possible. In this labora-
tory, not more than four samples are extracted simultaneously, and the
samples are assayed within 30-40 minutes after the start of extraction.
Standardization
The concentration of N A D H or N A D P H in the standard solution is
determined spectrophotometrically by preparing the following reaction
mixture in 1 cm ~ cuvettes: buffer, 1.98 ml; substrate mixture, 0.01 ml;
standard solution or distilled water, 0.50 ml.
Mix the contents of the cuvette, and record the initial optical density
at 340 m/~ (R1). Add 0.01 ml of lactate dehydrogenase or glutamate de-
hydrogenase and take readings until the reaction has reached completion
(R2). The optical density change upon addition of enzyme to a blank
cuvette containing distilled water instead of N A D ( P ) H is subtracted
from the difference R1 --Rz.
Adenine Nucleotides
A. Adenosine 5'-triphosphate--Determination with Hexokinase 64 and

G l u c o s e - f - p h o s p h a t e D e h y d r o g e n a s e 62
Principle
Hexokinase catalyzes the phosphorylation of glucose by ATP in the
presence of Mg +÷ according to Eq. (1).
M~ ++
D-Glucose + ATP , glucose 6-phosphate + ADP (1)
ATP :D-hexose 6-phosphotransferase, EC 2.7.1.1.

[65] FLUOROMETIIIC ASSAYS USING ENZYMATIC METHODS 489
The K,, for the yeast enzyme for both glucose and ATP is about 0.i
mM25 ITP also reacts with the enzyme26
Glucose-6-phosphate dehydrogenase catalyzes the oxidation of glu-
cose-6-phosphate by NADP ÷ Eq. (2).
D-Glucose 6-phosphate + NADP + --,
6-phosphogluconate + NADPH + H + (2)
The equilibrium constant for this reaction is greatly in favor of
NADPH formation, permitting quantitative measurement of ATP accord-
ing to the overall reaction described in Eq. (3).
Glucose + ATP + NADP + --,
ADP -t- NADPH + H + + 6-phosphogluconate (3)
The increase in fluorescence or optical density accompanying the conver-
sion gives a quantitative measure of ATP if glucose is in excess, or of
glucose if ATP is in excess.
Assay Reagents
Buffer: 50 mM triethanolamine-HCl (TRA), 10 mM MgC12, 5 mM
EDTA, pH 7.4. Adjust the pH of the buffer with KOH and store
at 2 - 4 ° .
Glucose, 1.0 M
NADP ÷, 10 mg/ml
Adenosine 5'-triphosphate standard, 0.1 mM. A stock solution of 10
mM ATP (sodium salt) may be prepared and stored frozen for
several weeks. This stock solution is diluted 1:100 with distilled
water.
Enzymes
a. Hexokinase, 2 mg/ml (140 U/rag). Dilute commercial hexo-
kinase (10 mg/ml) 1:5 with distilled water.
b. Glucose-6-phosphate dehydrogenase, 0.2 mg/ml (140 U/mg).
Dilute the commercial enzyme (1 mg/ml) 1:5 with distilled
water.
Assay Procedure
tion of 1.0 millimieromole of NADH (10 ~l of 0.1 mM solution) to 2.0
ml of buffer in a 1 cm 2 cuvette gives a deflection of 70-90% of the full
u p . K. Crane, in "Biochemists' Handbook" (C. Long, ed.), p. 401. Van Nostrand,

~A. Kleinzeller, Biochem. J. 36, 729 (1942).
External Standard and Enzyme Blank. Pipette into a 1 cm 2 cuvette:

2.0 ml of buffer (50 mM TRA, 10 mM MgC]~, 5 mM EDTA, pH 7.4);
10 ~l of glucose, 1.0M; l0 #l of NADP ÷, 10 mg/m]; 5 ~l of glucose-6-
phosphate dehydrogenase 0.2 mg/ml.
Mix thoroughly, place the cuvette in the fluorometer, and record the
minutes), add 5 ~l of hexokinase. A very small increase in fluorescence
will be recorded (external blank). Add 10 ~1 of ATP standard to the
cuvette. The increase in fluorescence will end within 1-3 minutes, and a
new baseline is established (external standard). The standard and/or
enzyme may be added a second time, and should'cause the same deflec-
tion as recorded originally.
A T P Measurements on Unknown Samples. Sample aliquots contain-
ing 0.2-6 millimicromoles of ATP are used. The corresponding volume of
Reset Reset_
Stir Hexokinose 0.96 m/~moleATP

FluorescenceIncrease~
Fro. 16. Determination of ATP by glucose-6-dehydrogenase and hexokinase. A
0.2 ml sample of neutralized perchloric acid extract from rat liver mitochondria was
used. Glucose-6-phosphate dehydrogenase was added to the cuvette prior to the
recording shown.

adjusted so that the volume of buffer plus sample is equal to 2.0 m].
Cuvettes are prepared as for the external standard and the reaction is
started by the addition of 5 ~l of hexokinase. Glucose 6-phosphate may
also be measured in the same cuvette by recording the baseline before
addition of 5 ~l of glucose-6-phosphate dehydrogenase. After a new base-
line has been recorded, 5 ~l of hexokinase is added. When the reaction is
complete, a second addition of 5 /~l of hexokinase is made (internal
enzyme blank) followed by addition of 10 ~l of ATP standard (internal
standard) (Fig. 16).
Discussion. Problems with the assay, characterized by a drift toward
reoxidation of NADPI-I after the reaction with hexokinase has ended,
indicate contamination of the enzyme with glutathione reductase or
[6S] FLUOROMETRIC ASSAYS USING ENZYMATIC METHODS 491
NADPH oxidase. A slow reaction indicates insufficient enzyme. To

localize the difficulty, the following procedure is recommended.
1. Place a cuvette containing 2.0 ml of buffer in the fluorometer. Add
10 ~l of NADP ÷, followed by 5 ~1 of glucose-6-phosphate dehydrogenase
and 10 ~l of 0.1 mM glucose 6-phosphate. Correct a slow reaction by
increasing the concentration of glucose-6-phosphate dehydrogenase, and
a backdrift or an enzyme blank greater than 5% of the glucose 6-phos-
phate standard by further dilution of the enzyme.
2. Add 10 ~l of 1.0 M glucose and 5 ~1 of hexokinase: no appreciable
change in fluorescence should occur. Add 10 ~l of 0.1 mM ATP standard.
If a slow reaction is observed, correct this by increasing the concentra-
tion of hexokinase.
3. If difficulty is encountered only in the presence of sample, the
procedure outlined in steps 1 and 2 above should be repe~,d in the
presence of sample. Inhibition of either reaction by the tissue..:' act may
be diminished by decreasing the sample volume and/or increasing the
concentration of the rate-limiting enzyme for the combined reaction.
Inhibition of hexokinase by ADP, glucose 6-phosphate, and Na ÷ ions
has been observed, e~ while glucose-6-phosphate dehydrogenase is inhibited
by high concentrations of Mg ÷÷, and is activated by EDTA.
Standardization
The concentration of ATP in the standard solution is determined
spectrophotometrically by following the optical density change at 340
m# using the following reaction mixture: buffer, 1.97 ml; glucose, 0.01 ml;
NADP +, 0.10 ml; ATP standard or distilled water, 0.50 ml; glucose-6-
phosphate dehydrogenase, 0.01 ml.
After mixing the sample, read the optical density at 340 m/~ (R1).
Add 0.01 ml of hexokinase and take readings until the reaction has
reached completion (R2). The optical density change upon addition of
0.01 ml of hexokinase to a blank cuvette containing distilled water instead
of ATP standard solution is subtracted from the difference R2 -- R1.
B. Adenosine 5t-triphosphate~Determination with Phosphoglycerate

K i n a s e 6~ a n d Glyceraldehyde-3-phosphate Dehydrogenase6s
Principle
Phosphoglycerate kinase catalyzes the transfer of phosphate from
ATP to 3-phospho-D-glycerate to form ADP, and 1,3-diphospho-n-glyc-
erate according to Eq. (1).
*~ATP: 3-phospho-a-glycerate 1-phosphotransferase, EC 2.7.2.3.
Uv-Glyceraldehyde-3-phosphate:NADoxidoreductase (phosphorylating), EC 1.2.1.12.
M g ++
ATP T 3-phosphoglycerate • ADP -{- 1,3-diphospho-D-glycerate (1)
Although the equilibrium constant for the reaction from left to right
is unfavorable, a quantitative conversion of ATP can be achieved by
reducing 1,3-diphosphoglycerate with glyceraldehyde-3-phosphate de-
hydrogenase and NADH (Eq. 2). The combined reaction (Eq. 3) is
followed by measuring the decrease of fluorescence or optical density of
NADH.
1,3-Diphosphoglycerate + NADH -}- H +

glyceraldehyde 3-phosphate + NAD + + P~ (2)
Mg+ +
A T P -{-3-phosphoglycerste % N A D H ~- H + v - '
A D P -{-glyeersldehyde 3-phosphate + N A D + + P~ (3)
This assay is used in preference to the method using hexokinase and
glucose-6-phosphate dehydrogenase (see Section A above) when trichloro-
acetic acid extracts are being measured, or when glucose 6-phosphate
levels are much higher than A T P levels.It has the disadvantage of being
nonspecific insofar as ITP, UTP, and G T P are also measured.
Assay Reagents
Buffer: 50 mM triethanolamine-HC1 (TRA), 10 mM MgCl~, 5 mM
EDTA, 5 mM mercaptoethanol, pH 7.4. Adjust the pH with KOH
and store at 2-4 °. Mercaptoethanol is omitted from the stock solu-
tion and added each day.
NADH, 2 mg/ml. Dissolve 2.0 mg of NADH in 1.0 ml of 0.1 M TRA,
pH 8.2.
3-phosphoglycerate, 0.5 M
ATP standard, 0.1 m M
Enzymes
a. Glyceraldehyde-3-phosphate dehydrogenase, 10 mg/ml (36 U/
rag).
b. Phosphoglyeerate kinase, 5 mg/ml (180 U/mg). Dilute the com-
mercial enzyme (10 mg/ml) 1:2 with distilled water.
Assay Procedure
of 1 millimicromole of NADH (10 #l of 0.1 mM solution) causes a deflec-
tion of 70-90% of the full scale on the recorder when added to 2.0 ml of
buffer in a 1 em ~ cuvette.
of buffer (50 mM TRA, l0 mM MgCI2, 5 mM EDTA, 5 mM mercapto-
ethanol, pH 7.4) ; 10 ~l of NADH, 2 mg/ml; 10 ~l of 3-phosphoglyceratc,

0.5 M; 5 ~l of glyceraldehyde-3-phosphate dehydrogenase, l0 mg/ml.
After mixing the contents, place the cuvette in the fluorpmeter, and
record the fluorescence level. When temperature equilibration is com-
plete (1-3 minutes), add 5 ~l of phosphoglycerate kinase. A small increase
in fluorescence will be recorded (external blank). Add 10 ~l of 0.1 mM
ATP standard to the euvette. Within 2-3 minutes the increase in fluores-
cence will end (external standard). The standard and enzyme may be
added a second time and should cause the same deflection as originally
recorded.
ATP Measurements in Unknown Samples. Sample aliquots contain-
ing 0.2-6.0 millimicromoles of ATP are used. The corresponding volume
of sample must be determined by trial and error. The buffer volume is
:3- P glycerate
Kinase
~ ~ , ~ - - ~8mp.moles ATP
-- Stir
I.
2min
Fluorescence IncreoseI
FIo. 17. Determination of ATP with 3-phosphoglycerate kinase. A 0.2 ml sample

of neutralized perchloric acid extract from rat liver mitochondria was used for assay.
started by the addition of 5 ~l of phosphoglycerate kinase. When the
reaction is complete, a second addition of phosphoglycerate kinase is
made (internal blank), followed by addition of 10 ~l of ATP standard
(internal standard) (Fig. 17).
Discussion. Problems with the assay, characterized by a slow reac-
tion, are usually caused by insufficient or inactive enzyme, insufficient
3-phosphoglycerate, or high phosphate concentrations in the extract.
Severe drifting after the reaction has ended is most frequently caused by
contamination of the enzymes.
Standardization
The concentration of ATP in the standard solution is determined
spectrophotometrically in 1 cm~ cuvettes using the following reaction
mixture: buffer, 1.88 ml; NADH, 0.05 ml; 3-phosphoglycerate, 0.05 ml;
glyceraldehyde-3-phosphate dehydrogenase, 0.01 ml; standard or dis-
tilled water, 0.50 ml.
Mix, read the optical density at 340 m~ (RI). Add 0.01 ml of phospho-
glycerate kinase, and take readings until the reaction is completed (R o).
The optical density change upon addition of 0.01 ml of phosphoglyceratc
kinase to a blank cuvette in which the ATP standard solution is replaced
by distilled water is subtracted from the difference R1 -- R2.
C. Adenosine S'-diphosphate and Adenosine 5"-monophosphate--

Determination with Pyruvate Kinase, ~9 Myokinase, 69 and
Lactate Dehydrogenase4°
Principle
Pyruvate kinase catalyzes the phosphosphorylation of adenosine 5'-
diphosphate by phosphoenolypyruvate according to Eq. (1).
• Mg++K+
ADP q- phosphoenolpyruvate ~ ~ • ATP ~- pyruvate (1)
The pyruvate formed is reduced to lactate by NADH in the presence of
lactate dehydrogenase according to Eq. (2).
Pyruvate + NADH q- H + -~ ~lactate + NAD + (2)
Myokinase (adenylate kinase) catalyzes the phosphorylation of
AMP by ATP to form two molecules of ADP according to Eq. (3).
Mg+ +
AMP T A T P . " 2 ADP (3)
Note that 1 mole of AMP is converted to 2 moles of ADP, which cause
the oxidation of 2 moles of NADH by reactions (1) and (2). The reaction
may be followed by recording the disappearance of NADH either fluoro-
metrically or spectrophotometrically.
Assay Reagents
Buffer: M/15 KH2P04, 5 mM MgCl~, pH 7.0. Neutralize the buffer
with KOH, and prepare freshly each day.
NADH, 2 mg/ml. Dissolve 2 mg NADtt (AMP-free) in 1.0 ml of
0.1M TRA, pH 8.2. Many commercially available samples of
NADH which were tested by us were found to contain AMP. An
exception is the highly purified coenzyme available from P-L
Biochemicals, Milwaukee, Wisconsin (cat. no. 6500). Contaminating
BgATP: AMP phosphotransferase,EC 2.7.4.3.

[65] FLUOIC.OMETRIC
AMP may be removed from N A D H by the method outlined by

Estabrook et al2 °
Phosphoenolpyruvate, tricyclohexylamine salt, 25 mg/ml. This solu-
tion is stable for several weeks when frozen.
Adenosine 5'-triphosphate, 1 mM. This solution is most stable at
a pH of about 9 and may be stored frozen for several weeks.
Adenosine 5'-diphosphate standard, 0.1 mM. A stock solution of 10
mM ADP may be prepared and stored frozen at a pH of about 9.
Dilute an aliquot 1:100 with distilled water.
Adenosine 5'-monophosphate standard, 50 #M. A stock solution of
10 mM AMP may be prepared and stored frozen at pH 6-7. Dilute
an aliquot 1:200 with distilled water.
Enzymes
a. Lactate dehydrogenase, 0.8 mg/ml (125 U/mg). Dilute beef heart
lactate dehydrogenase (40 mg/ml) 1:50 with distilled water.
b. Pyruvate kinase, 1 mg/ml (125 U/mg). Dilute pyruvate kinase
(10 mg/ml) 1:10 with distilled water.
c. Myokinase (MK), 5 mg/ml (360 U/rag).
Assay Procedure
Se~sitivity. Adjust the sensitivity of the fluoromcter so that the
addition of 1.0 millimicromole of N A D H (10 tL1 of 0.1 mM) to 2.0 ml of
buffer in a 1 cm 2 cuvette gives a deflection of 70-90% of the full scale
on the recorder.
External Standard and Enzyme Blank. Pipette into a cuvette: 2.0
ml of buffer (M/15 KHoP04, 5 mM MgC12, pH 7.0); 10 t~l of NADH,
2 mg/ml; 10/~l of phosphoenolpyruvate, 25 mg/ml; 5 td of lactate de-
hydrogenase, 0.8 mg/ml.
Mix, and place the cuvette in the fluorometer. When temperature
equilibration is complete (2-4 minutes), add 5/~l of pyruvate kinase. A
very small change in fluorescence will be recorded (external pyruvate
kinase blank) and a new baseline is established. Add 10 ~l of 0.1 mM
ADP standard to the cuvette. The decrease in fluorescence should end
within 1-3 minutes (external ADP standard). Add l0 ~l of 1 mM ATP.
Any reaction is due to ADP contamination in the ATP. Add 5/~l of myo-
kinase. Add 10/A of 6 0 / ~ / A M P standard to the cuvette. The reaction
should be complete after 2-3 minutes (external AMP standard). Make a
second addition of 5 ~l of myokinase (external myokinase blank).
A D P a~d A M P Measurements in Unk~wwn Samples. Sample aliquots
containing 0.2-6 millimicromoles of ADP are used. The corresponding
volume is decreased so that the volume of buffer plus sample is equal to
2.0 ml. Cuvettes are prepared as for the external standards, and the
496 SEPAP~TZON AND ASSAY METHODS [55]
reaction is started by the addition of 5/LI of pyruvate kinase. When the

reaction is complete, a second addition of enzyme is made (internal
pyruvate kinase blank) followed by addition of 10 #l of ATP (1 raM).
The AMP reaction is started by addition of 5/~l of myokinase. When the
reaction has reached completion, M K is added again (internal M K
blank). Finally, 10/~l of ADP standard is added to give the internal ADP
standard. This will give a correct standard only if the ADP is not con-
taminated with AMP. The external and internal ADP standards should
be similar (Fig. 18).
Discussion. Problems with assay usually arise as a result of con-
tamination of the enzymes or the nucleotides. Localization of specific
difficulties may be facilitated by proceeding as outlined below.
Py,uvote
Kinase
Myokinose___.__._~
Stir Reset--D 2ram
Fluorescence Increaset
Fie. 18. Determination of ADP and AMP using lactate dehydrogenase, pyruvate
kinase, and myokinase. A 0.2 ml sample of neutralized perehlor/c acid extract from
rat liver mitochondria was used for assay.
1. Place a cuvette containing 2.0 ml of buffer in the fluorometer, and

add 10/zl of N A D H (2 mg/ml). An increase in fluorescence equivalent
to 24 m~moles ADP standard should occur. Add 5/~l of lactate dehydro-
genase. Any decrease of fluorescence is due to contamination of the buffer
or N A D H solution with pyruvate. A pronounced drift after the addition
of lactate dehydrogenase is usually remedied by further dilution of the
enzyme. Add l0 ~1 of 0.1 mM pyruvate solution. The reaction should
require at least 20 seconds and not more than 2 minutes for completion.
Add 10/~l of phosphoenolpyruvate. If a large decrease in fluorescence is
observed, this is due to the decomposition of phosphoenolpyruvate to
pyruvate. Prepare a fresh solution of phosphoenolpyruvate. Add 5 ~l of
pyruvate kinase. A decrease of fluorescence is due to contamination of
one of the solutions by ADP. ADP standard may now be added, and the
reaction should be over after 1-3 minutes.
2. Add 10 ~l of I mM ATP. Any reaction is caused by the contamina-

tion of the ATP solution with ADP. Add 5 ~l of MK. A reaction may be
due to contamination of NADH, or ATP, or ADP solutions with AMP.
Contamination of the ATP may be checked by adding it to the reaction
cuvette a second time. The presence of AMP in the ADP standard solu-
tion is indicated by a larger ADP standard in the presence than in the
absence of myokinase. Addition of 10 ~l of AMP standard, followed by
a second addition of myokinase, should give a fluorescence change equal
to the first addition, if there is no AMP in any of the solutions.
3. Repeat steps 1 and 2 in the presence of sample. This reveals
contaminants in the enzymes, which have the effect of causing marked
drifting of the end point of the reaction. The presence of high concentra-
tions of Mg** or N H J (added with the enzymes) may cause precipitation
of phosphate salts. This can be eliminated by dialysis of the enzymes or
by decreasing the Mg +* concentration in the buffer.
Standardization
The concentration of ADP or of AMP in the standard solution is
determined spectrophotometrically in 1 cm ~ cuvette using the following
reaction mixtures: (a) ADP mixture: buffer, 1.88 ml; NADI-I, 0.05 ml;
phosphoenolpyruvate, 0.05 ml; lactate dehydrogenase, 0.01 ml; ADP
standard, 0.50 ml. (b) AMP mixture: buffer, 1.82 ml; :NADH, 0.05 ml;
phosphoenolpyruvate, 0.05 ml; ATP, 0.05 ml; lactate dehydrogenase,
0.01 ml; pyruvate kinase, 0.01 ml; AMP standard, 0.50 ml.
Mix, read the initial optical density at 340 rn~ (R~). Add 0.01 ml of
pyruvate kinase (or of myokinase in the case of the AMP standard) and
take readings until the reaction has reached completion (R2). The optical
density change upon addition of 0.01 ml of pyruvate kinase (or 0.01 ml of
myokinase) to a blank cuvette containing distilled water in place of the
ADP (or AMP) standard solution is subtracted from the difference
R1 -- R2.
CoA, Acetyl-CoA, and Long-Chain Fatty Acyl-CoA ~°

A. Determination with a-Ketoglutarate Oxidase and
Phosphotransacetylase 71
Principle
a-Ketoglutarate oxidase catalyzes the oxidative decarboxylation of
a-ketoglutarate in the presence of NAD ÷ and CoA to succinyl-CoA and
NADH according to Eq. (1).
,o p. K. Tubbs and P. B. Garland, this volume [72].
,t Acetyl-CoA:orthophosphate acetyltraneferase, E C 2.3.1~.
a-Ketoglutarate -{- NAD+ + CoA --~ succinyl-CoA -4- C02 A- NADH Jr H +

(1)
Additional cofactor requirements for this reaction are contained in the
protein structure of the enzyme, viz., thiamine pyrophosphatc and lipoic
acid. a-Ketoglutarate oxidase is a complex of enzymes catalyzing a num-
ber of steps included in the overall reaction described in Eq. (1). 72,~3
The equilibrium of the reaction lies far to the right, and the Km for CoA
is of the order of 0.1 #M. 74 This permits determination of very small
quantities of CoA.
The increase in fluorescence or absorption of NADH concomitant
with the conversion of CoA to succinyl-CoA permits quantitative deter-
mination of (a) soluble CoA present in the perchlorie acid extracts of
tissue, (b) long-chain fatty acyl-CoA, after alkaline hydrolysis of the
perchloric acid-insoluble material, (c) total CoA present in neutralized
sample after extraction with ethanolic KOH, and (d) aeetyl-CoA, when
coupled with the arsenolysis reaction catalyzed by phosphotransacetylase
shown in Eq. (2).
AsO4 3-
Acetyl-CoA , CoA -4- acetate (2)
Methods of tissue extraction suitable for the measurement of CoA and
derivatives are described under the heading Preparation of Samples.
Assay Reagents
Buffer: 50 mM KH2As04, pH 7.2. Adjust the pH with K O H and store
at 2-4 °. Fifty mM KH2PO, buffer may be used when acetyl-CoA
measurements are not required.
NAD ÷, 80 mg/ml.
a-Ketoglutarate, 0.1 M, pH 6 ± 0.5. Adjust pH with KOH and store
frozen up to 1 month.
Dithiothreitol, 0.1 M.
Standard solutions:
a. Coenzyme A, 0.1 mM. Dissolve in glass distilled water, adjust to
pH 4.0 and prepare freshly on the day of use.
b. Acetyl-CoA, 0.1 mM. Dissolve the lithium or sodium salt in
distilled water, and adjust the pH to 4.0. Either CoA or acetyl-
CoA may be used as standards in the combined assay, since they
produce equivalent fluorescence changes.
't D. R. Sanadi, J. W. Littlefield, and R. M. Bock, J. Biol. Chem. 197, 851 (1952).
,a S. Kaufman, C. Gilvarg, O. Cori, and S. Ochoa, J. Biol. Chem. 203, 869 (1953).
"V. Massey, BiocMm. Biophys. Acta 38, 447 (1960).
Enzymes
a. a-Ketoglutarate oxidase, 4 U/my. The enzyme is prepared
according to Sanadi, Littlefield, and Bock, 72 and 0.1 ml aliquots
are stored frozen in the presence of 1 mM dithiothreitoh The use-
ful lifetime of the enzyme is 4-6 months when stored frozen.
b. Phosphotransacetylase, 1 mg/ml (1200 U/my). Dilute the com-
mercial enzyme (10 mg/ml), 1:10 with distilled water.
Assay Procedure
addition of 1 millimieromole of N A D H (10 ~1 of 0.1 mM solution) to
2.0 ml buffer in a 1 em 2 cuvette, gives a deflection of 70-90% of the full
External Standards and Enzyme Blanks. Pipette into a 1 em 2 cuvette:
2.0 ml of buffer (50 mM KH=As04, pH 7.2) ; 10 #1 of NAD +, 80 mg/ml;
10 td of a-ketoglutarate, 0.1 M; 5 ~1 of dithiothreitol, 0.1 M.
Mix, and place the cuvette in the fluorometer. When temperature
equilibration is complete (1-2 minutes), add 5 ~1 of a-ketoglutarate
oxidase. A small increase in fluorescence occurs upon addition of the
enzyme (external a-ketoglutarate oxidase blank). Add 10 ~1 of CoA
standard to the euvette. Within 1-3 minutes the increase in fluorescence
will end (external CoA standard). Add 10 ~1 of phosphotransacetylase,
and note the external phosphotransacetylase blank. Add 10 #1 of acetyl-
CoA standard, and the reaction should end within 3-4 minutes (external
acetyl-CoA standard). The standards and/or enzymes may be added a
second time and should cause the same defection as originally recorded.
Measurements on Unknown Samples. Sample aliquots containing 0.2-
5.0 millimicromoles of CoA and/or acetyl-CoA are used. The corre-
sponding volume of sample must be determined by trial and error. The
buffer volume is decreased so that buffer plus sample volume is equal to
reaction is started by the addition of 5 ~l of a-ketoglutarate oxidase.
When the reaction is complete, a second addition of 5/~l of the enzyme is
made (internal a-ketoglutarate oxidase blank) followed by the addition
of 10 ~l of phosphotransacetylase. When the acetyl-CoA reaction has
ended, 10 #l of phosphotransacetylase is added a second time to record
the internal phosphotransacetylase blank, and the CoA or acetyl-CoA
standard is added to calibrate the reaction. Internal and external enzyme
blanks and standards should be approximately equivalent. However, large
volumes of highly fluorescent tissue extracts cause quenching of the
NADH fluorescence, resulting in lower internal than external standards.
It is therefore more accurate to use the internal standards (Fig. 19).
Discussion. Problems with the assay may be encountered, such as an

excessively slow rate of reaction, or a reaction ending with a marked
drift. A drift toward a fluorescence increase at the end of the reaction is
probably due to the breakdown of succinyl-CoA to free CoA. A slow rate
of reaction is caused by insufficient a-ketoglutarate oxidase. If the a-keto-
glutarate oxidase blank is also large, the enzyme has probably become
inactive. Generally, it is best to use a-ketoglutarate oxidase at the
highest possible dilution compatible with the CoA reaction reaching
completion in 2-3 minutes, a-Ketoglutarate oxidase loses about one-third
of its activity after 2 months at --10°. 72 Hence as the enzyme ages, a
higher concentration has to be used, until the above-mentioned problems
necessitate the preparation of fresh enzyme.
Reset
Phosphotransocetylase
F~eset~--~. ~ 1 ~
__ Stir __ ~_:
Stir_iL_L Oxidose I--I " -- I~J/--~--4~
FluorescenceIncreasel
Fro. 19. Determination of CoA and acetyl-CoA by a-ketoglutarate oxidase and
phosphotransacetylase. A 0.2 ml sample of neutralized perchloric acid extract from
perfused rat liver was used for assay (34 mg fresh wt).
Standardization
The concentration of CoA or acetyl-CoA in the standard solution is
determined spectrophotometrically by measuring the increase in optical
density at 340 m~ in 1 cm 2 cuvettes accompanying the appearance of
NADH in the following reaction mixture: buffer, 1.87 ml; a-keto-
glutarate, 0.05 ml; NAD ÷, 0.05 ml; dithiothreitol, 0.01 ml; CoA, acetyl-
CoA, or distilled water, 0.5 ml.
After mixing the contents of the cuvette, read the initial optical
density at 340 m~ against water (R1). Add 0.02 ml of a-ketoglutarate
oxidase, and take readings at 1 minute intervals until the reaction is
complete (R~). If the concentration of acetyl-CoA in the standard solu-
tion is also being determined, add 0.02 ml of phosphotransacetylase and
take readings until the reaction ends (R3). Enzyme blanks are determined
by adding the enzyme to cuvettes containing the above reaction mixture,
but with 0.5 ml distilled water replacing the standard CoA and acetyl-
CoA solution. After correction for the enzyme blank, the CoA concentra-
tion is determined from the optical density difference R 2 - R1, and
acetyl-CoA concentration from the difference R3 -- R2.
B. Acetyl-CoA--Determination with Citrate Synthase 75 and

Malate Dehydrogenase27
Principle
Citrate synthase catalyzes the synthesis of citrate from acetyl-CoA
and oxaloacetate according to Eq. (1).
Acetyl-CoA q- oxaloacetate --* citrate q- CoA (1)
Since this reaction consumes oxaloacetate, the disappearance of acetyl-
CoA may be linked to the malate dehydrogenase reaction (Eq. 2) with
the concomitant formation of NADH.
Malate q- NAD + ~---oxaloacetate q- NADH q- H + (2)
As oxaloacetate is utilized, reaction (2) is pulled from left to right. The
overall reaction, which has an equilibrium constant of 8.38 mM at pH
7.2 TM is shown in Eq. (3).
Aeetyl-CoA -t- malate -t- NAD + --* citrate q- CoA -t- NADH -t- H + (3)
Citrate synthase has a low K~ for acetyl-CoA (22 ~M). ~7 This permits
determination of millimieromole amounts of acetyl-CoA. The formation
of NADH that is associated with the removal of acetyl-CoA may be
measured fluorometrically or spectrophotometrically. However, the for-
mation of NADH is not stoichiometric with the amount of acetyl-CoA
(see below under discussion).
Assay Reagents
Buffer: M/15 KH~PO~, pH 7.2. Adjust the pH with KOH and store
at 2-4 ° .
NAD ÷, 40 mg/ml
L-Malate, 5 raM, Na ÷ or K ÷ salt
Acetyl-CoA standard, 0.1 raM. Dissolve the Li ÷ salt (P-L Chemicals,
Inc.) in distilled water, and adjust the pH to 4-6. Prepare daily,
and standardize before use.
,BCitrate oxaloacetate-lyase (CoA-acety]ating), EC 4.1.3.7.

~' J. R. Stem, S. Ochoa, and F. Lynen, J. Biol. Chem. 198, 313 (1952).
~J. R. Stern, in "Biochemists' Handbook" (C. Long, ed.), p. 461. Van Nostrand,
Enzymes
a. Citrate synthase (CS), 1 mg/ml (70 U/rag). Dilute commercial
enzyme (Boehringer, 2 mg/ml) 1:2 with distilled water.
b. Malate dehydrogenase (MDH), 1 mg/ml (720 U/mg). Dilute
commercial malate dehydrogenase (10 mg/ml) 1:10 with distilled
water.
Assay Procedure
addition of 1 millimicromole of NADH (10 ~l of 0.1 mM solution), to
2.0 ml of buffer in a 1 cm ~ cuvette gives a deflection of 70-90~ of the
full scale on the recorder.
External Standard and Enzyme Blank. Pipette into a cuvette: 2.0 ml
of buffer (M/15 KH2P04, pH 7.2); 5 ~1 of NAD *, 40 mg/ml; 10 ~l of
L-malate, 5 raM.
Mix, and place the cuvette in the fluorometer. When the temperature
equilibration is complete (1-2 minute), add 5/~l of MDH. An increase of
fluorescence is recorded as an equilibrium is established between malate,
oxaloacetate, NAD ÷, and NADH. When a new baseline is established,
add 5 ~l of citrate synthase. A small change in fluorescence is caused by
addition of the enzyme (external citrate synthase blank). Add 10 ~l of
aeetyl-CoA standard to the euvette. Within 1-3 minutes the increase of
fluorescence will end (external acetyl-CoA standard). The above proce-
dure is followed to ensure that the assay is working properly.
2.0 millimicromoles of acetyl-CoA are used. The corresponding volume of
Cuvettes are prepared as for the external standard and 5 /~l of malate
dehydrogenase is added. The fluorescence level increases to a new base-
line, and the acetyl-CoA reaction is started by the addition of 5 ~l of
citrate synthase. When the reaction is complete, a second addition of 5
~l of citrate synthase is made (internal citrate synthase blank), followed
by addition of 10 ~l of acetyl-CoA standard (internal acetyl-CoA stand-
ard) (Fig. 20). Internal and external standards are not equivalent in this
assay. Differences are caused by quenching of NADH fluorescence by
tissue extracts and by the presence of malate in the extracts. It is there-
fore necessary to run an internal standard with each sample (see Discus-
sion).
Discussion. The principal source of difficulty with this assay is the
nonstoichiometric relationship between the removal of aeetyl-CoA and
the formation of NADH. For this reason, and because of the convenience
[65] FLUOROMETRIC
of the combined assay of CoA and acetyl-CoA in the same cuvette,

measurement of acetyl-CoA as CoA after arsenolysis of acetyl-CoA by
phosphotransacetylase is preferred (Method A). However, since a-keto-
glutarate oxidase is not commercially available, measurement of acetyl-
CoA by citrate synthase offers a useful alternative, if the precautions
described in this section are followed. Reasons for the underestimation
of acetyl-CoA by the coupled assay [Eq. (3)], have been described fully
by Pearson, TM Buckel and Eggerer, TM and Bergmeyer and Moellering. 8° The
problem can be overcome either by adding NADH, e.g., final concentra-
Fie. 20. Determination of aeetyl-CoA by citrate synthase and malate dehydro-

g e n t . A 0.1 ml sample of neutralized perehlorie acid extract from rat liver was used
for assay (17 mg fresh wt).
tion 0.1 raM, to the cuvette prior to enzyme,TM or by measuring the NADH
change upon addition of malate dehydrogenase and citrate synthase and
applying a correction formula. TM Since the addition of large quantities of
NADH is not feasible with a fluorometric assay, the second method is
described here. The derivation of the correction factor is presented fully
by Buckel and Eggerer. TM The formula used to correct the NADH fluores-
cence is as follows:
( _~MDH
Acetyl-CoA = ~CS 1 + ACS + AMDH]
where ACS is the change in reading produced in the assay upon addition
of citrate synthase, and AMDH is the change produced upon addition of
malate dehydrogenase.
,a D. J. Pearson, Biochem. J. 95, 23C (1965).
W. Buckel and M. Eggerer, Biochem. Z. 343, 29 (1965).
80H. U. Bergmeyer and H. Moellering, Biochem. Z. 344, 167 (1966).
When acetyl-CoA is being measured by the spectrophotometric assay,

it is essential to use the above formula to obtain the correct acetyl-CoA
concentration. However, problems arise when it is applied to the fluoro-
metric assay, due to the necessity of calibrating the fluorescence change by
adding acetyl-CoA standards. Several alternatives, each resulting in some
error may be followed.
1. Use external acetyl-CoA standards, and apply the correction factor
to these reactions and to the reactions with sample. This method ignores
NADH fluorescence quenching due to the presence of the extract.
2. Use internal acetyl-CoA standards and apply the correction factor
first to the reaction with endogenous acetyl-CoA, and then to the com-
bined change of endogenous acetyl-CoA and internal acetyl-CoA stand-
ard. The correct change caused by the acetyl-CoA standard is then
obtained by difference. This method was found to give unreproducible
results when different volumes of sample were used.
3. Use a constant volume of sample throughout the assay, and run an
internal acetyl CoA-standard with each cuvette. If the sample volume
and sensitivity of the fluorometer are adjusted so that ACS is two to four
times as large as AMDH, good linearity is obtained over the range of
0-4 millimicromoles of acetyl-CoA by comparing the fluorescence change
upon addition of citrate synthase with the internal acetyl-CoA standard,
on a stoichiometric basis. This method usually results in a small under-
estimation of acetyl-CoA compared to the other methods, but is the
simplest alternative.
Standardization
The concentration of acetyl-CoA in the standard solution is deter-
mined spectrophotometrically by measuring the increase in optical
density in 1 cms cuvettes at 340 mfi accompanying the appearance of
NADH in the reaction mixture prepared as follows: buffer, 1.88 ml;
NAD +, 0.05 ml; malate, 0.05 ml; acetyl-CoA standard or distilled water,
0.50 ml.
Mix the contents of the cuvette, and read the initial optical density
at 340 m~ against water (R1). Then add malate dehydrogenase (0.01
ml) to the cuvette and take readings at 1 minute intervals until the reac-
tion is complete (R2). Add citrate synthase (0.01 ml), and take readings
again until the reaction has ended (Rs). The change in optical density
due to acetyl-CoA is calculated, according to the following formula:
R~ - RI)
Acetyl-CoA -- R3 - R~ 1 4- R,
[65] FLUOROMETRIC ASSAYS USING ENZYMATIC METIIODS 505
L-(--)-Carnitine and Long-Chain Fatty Acylcarnitine Derivatives sl

Determination with Acetylcarnitine Transferase, 82 Suceinate
Thiokinase, ss Pyruvate Kinase, 3~ and Lactate Dehydrogenase4°
Principle
Acetylearnitine transferase catalyzes the acetylation of L-(--)-carni-
tine by acetyl-CoA according to Eq. 1.
L-(-- )-Carnitine + acetyl-CoA ~ acetylcarnitine T CoA (1)
The reaction is readily reversible, with an equilibrium constant of
0.6. 8s This necessitates the removal of CoA to achieve quantitative re-
action of L-(--)-earnitine with excess acetyl-CoA. A number of methods
used for the assay of CoA 7° may be used. However, most of them give
problems when applied to the fluorometric assay, such as very slow
reactions which tail off into a large drift. A suitable sequence of coupled
reactions is shown in Eqs. (2) to (4).
CoA + succinate W ATP --* succinyl-CoA W ADP W P, (2)

ADP W phosphoenolpyruvate ~ ATP + pyruvate (3)
Pyruvate T NADH T H + --~ lactate T NAD+ (4)
CoA formed in the aeetylcarnitine transferase reaction interacts with
succinate and ATP in the presence of bacterial suceinato thiokinase to
form succinyl-CoA and ADP. ADP is then phosphorylated by phospho-
enolpyruvate, to yield ATP and pyruvate. The decrease in fluorescence
which results from the oxidation of NADH by pyruvate is used as a
quantitative indicator of the earnitine concentration.
The overall reaction is shown in Eq. (5).
L-(--)-earnitine + acetyl-CoA T suecinate T phosphoenolpyruvate T

NADH + H + --~ acetylcarnitine + succinyl-CoA + P, + lactate + NAD +
(5)
This assay procedure may be used to determine (a) soluble L-(--)-
carnitine present in acid extracts of tissues, (b) long-chain fatty acyl-L-
(--)-earnitine following alkaline hydrolysis of the deproteinized residue
remaining after perehloric acid extraction, and (c) the total carnitine
present in alkaline extracts of tissues, sl
BIj. F. A. Chase, this volume [60].

B,Acetyl-CoA: carnitine O-acetyltransferase, E C 2.3.1.7.
"I. B. Fritz, S. K. Schultz, and P. A. Stere, J. Biol. Chem. 2381 2509 (1963).
506 SEPARATION A N D ASSAY METHODS [65]
Assay Reagents
Buffer: 50 mM triethanolamine base (TRA), 10 mM MgS04, 5 mM
EDTA. Adjust the pH to 7.4 with HC1, and store at 2-4 °.
NADH, 2 mg/ml. Dissolve 2 mg of NADH in 1.0 nfl of 0.I M TRA,
pH 8.2.
Phosphoenolpyruvate, tricyclohexylamine salt, 25 mg/ml
ATP, sodium salt, 10 mM
Succinate, potassium salt, 0.1 M
Acetyl-CoA, lithium salt, l0 mM
L-(--)-Carnitine standard, 0.2 raM. A stock solution of 10 mM
L-(--)-carnitine is prepared and stored frozen. Dilute stock solu-
tion 1:50 with distilled water.
Enzymes
a. Lactate dehydrogenase, 0.8 mg/ml (125 U/rag). Dilute beef
heart lactate dehydrogenase (approximately 40 mg/ml) 1:50 with
distilled water.
b. Pyruvate kinase, 2 mg/ml (125 U/mg). Dilute commercial pyru-
vate kinase (10 mg/ml) 1:5 with distilled water.
c. Suceinate thiokinase, 0.6 mg/ml (35 U/mg). Succinie thiokinase
is not commercially available. It can be prepared from E. coli
according to the method of Bridger et al., .4 or from pig heart by
the method of Cha et al. 45 The mammalian enzyme is specific
for GTP, whereas the E. coli enzyme uses ATP.
d. Acetylcarnitine transferase, 0.6 mg/ml (54 U/mg). Acetyl carni-
tine transferase is prepared by the method of Chase et al. s4
Assay Procedure
tion of 2 millimicromoles of NADH (10/zl of 0.2 mM solution) to 2.0 ml
of buffer in a 1 cm 2 cuvette gives a deflection of 70-90% of the full
of buffer (50 mM TRA, 10 mM MgS04, 5 mM EDTA, pH 7.4) ; 10/~1 of
NADH, 2 mg/ml; 10 ~l of ATP, 10 raM; 10 ~l of succinate, 0.1 M; 10
/A of acetyl-CoA, 10 mM; 5/~1 of lactate dehydrogenase, 0.8 mg/ml; 5 ~l
of pyruvate kinase, 2 mg/ml; 10 ~I of succinate thiokinase, 0.6 mg/ml.
Mix the contents, place the cuvette in the fluorometer, and record the
minutes), add 10 /A of acetylcarnitine transferase. A small change of
8~j. F. A. Chase, D. J. Pearson, and P. K. Tubbs, Biochim. Biophys. Acta 96, 162
(1~).
[65] FLUOROMETRIC
fluorescence occurs due to the external acetylcarnitine transferase blank,

and a new baseline is established. Add 10 ~l of carnitine standard (0.2
raM) to the cuvette. Within 3--5 minutes the reaction ends (external
standard). The standard and/or enzyme may be added a second time and
should cause the same deflection as originally recorded.
L-(--)-Carnitine Measurements in Unknown Samples. Samples con-
taining 0.5-10.0 millimicromoles of carnitine are used. The corresponding
volume is decreased so that the volume of buffer plus sample is equal to
Acetylcornitine 2.9 mFmoles Cornitine
2,0 n - - F -
Acetylcorniline
Tronsferose
Fluorescence Increose~
FIO. 21. Determination of carnitine with acetylcarnitine transferase, succinate
thiokinase, pyruvate kinase, and lactate dehydrogenase. A 0.1 ml sample of neutral-
ized perchloric acid extract from perfused rat liver was used for assay (17 mg fresh
wt). Lactate dehydrogenase, pyruvate kinase, and succinate thiokinase were added
to the cuvette prior to the recording shown.
reaction is started by the addition of acetylcarnitine transferase (Fig. 21).
When the reaction is complete, a second addition of the enzyme is made
(internal acetylcarnitine transferase blank) followed by addition of 10 ~l
of carnitine standard (internal standard).
Discussion. Problems with the assay may arise from many sources.
Localization of specific difficulties may be facilitated by proceeding as
follows:
1. Place cuvette containing 2.0 ml of buffer into the fluorometer. Add
10 /~l of N A D H to give a fluorescence increase equivalent to 24 milli-
micromoles of carnitine. Add 5 ~l of lactate dehydrogenase. A decrease of
fluorescence is caused by contamination of the solutions with pyruvate,
which may be ignored if it is not extensive. Care should be taken not to
introduce pyruvate from fingers into the cuvette with subsequent addi-
tions. A drift of the baseline which occurs after addition of lactate dehy-
drogenase is usually overcome by dilution of the enzyme. Add 10/~l of 0.2
m M pyruvate. The reaction should require at least 20 seconds and not

more than 1 minute for completion.
2. Add 10 ~l of phosphoenolpyruvate. If a large decrease of fluores-
cence results, the phosphoenolpyruvate is contaminated with pyruvate
and must be replaced. Add 10 ~1 of pyruvate kinase; this should produce
a negligible fluorescence change. If the change is greater than a few chart
divisions, one of the solutions is contaminated with ADP. Add 10 ~1 of
0.2 mM ADP. The reaction should be complete in 2 minutes. If it is not,
add more pyruvate kinase until a rapid reaction is obtained. A further
addition of pyruvate kinase should produce a change in fluorescence
similar to that after the first addition, if there is no contamination by
ADP.
3. Add 10 ~l of ATP. A large decrease in fluorescence is caused by
the presence of ADP in the ATP solution; in this case the ATP solution
should be replaced. Add successively 10 ~l of succinate, 10 ~l of succinate
thiokinase, and 10 ~1 of 0.2 mM CoA. A slow reaction of this point is
caused by insufficient succinate, ATP, or succinate thiokinase. Excessive
drifting after completion of the reaction is caused by the breakdown of
succinyl-CoA, or contamination of suceinate thiokinase with NADH
oxidase. If a purer succinate thiokinase preparation is not available, it is
necessary to extrapolate the end point from the drift, and to use the
lowest concentration of succinate thiokinase compatible with a reasona-
ble rate of reaction.
4. Add 10 ~l of acetyl-CoA. A flesh solution must be prepared if a
large amount of CoA is present as a contaminant. When a constant base-
line is attained, add 10 ~l of acetylcarnitine transferase followed by 10
~I of standard carnitine solution (0.2 raM). A slow reaction is indicative
of insufficient acetyl-CoA or acetylcarnitine transferase. A drift is usually
caused by NADH oxidase, necessitating extrapolation of the end point
from the drift. The amount of acetylcarnitine transferase added should
be adjusted so that a reaction is achieved which terminates after about
5 minutes with a minimum drift.
5. The above procedure is repeated in the presence of sample. Drifts
at the end of the enzyme reactions can often be minimized by decreasing
the sample volume without undue loss of accuracy in the assay. A drif~
which disappears upon addition of an enzyme indicates contamination of
a previous solution with the enzyme last added; in this case the appro-
priate solution should be replaced.
Standardization
The concentration of carnitine in the standard solution is determined
spectrophotometrically by measuring the decrease in optical density at
[55] PLUOROMETRIC ASSAYS USING ENZYMATIC METHODS 509
340 m~ accompanying the disappearance of NADH in the following

reaction mixture: buffer, 1.96 ml; NADH, 0.05 ml; phosphoenolpyruvate,
0.05 ml; ATP, 0.05 ml; succinate, 0.05 ml; acetyl-CoA, 0.05 ml; lactate
dehydrogenase, 0.01 ml; pyruvate kinase, 0.01 ml, succinate thiokinase,
0.01 ml; L-(--)-carnitine standard (0.2 mM), 0.25 ml, or distilled water,
0.25 ml.
Mix the contents of the cuvette, then read the initial optical density
at 340 m~ against water (R1); add 0.01 ml of acetylcarnitine trans-
ferase, and take readings at 1 minute intervals until the reaction is com-
pleted (R2). The change in optical density of the blank upon addition of
acetylcarnitine transferase is subtracted from the difference R1 -- R2.
Alternatively, the L-(--)-carnitine solution may be standardized
using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to measure the CoA
liberated from acetyl-CoA upon addition of acetylcarnitine trans-
ferase2 ~,8~ Use the following reaction mixture: 2.0 ml of 0.1 M Tris-HC1
-~1 mM EDTA, pH 8.0; 0.02 ml of 15 mM acetyl-CoA; 0.25 ml of carni-
fine (approximately 0.2 mM) ; 0.02 ml of 15 mM DTNB.
Follow the optical density change at 412 m~ in a 1 cm = cuvette after
addition of 0.01 ml of acetylcarnitine transferase. The molar extinction
coefficient of 5-thio-2-nitrobenzoate at pH 8.0 is 13,600 cm-1.s8
Acetylcamitine81
Determination with Acetylcarnitine Transferase, 82 Citrate Synthase, 7~

and Malate D e h y d r o g e n a s e ~7
Principle
Acetylcarnitine transferase catalyzes the acetylation of CoA by
acetylcarnitine according to Eq. (1).
CoA -t- acetylcarnitine ~ acetyl-CoA ~- L-(-)-carnitine (1)
The equilibrium constant for this reaction is 0.62 ~ The reaction is pulled
from left to right by conversion of the acetyl-CoA to citrate (Eq. 2), a
reaction catalyzed by citrate synthase. The latter has a K,, for acetyl-
CoA of 22 ~M. 77
AcetyI-CoA -I- oxaloacetate --+ citrate -t- CoA (2)
To measure this conversion fluorometrically, it is linked to the NAD ÷
dependent malate dehydrogenase reaction (Eq. 3).
N. R. Marquis and I. B. Fritz, I. Lipid Res. 5, 184 (1964).
G. L. Ellman, Arch. Biochem. Biophys. 8"2, 70 (1959),
Malate -P NAD+ - . oxaloacetate + NADH Jr H + (3)

Under suitable conditions coupling of the three reactions leads to the
production of NADH, which is proportional to the quantity of acetyl-
CoA present. However, the amount of NADH formed is not stoichiometric
with the amount of acetylcarnitine originally present (see under Discus-
sion below).
Assay Reagents
Buffer: M/15 KH2P04, pH 7.2. Adjust the pH with KOH and store at
2-4 ° .
NAD*, 40 mg/ml
Malate, 5 mM
CoA, 10 raM. CoA is most stable at pH 4.0. Prepare the solution
each day.
Acetyl-L-(--)-earnitine standard, 0.1 raM. A 10 mM stock solution
may be prepared and stored frozen. Dilute 1:100 with distilled
water before use.
Enzymes
a. Acetylcarnitine transferase (ACT), 0.6 mg/ml (54 U/mg). The
ACT was prepared by the method of Chase, Pearson, and Tubbs24
b. Citrate synthase (CS), 1 mg/ml (70 U/rag). Dilute commercial
enzyme (2 mg/ml) 1:2 with distilled water.
c. Malate dehydrogenase (MDH), 1 mg/ml (720 U/mg). Dilute
commercial enzyme (10 mg/ml), 1:10.
Assay Procedure
of 1 millimicromole of NADH (10 ~1 of 0.1 mM solution) to 2.0 ml of
buffer in a 1 cm2 cuvette gives a deflection of 70-90% of the full scale
on the recorder.
of buffer (M/15 KH~P04; pH 7.2), 5 ~l of NAD ÷, 40 mg/ml; 10 ~l of
malate, 5 mM; 10 ~l of CoA, 10 raM; 5 ~l of malate dehydrogenase, 1
mg/ml; 5 ~l of citrate synthase, 1 mg/ml.
Mix thoroughly and place in fluorometer. When temperature equilibra-
tion is complete (1-2 minutes), add 10 #l of acetylcarnitine transferase.
A small change in fluorescence is caused by addition of the enzyme
(external acetylcarnitine transferase blank). Add 10 ~l of acetylearnitine
standard to the cuvette. Within 4-6 minutes the increase in fluorescence
will end, and a new baseline is reached (external standard). This proce-
dure ascertains that the rate of the ACT reaction is optimal before
proceeding with the assay in tissue samples.

5.0 millimicromoles of acetylcarnitine are used. The required volume of
sample must be determined by trial and error, and the volume of buffer
plus sample is made equal to 2.0 ml. Cuvettes are prepared as for the
external standard but without addition of any enzymes. When a constant
baseline is obtained, 5 #l of malate dehydrogenase is added and the
fluorescence change is recorded. Citrate synthase (5/~l) is then added, and
the reaction of endogenous acetyl-CoA is recorded (Fig. 22A). Then 10
~l of acetylcarnitine transferase is added, and the fluorescence change is
followed (Fig. 22B). The reaction should reach completion in 4-6 minutes.
Stir
__ - ~-r--
Mo~e Dehydrogenose, I,+f--~'/
~ _ Acetylcorniline
?-
Tronsferose / I J |
FIO. 22. Determination of carnitine with acetylcarnitine transferase, citrate syn-

thase, and malate dehydrogenase. A 0.2 ml sample of neutralized perchloric acid
extract from perfused rat liver was used for assay (34 mg fresh wt). Trace B is a
continuation of the recording shown in trace A.
A second addition of 10 ill of acetylcarnitine transferase gives a small
fluorescence change (internal acetylcarnitine transferase blank). The
fluorometer may be calibrated by the addition of either an acetyl-CoA
standard or an acetylcarnitine standard. Because of the nonstoichiomctry
between the NADH change and the concentration of acetyl-CoA or
acetylcarnitine, correction factors have to be used to calculate the results,
as described below.
D~scussion. The problems related to this assay have been discussed in
connection with acetyl-CoA determination (p. 502). Since measurement
of acetylcarnitine involves one more step than measurement of acetyl-
CoA, corrections to take into account the shift of the malate dehydro-
genase equilibrium are more involved. One method of calculation which
has been found suitable for the fluorometric assay was devised by Chase. sl
Essentially, it is necessary to calculate first the true acetyl-CoA content

from the observed fluorescence changes upon addition of malate dehy-
drogenase and citrate synthase, and then the total acetyl-CoA plus
acetylcarnitine content from the combined changes upon addition of
malate dehydrogenase, citrate synthase, and acetylcarnitine transferase.
The acetylcarnitine content is obtained by difference. The calcula-
tion method described by Buckel and Eggeref 9 is given below. However,
the two methods are equivalent.
The following formula is used to calculate the fluorescence change
proportional to the acetyl-CoA content:
( zXMDH
Acetyl-CoA = ACS 1 + ,~CS-~-"~-MDH]
where ACS is the fluorescence change observed after addition of citrate

synthase (after correction for the enzyme blank), and AMDH is the
fluorescence change observed after addition of malate dehydrogenase
(after correction for the enzyme blank).
The combined fluorescence change due to the reaction of both acetyl-
CoA and acetylcarnitine is calculated as follows:
Acetyl-CoA + acetylcarnitine
-- ACS -t- AACT 1 + z~ACT -t- ACS -t- z~MDH

where AAGT is the fluorescence change upon addition of acetylcarnitine
transferase (after correction for the enzyme blank).
The acetyl-CoA standard is more convenient to use than the acetyl-
carnitine standard to calibrate the fluorometer. The calculations are
made simpler if an external standard is used. This involves no great error
due to N A D H fluorescence quenching of the tissue extract if small
sample volumes are used. The number of divisions of fluorescence equiva-
lent to a known amount of acetyl-CoA standard is calculated as above,
and this value may be used for the calibration of the unknown acetyl-
carnitine values. Using the correction factors described above, the rela-
tionship between the acetylcarnitine content and the sample volume was
found to be linear over the range 0.05-0.3 ml.
If the contents of acetyl-CoA and acetylcarnitine in the tissue extract
are within the same order of magnitude, both compounds can be measured
in the same cuvette. However, due to the lack of stability of CoA and
acetyl-CoA in neutralized perchloric acid extracts, it is generally prefera-
ble to determine CoA and acetyl-CoA in the same cuvette by the a-keto-
glutarate oxidase method (described earlier), and to determine acetyl-
carnitine separately, at a later time.
[66] CHEMICALMETHODS FOR CITRATE .AND ACONITATE 513
Standardization
The concentration of acetylcarnitine in the standard solution is
density at 340 m~ accompanying the appearance of N A D H in the
reaction mixture, as follows:
Pipette into a 1 cm 2 cuvette: buffer, 1.82 ml; NAD ÷, 0.05 ml; malate,
0.05 ml; CoA, 0.05 ml; acetylcarnitine standard or distilled water,
0.50 ml.
The contents of the cuvettes are mixed, and the initial optical density
at 340 m~ is read against water (R1). Then 0.01 ml of malate dehydro-
genase is added to the cuvettes and readings are taken until the reaction
is complete (R2). If the solution contains no acetyl-CoA, 0.01 ml of
citrate synthase and 0.01 ml of acetylcarnitine transferase may be added
together and readings taken until the reaction is complete (R3). The
change in optical density due to acetylcarnitine (AAC) is calculated from
the formula given below, after suitable correction of the optical density
changes for the enzyme blanks.
AAC = R3 - R~ 1 + ----L-RR1R3
[66] Chemical M e t h o d s for Citrate and Aconitate

By JOHN M. LOWENSTEIN
Although enzymatic methods for the estimation of citrate and aconi-

tate are available (this volume [65]), the chemical methods described
below are useful in a number of situations, for instance when a large
number of analyses must be performed on a routine basis. The simplest
chemical method for citrate, that of Saffran and Denstedt, 1 fell into dis-
repute because of its alleged lack of accuracy. 2 We have found that this
method yields very reproducible results when performed according to the
original instructions. 8 The method lacks specificity insofar as citrate,
isocitrate, c/s-aconitate, and t r a n s - a c o n i t a t e all yield colored compounds
with similar absorption spectra. However, in many experimental situa-
tions this is of little concern because citrate predominates over the other
compounds.
1M. Saffran and O. F. Denstedt, J. Biol. Chem. 175, 849 (1948).
' See J. R. Stem, Vol. III, p. 425.
'A. F. Spencer and J. M. Lowenstein, Biochem. J. 103, 324 (1967).
[66] CHEMICALMETHODS FOR CITRATE .AND ACONITATE 513
Standardization
The concentration of acetylcarnitine in the standard solution is
density at 340 m~ accompanying the appearance of N A D H in the
reaction mixture, as follows:
Pipette into a 1 cm 2 cuvette: buffer, 1.82 ml; NAD ÷, 0.05 ml; malate,
0.05 ml; CoA, 0.05 ml; acetylcarnitine standard or distilled water,
0.50 ml.
The contents of the cuvettes are mixed, and the initial optical density
at 340 m~ is read against water (R1). Then 0.01 ml of malate dehydro-
genase is added to the cuvettes and readings are taken until the reaction
is complete (R2). If the solution contains no acetyl-CoA, 0.01 ml of
citrate synthase and 0.01 ml of acetylcarnitine transferase may be added
together and readings taken until the reaction is complete (R3). The
change in optical density due to acetylcarnitine (AAC) is calculated from
the formula given below, after suitable correction of the optical density
changes for the enzyme blanks.
AAC = R3 - R~ 1 + ----L-RR1R3
[66] Chemical M e t h o d s for Citrate and Aconitate

By JOHN M. LOWENSTEIN
Although enzymatic methods for the estimation of citrate and aconi-

tate are available (this volume [65]), the chemical methods described
below are useful in a number of situations, for instance when a large
number of analyses must be performed on a routine basis. The simplest
chemical method for citrate, that of Saffran and Denstedt, 1 fell into dis-
repute because of its alleged lack of accuracy. 2 We have found that this
method yields very reproducible results when performed according to the
original instructions. 8 The method lacks specificity insofar as citrate,
isocitrate, c/s-aconitate, and t r a n s - a c o n i t a t e all yield colored compounds
with similar absorption spectra. However, in many experimental situa-
tions this is of little concern because citrate predominates over the other
compounds.
1M. Saffran and O. F. Denstedt, J. Biol. Chem. 175, 849 (1948).
' See J. R. Stem, Vol. III, p. 425.
'A. F. Spencer and J. M. Lowenstein, Biochem. J. 103, 324 (1967).
Although the pentabromoacetone method is more specific for citrate, ~

it is more complicated and more time-consuming than the acetic anhy-
dride-pyridine method of Saffran and Denstedt. Under the conditions
described previously the pentabromoacetone method is difficult to use
reproducibly. The method given below includes modifications devised by
Dr. R. J. Rubin which overcame this problem.
Acetic Anhydride-Pyridine Method 3

The method is essentially that described by Saffran and Denstedt, I
except that one-fifth of their recommended volumes are used. The final
concentration of trichloroacetic acid in the assay mixture is critical~ and
the recommended concentration should be used whenever possible.
Changes in this parameter affect the yield of the colored compound, which
is measured spectrophotometrically.
Reagents
Acetic anhydride
Pyridine
Procedure. The extract is prepared to contain a final concentration
of 5 ~ (w/v) trichloroacetie acid. Acetic anhydride (1.6 ml) is added to
0.2 ml of the extract prepared as above. The mixture is heated at 60 ° for
10 minutes and cooled to room temperature by immersion in cold water.
Pyridine (0.2 ml) is added, the tube is sealed with a glass stopper and is
heated at 60 ° for 40 minutes. The tube is then cooled in ice and the
extinction of the solution is determined at 425 m~ (light path 1 cm).
The specificity of the method is shown in the table. Under the above
conditions, trans-aconitate gives an extinction coefficient that is 15~
higher, and c/s-aconitate one that is 35% lower, than that for citrate.
Isocitrate gives an extinction coefficient that is only 25% that of citrate.
The aconitase equilibrium at pH 7.4 and 25 ° is 90.07~ of citrate, 2.9~ of
c/s-aeonitate and 6.2% of isocitrate2 Under equilibrium conditions cis-
aconitate will yield about 2 ~ of the color obtained with citrate, and
isocitrate will contribute less than 2 ~ of the color. However, equilibrium
conditions may not be attained in living cells. Unusual conditions can
be guarded against by measuring the amount of isocitrate present with
isocitrate dehydrogenase (this volume [65]); aconitate can be measured
separately by carrying out the acetic anhydride--pyridine method at 0 °.
4G. W. Pucher, C. C. Sherman, and H. B. Vickery, Y. Biol. Chem. 113, 235 (1936);
S. Natelson, J. B. Pincus and J. K. Lugovoy, ibid. 175, 745 (1948).
sH. A. Krebs, Biochem. J. 54, 78 (1953).
[66] CHEMICAL METHODS FOR CITIL~TE AND ACON1TATE 515
Under these conditions aconitate gives the color reaction whereas citrate
gives little or no color. Tartrate and glutaconate are the only other com-
pounds that yield significant extinctions at 425 m~, the molar extinction
coefficients being about 8% of that for citrate. Tartrate yields a colored
compound with an absorption spectrum virtually the same as that ob-
tained with citrate, whereas glutaconate yields a totally different absorp-
tion spectrum with a maximum at 480 n~.
Pentabromoacetone Method ~
Eeagents
H2S0,, 18 N
Bromine water
Mn02 suspension (prepared by mixing together 1 vol of 1 M MnCl,,
1 vol of 1 M KMn04, and 0.8 vol of 18 N H~S04)
H~O~, 6 ~ (v/v)
KMnO~, 50 mM
Heptane
Thiourea, 4 ~ (w/v)
Sodium borate, 2 ~ (w/v)
Procedure. The unknown solution is deproteinized by addition of

trichloroacetic acid to a final concentration of 5 ~ (w/v). To 5.0 ml of
this solution is added 0.2 ml of 18 N H~SO~ and a boiling chip. The solu-
tion is heated in an oil bath at 120-130 ° until its volume is reduced to
about 2 ml; it is then cooled to room temperature. Bromine water (0.8
ml) is added, the solution is shaken vigorously, 2.0 ml of a suspension
of Mn02 is added, and the mixture is again shaken vigorously and al-
lowed to stand at room temperature for 15 minutes. The mixture is cooled
to 0 °, and 6% (v/v) hydrogen peroxide is added dropwise until the sus-
pension becomes colorless. Any slight excess of peroxide is destroyed by
cautious addition of 50 mM KMnO~ until a faint.yellow color appears.
Next 0.2 ml of 50 m M KMnO~ is added, the volume is adjusted to 5 ml
and 4 ml of heptane is added. The tube is stoppered and shaken on a
wrist-action shaker for 10-15 minutes. Part of the heptane layer (3.5
ml) is withdrawn and added to 3.0 ml of a solution containing 47~ of
thiourea and 2 ~ of sodium borate. The mixture is shaken in a glass-
stoppered tube for 10 minutes, then the aqueous layer is withdrawn and
its extinction is measured at 430 m#. Under these conditions 0.1 micro-
mole of citrate yields an extinction at 430 n ~ of 0.126 (1 cm light path),
corresponding to an extinction coefficient of 5040 mole -1 cm -1.
SPECIFICITY OF ACETIC ANHYDRIDE--PYRIDINE METHOD

FOR DETERMINATION OF CITRATEa'b
Molar
Amounts tested extinction
~moles/2.0 ml of coefficient
Compound reaction mixture) (425 mg)
Citrate 0.05, 0.1, 0.2 3620

Isocitrate 0.2, 0.4, 1.0 900
c/s-Aconitate 0.05, 0.1, 0.2 2360
trans-Aconitate 0.05, 0.1, 0.2 4160
Tricarballylate 0.2, 2.0 0
Benzene-l,2,3-tricarboxylate 0.2, 2.0 0
Oxalate 5, 20 0
Malonate 100, 200 3
Succinate 5, 20 0
Glutarate 0.2, 2.0 0
~-Methylglutarate 0.2, 2.0 3
Oxaloacetate 1.25, 2.5c 90
a-Oxoglutarate 0.2, 2.0 0
Malate 5, 10, 20 25
Citramalate 0.2, 2.0 <1
~-Hydroxy-#S-methylglutarate 2, 5, 10, 20 42
v~-Tartrate 0.25, 0.5, 1.0 286
Fumarate 1.25, 2.5, 5 132
Maleate 5, 10, 15 38
Citraconate 0.2, 2.0 2
Mesaconate 0.2, 2.0 0
Itaconate 10, 20, 50 3
Glutaconate 1.25, 2.5 310
Dihydroxymaleate 5, 20 0
Pymvate 5, 20 0
Acetvacetate 5, 20 0
Lactate 5, 20 0
#S-Hydroxybutyrate 5, 50 0
Crotonate 5, 50 0
Glyoxylate 5, 20 <1
Ascerbate 1.25, 3.5, 5.0 58 a
Oxaloglycolate 0.2, 2.0 0
o The method used is described in the text. The final volume of the reaction mixture
was 2.0 ml.
Reproduced from A. F. Spencer and J. M. Lowenstein, Biochem. J. 108, 342 (1967).
Higher concentrations give anomalous results.
J From the lowest amount tested; larger amounts gave lower values.
[67] DETERMINATION OF CITRIC ACIDS WITH CITRATE LYASE 517
[67] D e t e r m i n a t i o n of C i t r i c A c i d b y M e a n s of C i t r a t e L y a s e
[EC 4.1.3.6 Citrate oxaloaeetate-lyase]
B y S. DAfiLEY
Aerobacter aerogenes (Klebsiella aerogenes) can grow with citrate as
sole source of carbon in a mineral salts medium. When the culture is
aerated, citrate is metabolized by reactions of the tricarboxylic acid
cycle, but when oxygen is withheld, citrate lyase [EC 4.1.3.6] is dere-
pressed and the growth substrate is cleaved to acetate and oxaloacetate. 1
This aldolase, which has also been referred to as citratase, 2 is activated
by a divalent metal ion (Mg**, Zn÷÷, Mn ÷÷, Fe z*, or Co +*) but coenzyme
A is not required? Extracts of A. aerogenes grown anaerobically or
semianaerobically with citrate also contains high concentrations of
oxaloacetate decarboxylase [EC 4.1.1.3], which is not separated readily
from citrate lyase: accordingly, pyruvate arises from the joint action of
these two enzymes
Citrate ~ oxaloacetate -~ acetate

0xaloacetate -~ pyruvate + carbon dioxide
Previous methods for citrate assay have depended upon the determina-
tion of pyruvate so formed,4,5 and extensive purification of citrate lyase
was neither necessary nor desirable. However, these methods used Mg**
ions as cofactor and suffered from the disadvantage that only a limited
amount of citrate was decomposed before citrate lyase became inacti-
vated completely. In the present modification~ Zn+~ ions replace Mg ~+
ions since the enzyme remains active for much longer in the presence of
Zn++,~ probably because of the formation of a zinc-oxaloacetate complex
which is less inhibitory to the enzyme than that formed between Mg **
and oxaloacetate.~
Method
Principle. Citrate is converted into acetate and pyruvate when
incubated with a cell-free extract containing citrate lyase and oxalo-
S. Dagley and E. A. Dawes, J. Bacteriol. 66, 259 (1953).

"~H. H. Daron and I. C. Gunsalus, Vol. V [85].
S. Dagley and E. A. Dawes, Biochim. Biophys. Acta 17, 177 (1955).
4S. Dagley and E. A. ]:)awes, Enzymologia 16, 226 (1953).
~S, Dagley, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), p. 313.
H. Moellering and W. Gruber, Anal. Biochem. 17, 369 (1966).
7W. Gruber and It. Moellering, Biochcm. Z. 346, 85 (1966).
518 SEPARATION" AND ASSAY METHODS [67]
acetate decarboxylase. Pyruvate is determined by following the increase

in optical density at 340 mt~ which accompanies the reduction of NAD
on addition of lactate dehydrogenase2 Sufficient oxaloacetate decarboxyl-
ase is normally present to ensure the complete conversion of 1 mole of
citrate into pyruvate with the concomitant oxidation of 1 mole of
NADH, but any trace of unchanged oxaloacetate may be reduced
enzymatically by addition of malate dehydrogenase.
Reagents
Buffer: triethanolamine, 0.1 M pH 7.6
NADH, 10 mM
Zinc chloride, 3 mM
Lactate dehydrogcnase (muscle), 2 mg of protein/ml (360 IU/mg)
Malate dehydrogenase, 2 mg of protein/ml (720 IU/mg)
Enzyme preparation, 10 mg of protein/ml
Procedure. In a cuvette of 1 cm light path and 3 ml capacity, place
the reagents in the following order: 2.66 ml of buffer, 0.06 ml of NADH,
0.20 ml of zinc chloride, 0.05 ml of citrate solution (0-0.01 M, pH brought
to 7.5 with NaOH), 0.01 ml of lactate dehydrogenase, 0.01 ml of malate
dehydrogenase, 0.01 ml of enzyme. The change in optical density is
complete in 5-10 minutes at 25 ° . When phosphate or carbonate is present
in samples, the amount of zinc chloride is limited by the tendency of the
zinc to precipitate. The amount of zinc chloride added may be reduced
and the volume of enzyme increased in proportion.
The growth conditions and preparation of a cell-free extract have
been described for a strain of Aerobacter aerogenes suitable for use in
this determination3 Any strain of the organism that grows well without
aeration in a citrate-mineral salts medium may be used; the above
organism is now listed as Klebsiella aerogenes NCIB 418 by the Torry
Research Station, Aberdeen, Scotland. The enzyme need not be fraction-
ated, bu~ crude extracts contain N A D H oxidase which must be removed.
Place 10 ml of extract (10 mg of protein/ml) in a dialysis sac and stir for
5 minutes at 50 ° in 800 ml of distilled water. Cool the contents of the sac
in ice water and add 10 ml of C~-alumina gel;9 centrifuge the suspension
until the enzyme preparation is clear. The extract keeps its activity for
several weeks if stored in the frozen state and may be frozen and thawed
with little loss of activity. The enzyme is specific for citrate, and extracts
are essentially free from aconitase (see also this volume [28]).
8 A. Kornberg, Vol. I [67].
9S. P. Colowick, Vol. I [11].
[68] PURITY AND STABILITY OF ~-KETO ACIDS 519
[ 6 8 ] P u r i t y a n d S t a b i l i t y of P y r u v a t e a n d a - K e t o g l u t a r a t e
By R. W. Vo~¢ KORFr
The purity and stability of pyruvate, of radioactive pyruvate in
particular, have been a source of concern to many investigators. ~-7 The
tendency of alkaline solutions of a-keto acids to undergo an aldol-type
condensation to form polymers is a maior source of difficulty. Pyruvate
forms a dimer, v-methyl-~,-hydroxy-a-ketoglutarate, or parapyruvate, as
one of the products. This dimer is an inhibitor of a-ketoglutarate dehy-
drogenase s and interrupts Krebs cycle oxidations. Pyruvate oxidation by
rat heart mitochondria in the presence of parapyruvate is blocked unless
a dicarboxylic acid is present in which ease a-ketoglutarate accumulates, s
Pyruvate oxidation by rabbit heart mitochondria in the presence of
parapyruvate yields acetate as the major oxidation product even when
a dicarboxylic acid is present2
The polymerization of pyruvate is catalyzed by base. Since the
average pKa of the acidic groups of the products is higher than that of
pyruvate, polymerization tends to become autocatalytic. The pH of a
solution of sodium pyruvate increases as a result of this polymerization.
Pure pyruvic acid or acidic aqueous solutions are relatively stable
when stored at --200. ~,5-7 Acidic solutions of radioactive pyruvate do not
form parapyruvate, but traces of acetate may arise on long storage 1° (3
months to 1 year) possibly as a result of radiation-induced reactions.
Solutions of sodium pyruvate are unstable, particularly when frozen
~nd stored at --20 °. At alkaline pH in the presence of ammonium salts
or of tris(hydroxymethyl)aminomethane, the rate of pyruvate disap-
pearance is increased. 7,~
The destruction of a-ketoglutarate due to a similar base-catalyzed
reaction does not appear to have been reported. Although this reaction
might be predicted, the author neglected to look for such a reaction in
D. P. Groth and G. A. Le Page, J. Am. Chem. Soc. 77, 1681 (1955).
"~C. M. Montgomery and J. L. Webb, Science 120, 843 (1954).
tI. Goldfine, Biochim. Biophys. Acla 40, 557 (1960).
' M. F. Utter and D. B. Keeeh, J. Biol. Chem. 238, 2603 (1963).
~E. Silverstein and P. D. Boyer, Anal. Biochem. 8, 470 (1964).
R. W. Von Korff, AT~aI. Biochem. 8, 171 (1964).
O. H. I,owry, J. V. Pmssoneau, F. X. ttasselberger, and D. W. Sehulz, d. Biol. Chem.
239, 18 (1964).
C. M. Montgomery and J. L. Webb, J. Biol. Chem. 221, 359 (1956).
R. W. Von Korff, d. Biol. Chem. 240, 1351 (1965).
,o R. W. Von Korff, unpublished observations, 1965.
"A. D. Winner and G. W. Schwert, J. Biol. Chem. 231, 1065 (1958).
the case of a sample of a-ketoglutaratc-5-~'C reported on previously."

When assayed with glutamate dehydrogenase, this sample assayed only
36% of the expected value and was reported to be free of radioactive
contaminants as judged by column chromatography. On a later occasiol~,
a solution of potassium a-ketoglutarate which had been frozen for many
months was observed to contain an appreciable amount of insoluble
material. The a-ketoglutarate content as measured with glutamate de-
hydrogenase had decreased markedly. These observations led us to re-
examine the sample of radioactive ,~-ketoglutarate. The concentration
had fallen from 36% of the calculated value, as originally reported, to
16%. This finding suggested that radioactive contaminants might have
been missed as a result of incomplete elution during column chromatog-
raphy. This was found to be the case (see Fig. 3). In addition, an
increase in succinate content occurred during the two year storage period.
The formation of succinate 12 from a-ketoglutarate is similar to the
formation of acetate observed in the case of radioactive pyruvate.
In dilute aqueous solutions of the free acid or in solutions neutralized
to a pH of 6.0-7.7, no loss of a-ketoglutarate was observed in 22 days at
--20 °. Although a solution at pH 8.8 showed no loss during the first 10
days, there was a 20% decrease in a-ketoglutarate in the next 12 days.
At a high pH (10.8), there was a rapid loss of a-ketoglutarate (67%,
78%, and 84% loss in 10, 15, and 22 days, respectively).
Double or triple distillation under nitrogen at reduced pressure
generally is recognized as the method of choice for the purification of
pyruvic acid. Preparation of sodium pyruvate by neutralization of pyr-
uvic acid in alcohol or recrystallization of sodium pyruvate from al-
cohol '3 may actually result in an enrichment of the product in parapyru-
vate. lo
The simplest and most rapid check on the purity of pyruvate or of
a-ketoglutarate is to assay using lactate debydrogcnase or glutamate
dehydrogenase. These assays, when possible, should be compared with
calculated values based on dry weight or neutralization equivalent. The
assays may be conducted in a matter of minutes and are based on rela-
tively common procedures. '4,'~
':The conversion of a large fraction of radioactive a-ketoglutarate to succinate has
been observed when a-ketoglutarate fractions (eluted from Dowcx 1 columns with
ttCl) are neutralized and concentrated to dryness on a rotatory evaporator.
C. Bauer, M.S. Thesis, University of Minnesota, Minneapolis, Minnesota, 1966.
'~ "Manometric Techniques," 3rd ed., p. 295. Burgess Publ., Minneapolis, MinnesGta,
1957.
"A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 193, 481 (1951).
"D'. U. Bergmeyer (ed.), "Methods of Enzymatic Analysis," p. 324. Academic Press,
New York, 1963.
[58] PURITY AND STABILITY OF a-KETO ACIDS 521
Assay for Pyruvate
Reagents
Reduced nicotinamide adenine dinueleotide (NADH), 4 mg/mi
Lactate dehydro~enase, preferably at a specific activity of at least
100 International Units/rag, 1 mg/ml in 0.1 M phosphate buffer
pH 7.4 or in 0.1% bovine serum albumin solution
Procedure. To each of three cuvettes add phosphate buffer, 0.30 ml;
water to yield a final volume of 2.99 ml; NADH, 0.10 ml; and pyruvate
solution diluted so as to be approximately 1 raM, 0.10, 0.20, and 0.30 ml
in successive cuvettes. Mix well and read A34o against a blank tube con-
taining water. Add 0.01 ml of lactate dehydr0genase and read the A~,~
until a constant value is obtained (this should not require more than 2-3
minutes). The pyruvate content (in micromoles) of each cuvette is
calculated from the product, (AA34oX 0.483). The concentration of the
diluted solution (in micromoles/ml) is given by the expression (5A34o X
0.483/volume of pyruvate solution added to the cuvette).
Assay for a-Ketoglutarate
Reagents
Reduced nicotinamide adenine dinucleotide, 4 mg/ml
Ammonium chloride, 1 M
Glutamate dehydrogenase, about 3 International Units/mg, 10
mg/mI. Since a large excess of ammonium ions is added in the
procedure, either, enzyme suspended in ammonium sulfate or
enzyme in glycerol and free of ammonium ion may be used
Procedure. To each of three cuvettes add phosphate buffer, 0.3 ml;
water to yield a final volume of 2.99 ml; ammonium chloride, 0.10 ml;
NADH, 0.10 ml; and ~-ketoglutarate solution, diluted so as to be
approximateIy 1 raM, 0.10, 0.20, and 0.30 ml in successive cuvettes. Mix
and read A34o against a blank containing water only. Add 0.01 ml of
glutamate dehydrogenase and read the As4o until constant. This should
not require over 4-5 minutes. Calculation of the a-ketoglutarate is done
exactly as described for pyruvate.
Tests for Purity Using Column Chromatography on Dowex 1 X-8 Resin
The presence of impurities such as parapyruvate and acetate in radio-
active pyruvate and of polymers of a-ketoglutarate and, of succinate in
60(~ I .........
;!
50~- ,
-~ ',
i
'2 4 0 - 4,
i
* f : i
,o
I0 20 30 40 50 60 70 80 90
Tube no.
Fie. 1. Instability of sodium pyruvate-l-"C during storage of a frozen aqueous
solution at --20°. Chromatograms on Dowex 1 [C1] using the procedure described in
the text. Aliquots of neutralized fractions were plated on planchets and counted with
a D-47 gas flow detector. - . . . . . , Chromatogram of freshly prepared 20 mM
solution. ~ , Chromatogram of the same solution after storage for 14 weeks
at --20 ° .
r a d i o a c t i v e ~ - k e t o g l u t a r a t e is e a s i l y a c c o m p l i s h e d u s i n g a m o d i f i c a t i o n
of t h e p r o c e d u r e d e s c r i b e d in t h i s v o l u m e [63]. F o r these c h r o m a t o g r a m s
a D o w e x 1 X - 8 c o l u m n 1 X 7 c m is used a n d t h e g r a d i e n t is p r o v i d e d b y
p u m p i n g 0.100 N H C I into a r e s e r v o i r c o n t a i n i n g 250 m l of w a t e r . F o r
s a m p l e s a t a specific a c t i v i t y of 1-5 ~ C / ~ m o l e , 0.05 m i e r o m o l e of s a m p l e
0.9 -
3.8
0.7
0.6
).~ 0.5
Acetate
I l1 0.4 A ,-
-.. ) 2 - -
).~ K =13- f
J 0
150 ml I 0 0 ml 5 0 ml 2 6 rnl
Fro. 2. Stability of an aqueous solution of pyruvic acid-2-14C against polymeriza-

tion during storage at --20 ° for a period of 14 months. In this case, the eluate was
passed through an anthracene scintillation flow cell assembly. Full scale sensitivities
in thousands of counts are indicated by K values.
[68] PURITY AND STABILITY OF oz-KETO ACIDS 523
is usually sufficient for the analysis. The column effluent may be passed
through a scintillation flow cell for counting or collected in 2 ml fractions
and counted after neutralization and plating on planchets. Acetate is
eluted in the region of 22-30 ml, pyruvate in the region of 65--100 ml,
and parapyruvate in the region of 120-160 ml.
Figure 1 (taken from reference cited in footnote 6) shows an increase
in parapyruvate after storage of a neutral solution of sodium pyruvate
for about 4 months at --20 °.
Figure 2 shows a chromatogram of an acidic solution of pyruvic
acid-3-1'C after 14 months of storage at --20 °. Although a small amount
of acetate as well as a trace of neutral radioactive material are present,
parapyruvate is absent.
As noted by Montgomery and Webb, s parapyruvate yields a positive
reaction in the Ettinger, Goldbaum, and Smith TM modification of the
pentabromoacetone procedure for citrate assay. The presence of para-
pyruvate in pyruvate can be detected using this procedure.
Figure 3 illustrates a chromatogram (run August, 1965) of the sample
of a-ketoglutarate-5-1,C reported on previously2 The presence of sue-
cinate-l'C was confirmed by cochromatography with nonradioactive suc-
Succ~no~e a - Ketogiulorefe I . . . .
Impurif y --
K=IOILJ
'1.] 34Ii m[ - ~ *~ [
~j. K=IO0 £' t '
"tlli.,ith,,J If~,~Jl .~..~, ,-,-:m'rrT~:_ :.: .-~,,liimhl&~, - ..... j . . . . . l~;,t,;, :,L,~ ................ h,qn,
Fie. 3. Formation of succinate and polymeric impurity following storage of an

aqueous solution of sodium a-ketozlutarate-5-1'C solution at --20 °. From tube No. 70
(140 ml) 0.1 N HC1 was passed directly through the column to elute the polymeric
material. The effluent was counted using an anthracene scintillation flow cell.
cinate on silicic acid (this volume [63]). The presence of a major amount
of a component eIuting at a relatively high acidity is clearly evident.
Buffered solutions of the a-keto acids at neutral pH are more stable
than unbuffered solutions. The greatest stability appears to be obtained,
however, in acidic solutions frozen and stored at --20 ° . Neutral solutions
are best prepared as needed from the acidic stock solutions.
~R. H. Ettinger, L. R. Goldbaum, and L. H. Smith, Jr., J. Biol. Chem. 199, 531
(1952).
[ 6 9 ] D e t e r m i n a t i o n of S u c c i n a t e w i t h
Suceinate Dehydrogenase
By C. VEEGER and W. P. ZE~LEMAKER
The method is identical with the one published by Massey. 1 Although
other methods have been described,2 their sensitivity is about twenty
times lower. The method is based on the oxidation of succinate by suc-
cinate dehydrogenase, followed by oxidation of the reduced enzyme by
phenazine methyl sulfate (PMS) and cytochrome e. PMS acts here as an
electron carrier between enzyme and cytochrome c:
succinate
Suceinate + PMS , fumarate -~ PMSH2
dehydrogenase
PMSH2 ~- 2 cyt c3+ -* PMS -~ 2 cyt c2+ ~- 2 H +
Although the reduced form of PMS is very autoxidizable, oxygen does
not affect the determination. 2,6-Dichlorophenol indophenol can be used
instead of cytochrome c, but the reported values for the molar extinction
coefficient show a large variation (see also footnote 3).
Reagents
Perchloric acid, 70~
Potassium carbonate, 1 M
Phosphate buffer, 0.3 M pH 7.6
Bovine serum albumin, 2 ~ (w/v), in H20
EDTA, 20 mM pH 7.6
Purified cytochrome c, 1 ~ (v/w), in H20
Phenazine methyl sulfate, 0.01~'o, thoroughly protected from light,
freshly diluted from a 1 ~ stock solution
Succinate dehydrogenase, 2 mg/ml, soluble, purified (see footnote 4
for purification procedure)
Procedure
Deproteinization. Perchloric acid in a final concentration of 5 ~ is
added to the sample. The sample is centrifuged, and potassium carbonate

2T. P. Singer, P. Bernath, and C. J. Lusty, in "Methods of Enzymatic Analysis"
(H. U. Bergmeyer, ed.), p. 340. Academic Press, New York, 1963.
a T. E. King, J. Biol. Chem. 238, 4032 (1963).
[69] ENZYMATIC DETERMINATION OF SUCCINATE 525
solution is added to a portion of the supernatant up to p H 7-8. The

mixture is allowed to stand at 0 ° for 15 minutes and centrifuged at 0 °.
The supernatant is used in the assay.
Assay. Cuvettes with a 1 cm light p a t h and final volume of 2 ml are
used. The absorbance is measured against H20 at 550 m~. The reagents
are added to two cuvettes incubated at 25 ° (see tabulation).
Assay Blank
Component (ml) (n~)
I-I20 to 1.85 to 1.85

Phosphate buffer 0.4 0.4
Bovine serum albumin 0.1 0.1
EDTA 0.1 0.1
Cytoehrome c 0.1 0.1
Sample (containing 2-30 nanomoles succinate) -F
Mix and note the absorbance of both euvettes (A~ and A2), then add:
Phenazine methyl sulfate 0.05 0.05
Enzyme 0.1 0 1
Mix and record the absorbance of both cuvettes until the difference
between the assay cuvette and the blank cuvette is constant (As and A,).
The m a x i m u m amount of suceinate which can be determined is about 40
nanomoles, but it is recommended to use a smaller sample when the
aliquot contains more t h a n 30 nanomoles.
Calculation. The initial absorbances of the assay cuvette (A~) and of
the blank cuvette (A2) m u s t be corrected b y a factor 1.85/2.00 = 0.92.
Uncorrected absorbance increase in assay cuvette: A3 - - 0.92 A~; absorb-
ance increase in blank cuvette: A ~ - - 0 . 9 2 A~; corrected absorbance in-
crease in assay cuvette: A 3 - A , - 0.92 ( A 1 - A2). I n this determina-
tion 2 moles of cyt c 3~ are reduced per mole of succinate oxidized:
A~ = ~(cyt c2+) - ~(cyt c3+) = 2.1 X 104M -1 cm -1
The succinate content of the sample is:
2 X {Aa - A, - 0.92(A1 - As)} X 100

nanomoles
2X2.1
Specificity. The method is specific for succinate. L-Malate has been
reported to be a substrate for suceinate dehydrogenase. * However, the
reaction rate with L-malate is low even at high concentration of the
latter. Therefore, L-malate does not interfere significantly with the re-
sults, especially since the p r o d u c t - - o x a l o a c e t a t e (KD = 3 /J3t/)--is a
competitive inhibitor and blocks the reaction completely.
• C. Veeger, D. V. DerVartanian, and W. P. Zeylemaker, this volume [16].
[70] Fluorometric Assay of Malic Acid and

Its a-Substituted Derivatives 1
By MuR~AY STRASSMAN a n d L 0 ~ I s CECI
Principle. This fluorometric assay is based on the degradation of

substituted malic acids to fl-keto acids, which condense with resorcinol to
form derivatives of the highly fuorescent 7-hydroxycoumarin (umbelli-
ferone). Malic acid itself condenses with orcinol in the presence of hot
sulfuric acid to yield 7-hydroxy-5-methylcoumarin (homoumbelli-
ferone).2.3 This procedure is not applicable to a-substituted malic acids
because they are degraded extensively in hot acid. The use of cold
concentrated sulfuric acid limits the degradation of a-substituted malic
acids to fl-keto acids. Acetoacetic acid, the simplest fl-keto acid, con-
denses with resorcinol in the presence of acid at room temperature to
form 7-hydroxycoumarin.4 Other fl-keto acids undergo analogous con-
densations to yield fluorescent products. Hence, ~-keto acids and com-
pounds that are converted easily to p-keto acids, such as a-substituted
malie acids, can be assayed fluorometrically after condensation with
resorcinol.5 Malic acids monosubstituted in the fl-position may be simi-
larly assayed but are considerably less fluorogenic.
Reagents
Conc. H2S04, analytical grade, 96.1%
Conc. HC1, analytical grade
Resoreinol solution. Dissolve 1 g of resorcinol in 10 ml of H20 just
before use.
Sodium carbonate solution. Dissolve 28 g of anhydrous sodium
carbonate in 100 ml of H20.
Borate buffer. Dissolve 7.32 g of boric acid in 100 ml of the sodium
carbonate solution, dilute to 900 ml, adjust to pH 10 with 50%
NaOH, and then dilute to 1 liter
Standards. Stock solutions of substituted malie acids are prepared
by dissolving 1 mg of compound in 10 ml of ether at 0 °
Water, redistilled 3 times from a glass still
1Manuscript prepared by A. F. Tucci.

sj. p. Hummel, J. Biol. Chem, 180, 1225 (1949),
sO. H. Lowry, N. R. Roberts, M. Wu, W. S. Hixon, and E. J. Crawford, J. Biol.
Chem. 207, 195 (1954).
~G. Leonhardi and I. von Glasenapp, Z. Physiol. Chem. 286, 145 (1951).
M. Strassman and L. N. Ceci, J. Biol. Chem. 238, 2445 (1963).
[70] FLUOROMETRIC ASSAY OF MALIC ACID 527
Special Apparatus
Fluorometer (G. K. Turner Associates, Palo Alto, California),
model 110 null-balancing filter fluorometer. Primary filter :No.
110-811 [7-60 (365 mtL)]; a secondary filter No. 110-817 [8
(485 m~ sharp cut)]; plus a No. 110-823 [N D 1% (100-fold
reduction)]; and a 110-851 far UV lamp with the range selector
(intensity) set at 1X for a-substituted malic acids, 10X for
fl-substituted malic acids, and 30X for malic acid
Cuvettes are round, quartz, and nonfluorescent
Procedure2 A reaction mixture may be freed of proteins by addition
of a precipitant which is not soluble in ether (such as tungstic acid), and
sufficient sulfuric acid to bring the mixture to pH 1-2. The solutions are
clarified by filtration or centrifugation and extracted with ether in a
continuous liquid-liquid extractor for 20 hours. The ether extracts are
evaporated to dryness, and the residues are dissolved in 2 ml of anhy-
drous ether. Ether solutions arc pipctted and aliquoted at 0% Aliquots
(0.1--0.2 ml) of the extracts and of the appropriate standard solutions are
pipetted into 15 ml tapered glass-sti)ppered centrifuge tubes. The blank
tube receives no sample. The solutions are evaported in a hood in a warm
water bath at 32 °, and completely dried at 65 °. The tubes are cooled to
room temperature, and 0.6 ml of conc. H~SO~ is added to each; they arc
stoppered, and mechanically shaken for 30 minutes. Then 0.5 ml of
resorcinol solution is added, the tubes are shaken briefly, 1 ml of conc.
HC1 is added, and the tubes are shaken again. The tubes are stoppered
and placed in the dark for 18-20 hours.
The contents of the tubes are transferred to 30 ml glass-stoppered
round-bottom tubes. To make the transfer quantitative the small tubes
are rinsed with 3 portions of 1 ml each of sodium carbonate solution,
and these are added gradually to the larger tubes with shaking. The
addition must be made slowly because vigorous effervescence occurs.
Sufficient sodium carbonate solution is added to bring the solutions to pH
7.6 (a total of 7-8 ml). Borate buffer is added to bring the total volume
to 12.5 ml. The final solution is at pH 8.5--9.0. Solutions are shaken and
read in the fluorometer from immediately after preparation to within an
hour thereafter.
Remarks. Fluorescence is directly proportional to concentration, with
no evidence of quenching from 1 to 100 millimicromoles of a-substituted
malic acids. Fluorescence is also dependent on the amount of sulfuric
acid, with maximum fluorescence at 0.6 ml of sulfuric acid for 30
minutes, a-Substituted malic acids such as a-ethyl malic, a-methyl malie,
a-isopropylmalic and citric acids show a much higher degree of fluores-
cence than B-substituted malic acids such as B-methyl malic and/~-ethyl

malic acid. Malic acid shows a still lower degree of fluorescence. The
relationship of fluorescence to concentration is linear for all these com-
pounds, a-Ketoisovaleric, a-ketoglutaric, succinic, homocitric, and oxalo-
acetic acid give no evidence of fluorescence.
[ 7 1 ] T h e D e t e r m i n a t i o n of Specific R a d i o a c t i v i t i e s o f Citric
Acid Cycle Intermediates by Enzymatic Decarboxylation
By F. A. MCELRoY and O. R. WILLIAMS
For the effective use of labeled precursors in studies of the citric acid
cycle, accurate determinations of the specific radioactivities of the cycle
intermediates are required. Sensitive fluorimetric methods are available
for the estimation of the total amount of the individual intermediates
present in a mixture (see also this volume [65]). Several procedures
involving an initial separation of the various intermediates have pre-
viously been employed to determine the total radioactivity content of
each component. For example, Von Korff1 has used the butanol-chloro-
form-silica gel system of Bulen, Varner, and Burrell, 2 Stuart and
Williams s have used the paper chromatographic procedure of Lugg and
Overell,' and Walter, Paetkau, and Lardy 5 have employed electrophoresis
to separate the cycle intermediates. These methods are frequently time-
consuming, require considerable quantities of starting material and, in
addition, give no information concerning the precise location of the
radioactivity in the compounds under consideration. An interesting ap-
proach to the latter problem has been taken by Bidwell, 6 who used the
ninhydrin reaction to estimate the radioactivity content of the carboxylic
group of amino acids. Enzymes have been used to a limited extent in
studies of the location of labeling in dicarboxylic acids2 ,~
In the present paper a method is described which takes advantage of
the specificity of enzyme reactions to determine accurately the radio-
activity content of specific positions in the intermediates of the citric
acid cycle. Not only is this method rapid and highly sensitive, but it
1R. W. Von Korff, J. Biol. Chem. 240, 1351 (1965).

W. Bulen, J. E. Varner, and R. C. Burrell, Anal. Chem. 24, 187 (1952).
s S. C. Stuart and G. R. Williams, Biochemistry 5, 3912 (1966).
"J. W. H. Lugg and B. T. Overell, Australian J. Sc/. 1, 98 (1948).
*P. Walter, V. Paetkau, and It. A. Lardy, J. Biol. Chem. 241, 2523 (1966).
"R. G. S. Bidwell, Can. J. Botany 41, 1623 (1963).
' R. 12. Haynes, Jr., J. Biol. Chem. 240, 4103 (1965).
cence than B-substituted malic acids such as B-methyl malic and/~-ethyl

malic acid. Malic acid shows a still lower degree of fluorescence. The
relationship of fluorescence to concentration is linear for all these com-
pounds, a-Ketoisovaleric, a-ketoglutaric, succinic, homocitric, and oxalo-
acetic acid give no evidence of fluorescence.
[ 7 1 ] T h e D e t e r m i n a t i o n of Specific R a d i o a c t i v i t i e s o f Citric
Acid Cycle Intermediates by Enzymatic Decarboxylation
By F. A. MCELRoY and O. R. WILLIAMS
For the effective use of labeled precursors in studies of the citric acid
cycle, accurate determinations of the specific radioactivities of the cycle
intermediates are required. Sensitive fluorimetric methods are available
for the estimation of the total amount of the individual intermediates
present in a mixture (see also this volume [65]). Several procedures
involving an initial separation of the various intermediates have pre-
viously been employed to determine the total radioactivity content of
each component. For example, Von Korff1 has used the butanol-chloro-
form-silica gel system of Bulen, Varner, and Burrell, 2 Stuart and
Williams s have used the paper chromatographic procedure of Lugg and
Overell,' and Walter, Paetkau, and Lardy 5 have employed electrophoresis
to separate the cycle intermediates. These methods are frequently time-
consuming, require considerable quantities of starting material and, in
addition, give no information concerning the precise location of the
radioactivity in the compounds under consideration. An interesting ap-
proach to the latter problem has been taken by Bidwell, 6 who used the
ninhydrin reaction to estimate the radioactivity content of the carboxylic
group of amino acids. Enzymes have been used to a limited extent in
studies of the location of labeling in dicarboxylic acids2 ,~
In the present paper a method is described which takes advantage of
the specificity of enzyme reactions to determine accurately the radio-
activity content of specific positions in the intermediates of the citric
acid cycle. Not only is this method rapid and highly sensitive, but it
1R. W. Von Korff, J. Biol. Chem. 240, 1351 (1965).

W. Bulen, J. E. Varner, and R. C. Burrell, Anal. Chem. 24, 187 (1952).
s S. C. Stuart and G. R. Williams, Biochemistry 5, 3912 (1966).
"J. W. H. Lugg and B. T. Overell, Australian J. Sc/. 1, 98 (1948).
*P. Walter, V. Paetkau, and It. A. Lardy, J. Biol. Chem. 241, 2523 (1966).
"R. G. S. Bidwell, Can. J. Botany 41, 1623 (1963).
' R. 12. Haynes, Jr., J. Biol. Chem. 240, 4103 (1965).
[71] DETERMINATION OF SPECIFIC RADIOACTIVITIES 529
also avoids the need for a preliminary separation of the intermediates

before radioactivity determinations. In addition, by means of successive
enzymatic incubations, it is possible to obtain information concerning the
labeling of scvcral intermediates from the same small sample.
Principle
Decarboxylating enzymes, employed alone or in conjunction with
enzymes leading to a dccarboxylation reaction, can be used to release the
radioactivity present in a specific position of a cycle intermediate as
'4C02. This 14C02 can be collected with a suitable trapping agent using
microdiffusion techniques and the radioactivity then determined by
conventional means. The positions which can be studied by this method
(shown in italics) and the enzymes required are as follows:
H00C--CH(0H)--CH2--C00H malic enzyme
H00C--CH~CH--C00H fumarase -{- malic enzyme
0
II
HOOC--C--CH2--COOH oxaloacetic decarboxylase
or phosphopyruvate carboxykinase
CH~--COOH CH2--COOH CH~--COOH
IIO--C--COOH HC--COOH CH~
CH2--COOH HO--CH--COOH O-~C--COOH
Aconitase ~ ICDH Isocitrate dehydro- a-Ketoglutarate
genase (ICDH) dehydrogenase
Although not all carbons are approachable by this method, sufficient
information is obtainable in most cases especially if suitable precursors,
such as carboxyl-labeled succinic acid, are used.
Equipment
The reactions are carried out in Conway microdiffusion dishes (44
mm diameter, 0brink modification, available from Fisher Scientific). The
central chamber in each case is fitted with a glass insert (12 mm outer
diameter, 7 mm in height) to facilitate the rapid and quantitative re-
moval of the center well contents. The use of similar glass inserts has
been shown to be profitable by Snyder and Godfrey2 A small hole is
drilled in each lid which allows additions to be made to the outer
diffusion chamber and which is plugged with plasticine when a closed
system is required.
SF. Snyder and P. Godfrey, J. Lipid Res. 2, 195 (1961).
Procedure
1 M hydroxide of Hyamine 10-X, 0.35 ml, is placed in the glass insert
in the central chamber to act as the '4C02-trapping agent. The corrosive
effects of other materials (phenethylamine, Nuclear Chicago solubilizer)
on the lids of the Conway dishes preclude their use in the present situa-
tion. It is also advisable to avoid, when possible, the use of acetone in
enzyme fractionation, since it has been found that acetone in trace
amounts causes extensive discoloration of the Hyamine and hence inter-
feres with counting procedures.
A reaction mixture appropriate to the enzymes being used and
containing the radioactive compound under study is placed in the outer
diffusion chamber and the final volume is adjusted to 1.50 ml. With
aconitase and I C D H the reaction mixture contained the following com-
ponents: Tris-HC1 buffer, pH 7.4, 25 mM; MnC12, 0.3-0.6 mM; NADP,
0.2 raM; citrate, 0.1 mM. The cofactor-buffer solution of Nossal a was
used with the Lactobacillus plantarum suspension, and the reaction mix-
ture recommended by Rutter and Lardy '° was employed with the pigeon
liver malic enzyme. In all cases, 0.1 ml of toluene is included in the
reaction mixture to prevent microbiological activity which could cause
spurious results. This has had no effect on the enzymes so far investi-
gated. Two milliliters of a solution similar to that used in the outer
diffusion chamber but without enzymes or radioactive compounds is
added to the sealing chamber.
Reactions are initiated at timed intervals by the addition of enzyme.
Optimal mixing is achieved by rapidly stirring the enzyme with the
reaction mixture before placing the lid and sealing the system. It has
been found that no 14CO2 is lost during the 10-15 seconds required for
this procedure. Reactions are stopped by the addition of perchloric acid
(0.2 ml of 3 M ) by syringe through the hole in the lid and, after
resealing, adequate time is allowed for complete 14C0z diffusion. Experi-
ments have shown that 90% of the ~4COz has diffused in 1 hour but that
3 hours are required for complete diffusion. These findings compare
favorably with those reported by Conway. 1~ After the diffusion period
the glass inserts are removed and placed in counting vials' containing
10 ml of scintillation solution [0.4% (w/v) diphenyloxazole and 0.01%
(w/v) 1,4-bis-2-(5-phenyloxazolyl)benzene in toluene] and 0.15 ml of
Hyamine. The vials are placed for 30 minutes on a shaking table to
ensure complete solution of the center well contents, which have becomc
~P. M. l~ossal, Biochem. J. 50, 349 (1952).
'~ W. g. Rutter and H. A. Lardy, J. Biol. Chem. 233, 374 (1958).
,1E. J. Conway, "Microdiffusion Analysis and Volumetric Error," 3rd ed. Lockwood,
London, 1950.
[71] DETERMINATION
OF SPECIFIC RADIOACTIVITIES 531
semisolid. The glass inserts are routinely left in the vials during count-
ing, but their removal does not alter the results.
Several other diffusion and trapping techniques have been investi-
gated in other laboratories, s'~-~5 Presumably these also could be em-
ployed effectively in the present context.
Sources of E n z y m e s
For the successful application of this procedure it is important that
the enzyme preparations employed be as free as possible of other en-
zymes which might lead to the decarboxylation of labeled substrates
other than the particular one under study. For example, it is necessary
to check the malic enzyme activity of the aconitase and I C D H prepara-
tions and the fumarase activity of the malic enzyme source.
For the results reported here, the following enzyme sources were used.
Aconitase was prepared from pig heart according to the procedure of
Morrison 16 except that, following the suggestions of Goldberg, Passonneau,
and Lowry, 17 the extraction was carried out at pH 7.5 and tricarballylic
acid was used as a stabilizer. Purification was carried to the second
ethanol fractionation. The 23-45% fractio~l was found to be most useful
since its malie enzyme activity was very low. L a c t o b a c i l l u s p l a n t a r u m
NRC-700 is (designated previously L a c t o b a c i l l u s arabinosus, Wisconsin
strain 17-5) was grown under the conditions described by Singer and
Lusty ~9 and the lyophilized cells were resuspended by homogenization in
cold water when needed. The crystalline malic enzyme was prepared
from pigeon liver and kindly supplied by Dr. H. A. Lardy. Isocitrate
dehydrogenase (Type IV, from pig heart) and crystalline fumarase were
obtained from Boehringer, Mannheim.
Duration of Incubation with Enzymes

The time required for the incubation period varies with the particular
enzyme source and should be ascertained in each ease using labeled
standards. Figure 1, for example, shows the results of time course studies
1--G. Moss, Intern. J. Appl. Radiation Isotopes 11, 47 (1961).

13D. A. Buhler, Anal. Biochem. 4, 413 (1962).
~R. W. Albers and R. O. Brady, ]. Biol. Chem. 234, 926 (1959).
1~D. Seligson and H. Selig~n, g. Lab. Clin. Med. 38, 324 (1951).
l~J. F. Morrison, Biochem. J. 56, 99 (1954).
~7N. D. Goldherg, J. V. Passonneau, and O. H. Lowry, J. Biol. Chem. 241, 3997
(1966).
'"Obtained fi'om the Division of Bioscienees, National Research Council, Ottawa,
Canada.
"0T. P. Singer and C. J. Lusty, in "Methods of Enzymatic Analysis" (H. V. Berg-
meyer, ed.), p. 346. Academic Press, New York, 1963.
532 S~PA~TIos AND ASSAY ME~HODS [71]
for an aconitase preparation and a L. plantarum suspension. In the

former case, there is no advantage to an incubation period extending
beyond 10 minutes, whereas in the latter case this time period would be
inadequate. In general, it is preferable to use as short an incubation as
is feasible in order to minimize nonspecific radioactivity transfer (see
later discussion of this problem) and to prevent the slow action of trace
amounts of interfering enzymes.
'°°t
soL x '- .... '
i 6O
"6
o~ 40
2O
I
I0 20 30 " 40 50 60
Time of incubotion(minufes)
Fro. I. Time course of 1°CO~ release from labeled standards. X - - X , "CO, released
from citric acid-6-~4C by incubation with aconitase (0.25 IU) and ICDH (0.20 IU).
Q--'O, ~'CO~released from ~,-malic acid-'C (U) by incubation witth Lactobacillus
plantarum suspension (6 mg). Conditions of incubation and diffusion were as out-
lined in text.
Evaluation of the Method Using Synthetic Radioactive Intermediates

The table gives results obtained using this procedure with several
synthetically prepared labeled cycle intermediates obtained from com-
mercial sources. The 1~C02 recovered in each case approaches the theo-
retical as judged by counting the labeled substrates under the same
conditions as the 14C02 samples and assuming 100% purity. Controls
carried out in the absence of enzymes showed a diffusion of less than
0.1% of the radioactivity into the center well. In early experiments when
periods of up to 3 hours were used for incubation with enzymes, con-
siderably more radioactivity was detected in these controls. These find-
ings emphasize the advisability of restricting the incubation time to a
minimum. Although the exact source of this nonspecific t~:ansfer of
[71] DETERMINATION O F SPECIFIC RADIOACTIVITIES 533
APPLICATION OF ENZYMATIC DECARDOXYLATION TO RADIOACTIVE STANDARDS
Dpm Percentof
Radioactive Dpm releasedas expected
compound Enzymes useda added ~C02 value
Citric acid-&'*C Aconitase + ICDH 53,400 50,700 95

Sodium DL-isocitrate- ICDH 50,600 11,500 91
5,6-1,C
Fumaric acid-l,4-*4C L. plantarum 37,100 18,000 97
suspension +
fumarase
~-Malic acid-~*C (U) (i) malic enzyme 226,200 59,000 104
from pigeon liver
(ii) L. plantarum 121,200 27,200 90
suspension
" The quantities of enzymes and the times used for incubation were as follows:
aconitase, 0.10 IU, plus ICDH, 0.125 IU, 10 minutes; ICDH, 0.125 IU, 10 minutes;
LadobaciUus plantarum suspension,8 mg, with or without fumarase,20 ~g, 30 min-
utes; pigeon liver malicenzyme,40 ~g, 30 minutes. Other conditionsof incubation
and diffusionwere as outlined in text.
radioactivity into the center well is not known, the fact that it is greater
at pH 7.4 than at pH 5.0 and that it is negligible afer the addition of
perchloric acid, taken together with the observation that it is largely
prevented by the inclusion of a small amount of toluene in the reaction
mixture, suggests that it may be in part due to microbiological activity.
Application of the Method to Metabolically

Generated Radioactive Intermediates
For the results reported here, samples containing metabolically gen-
erated labeled cycle intermediates were obtained by incubating rat heart
mitochondria metabolizing pyruvate (10 raM) as major substrate with
succinate-l,4-1*C (20 /xM) in the presence (state 3) or absence (state
4) of ADP (2.5 raM). In order that the enzymatic decarboxylation tech-
nique be applicable to analyzing cycle intermediates obtained by this
type of incubation, it is essential that the resulting samples be completely
freed of "CO, formed during the period of incubation with mitochondria.
This can be accomplished readily by bubbling air through the acidified
samples until all the ~*CO~ is driven off. A 10-minute bubbling period
was found to be adequate. If required, the total '*CO2 produced during
the mitochondrial incubation can be measured at this stage by using the
~4CO,-trapping procedure described by Stuart and Williams*° provided
that a closed system is maintained at all stages. The samples are sub-
S. C. Stuart and G. R. Williams, see Vol. X [100].
sequently freed of precipitated protein, the pH is adjustcd to 5.8-6.2 with
KOH, and the resulting KHCI04 is removed by centrifugation prior to
storage of the samples in the frozen state.
A comparison is given in Fig. 2 of the results obtained on the analysis
of metabolically generated intermediates by the enzymatic decarboxyla-
2 0 L- malate + / 20 citrate +
/
_ I fumarate / / _o
,
isocitrate /
x I6 // x 16 / o
/
"U / / o CL o./
x /
12 ,'° ..~ 12 x /
o /
/
x//
/
/ o X/
/
o
f /
/" ~
8
4
/
/
/
/
~. ]
2
...I
4 6
I I
8
I
I0
o
n
I
]
2 4
r 1
6
I
8
l,
I0
Decorboxylalion-diffusion(dpm x 10"31 Decarboxylation-diffusion(dpm x I0 "z)
(a) [b)
FIG. 2. Metabolically generated intermediates--a comparison of results obtained
by decarboxylation-diffusion and by paper chromatography. Samples for analysis
were prepared by incubating rat heart mitochondria with succinic acid-l,4-~C (0.1
#C) under state 3 ( × ) or state 4 (O) conditions as outlined in text. Paper chroma-
tographic separations were carried out according to the procedure used by S. C.
Stuart and G. R. Williams [Biochemistry 5, 3912 (1966)]. The theoretical line ( - - - )
was drawn on the assumption that 50% of the 1'C present in the intermediates
generated from carboxyl-labeled succinate would be released by the enzyme prepara-
tions used.
Two successive enzymatic decarboxylations were performed. The samples were
initially incubated for 3 hours with Laclobacillus plantarum (8 mg). The incubation
medium was that of P. M. Nossal [Biochem. Y. 50, 349 (1952)] except that [Mn ÷*]
was lowered to 0.4 mM to prevent a subsequent inhibition of aconitase. The 14C
content of the center wells was measured to give the value malate-4-14C -}- fumarate-
4-14C (graph a). The pH of the contents of the outer diffusion chambers was adjusted
to 7.4 with 1.0 M Tris, new glass cups containing Hyamine were inserted, and citrate
(0.15 micromole) and N A D P (0.50 micromole) were added to a final volume of 1.65
ml. Incubations were carried out with aconitase (0.33 IU) and ICDH (0.13 IU) for
15 minutes; the reactions were stopped and diffusion was carried out as usual. The
results gave the values for citrate-6-~4C-b isocitrate-6-~'C (graph b).
tion technique described here and those obtained using the paper
chromatographic technique used by Stuart and Williams2 The results
obtained are in reasonable agreement. The failure of the experimentally
determined points to lie on the theoretical line is due to the sum of the
discrepancies of the two methods and thus does not indicate the degree
of accuracy attainable by the microdiffusion procedure. Further evidence
[72] ASSAY O F COA AND ACYL DERIVATIVES O F COA 535
for the reliability of the latter method is given by the finding that over
95% of a labeIed standard is decarboxylated when added to a metabolic
sample treated as previously described.
It should be noted that in the experiment illustrated in Fig. 2 both
the malate-14C q- fumarate-l*C and the citrate-14C q- isocitrate-14C
values were obtained from the same sample by means of two successive
incubations. An initial incubation with a Lactobacillus plantarum sus-
pension gave a measure of the radioactivity in malate q- fumarate. Since
this incubation was carried out at pH 5.0, quantitative ~*CO~ trapping
was achieved without further acidification. Although others ~ have re-
ported that this type of L. plantarum preparation contains insignificant
amounts of fumarase activity, the preparation used in the experiment
of Fig. 2 was capable of decarboxylating essentially all the fumarate
present, even in the absence of added fumarase, under the conditions
required for maximum malate decarboxylation (see the table). There-
fore, to obtain a measure of malate-~4C alone, a more highly purified
malie enzyme preparation is required. Methods are available for this
type of preparation from L. plantarum, 21 pigeon liver, 1°,22 and wheat
germ. 2~ Subsequent incubation with aconitase and I C D H gave a measure
of citrate-~4C q-isocitrate-*4C. Since the samples had been freed previ-
ously of labeled malate q-fumarate, there was no chance of even small
amounts of malic enzyme activity in these enzymes causing erroneous
results. Incubations with I C D H alone would allow the estimation of
isocitrate-l*C and hence citrate-~*C separately, but, with the samples
studied, the radioactivity present in isocitrate was too low for accurate
determinations.
~S. Kaufman, S. Korkes, and A. del Campillo, J. Biol. Chem. 192, 301 (1951).
2..,S. Ochoa, see Vol. I, p. "/39.
[ 7 2 ] A s s a y of C o e n z y m e A a n d S o m e A c y l D e r i v a t i v e s
By P. K. TUBBS and P. B. GARLAND
A rather long period elapsed between the discovery of coenzyme A
and its thioesters, the recognition of the importance of these compounds
in metabolism, and tlle investigation of the in vivo acylation state of the
coenzyme. However, the problems of metabolic control at the subcellular
lever are now being explored, and sensitive enzymatic assays have been
developed for the determination of many compounds of interest, including
CoA and its thiocsters.
[72] ASSAY O F COA AND ACYL DERIVATIVES O F COA 535
for the reliability of the latter method is given by the finding that over
95% of a labeIed standard is decarboxylated when added to a metabolic
sample treated as previously described.
It should be noted that in the experiment illustrated in Fig. 2 both
the malate-14C q- fumarate-l*C and the citrate-14C q- isocitrate-14C
values were obtained from the same sample by means of two successive
incubations. An initial incubation with a Lactobacillus plantarum sus-
pension gave a measure of the radioactivity in malate q- fumarate. Since
this incubation was carried out at pH 5.0, quantitative ~*CO~ trapping
was achieved without further acidification. Although others ~ have re-
ported that this type of L. plantarum preparation contains insignificant
amounts of fumarase activity, the preparation used in the experiment
of Fig. 2 was capable of decarboxylating essentially all the fumarate
present, even in the absence of added fumarase, under the conditions
required for maximum malate decarboxylation (see the table). There-
fore, to obtain a measure of malate-~4C alone, a more highly purified
malie enzyme preparation is required. Methods are available for this
type of preparation from L. plantarum, 21 pigeon liver, 1°,22 and wheat
germ. 2~ Subsequent incubation with aconitase and I C D H gave a measure
of citrate-~4C q-isocitrate-*4C. Since the samples had been freed previ-
ously of labeled malate q-fumarate, there was no chance of even small
amounts of malic enzyme activity in these enzymes causing erroneous
results. Incubations with I C D H alone would allow the estimation of
isocitrate-l*C and hence citrate-~*C separately, but, with the samples
studied, the radioactivity present in isocitrate was too low for accurate
determinations.
~S. Kaufman, S. Korkes, and A. del Campillo, J. Biol. Chem. 192, 301 (1951).
2..,S. Ochoa, see Vol. I, p. "/39.
[ 7 2 ] A s s a y of C o e n z y m e A a n d S o m e A c y l D e r i v a t i v e s
By P. K. TUBBS and P. B. GARLAND
A rather long period elapsed between the discovery of coenzyme A
and its thioesters, the recognition of the importance of these compounds
in metabolism, and tlle investigation of the in vivo acylation state of the
coenzyme. However, the problems of metabolic control at the subcellular
lever are now being explored, and sensitive enzymatic assays have been
developed for the determination of many compounds of interest, including
CoA and its thiocsters.
536 SEPARATION AND ASSAY METHODS [7~]
In view of the number of procedures that have been published we

have had to conserve space by incorporating the principles into the form
of tables, and in many cases the details must be sought in the original
papers; even so the list is incomplete, although it is hoped that the most
suitable methods in each case have been given. In addition to the tables,
some assay methods which the authors have found especially useful are
described in detail. No attempt has been made to repeat the information
given in earlier volumes of this treatise; for example, the optical and
chemical properties of thioesters. 1 (See also sources cited in footnotes
2 and 3.)
In general, tissue contents of CoA and its derivatives are derived from
extracts prepared from samples which have been deproteinized with acid
{most conveniently HC104 followed by neutralization with KOH, K2C03,
or potassium acetate). Attention is drawn to the necessity for "instant"
freezing 4 of tissue samples, since the acylation state of CoA can change
very rapidly2 In vitro incubation systems s are conveniently quenched by
rapid mixing with dilute HC10, (final concentration 2--5~, w/w).
Stoichiometric assays are normally to be preferred to catalytic pro-
cedures; although the latter are in principle more sensitive, elaborate
controls may be needed and a stoichiometric assay is needed for calibra-
tion purposes. The use of oxoglutarate dehydrogenase, which has a K=
for CoASH of less than 0.1 ~ , ~ permits assay of very small amounts of
CoA (less than 1 millimicromole),e especially since fluorimetric methods
ear be employed. We also draw attention to the convenience of 5,5t-
dithiobis-(2-nitrobenzoate) s as a reagent, and to the fact that the
specificities of citrate synthase (acetyl-CoA only) and carnitine acetyl-
transferase (acetyl- to decanoyl-CoA) permit differentiation between
acetyl-CoA and other short-chain acyl-CoA compounds. The latter
enzyme may be coupled to methods described in this article, so giving
assays for carnitine and its acyl derivatives, s~
The names of enzymes commercially available in purified form
(March, 1969) are indicated by asterisks.
1E. R. Stadtman, Vol. III, p. 931.

~H.-U. Bergmeyer (ed.), "Methods of Enzymatic Analysis." Verlag Chemic, Wein-
helm, and Academic Press, New York, 1963.
, L. Jaenicke and F. Lynen, in "The Enzymes" (P. D. Boyer, H. Lardy, and K.
Mj~rback, eds), Vol. 3, p. 3. Academic Press, New York, 1960.
A. Wollenberger, 0. Ristau, and G. Schoffa, Arch. Ges. Physiol. 270, 399 (1960).
SD. J. Pearson and P. K. Tubbs, Biochem. J. 105, 953 (1967).
, p . B. Garland, D. Shepherd, and D. W. Yates, Biochem. J. 97, 587 (1965).
~Y. Massey, Biochim. Biophys. Acta 38, 447 (1960).
I G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959).
~°Article by Pearson el al., Vol. 14, in preparation.
[72] ASSAY OF CO). AND ACYL DERIVATIVES OF COA 537
Assay of Coenzyme A (CoASH)

Procedures are given in Table I. For methods other than those
described below see original references.
Table I, Procedure (7)

Sorbate T CoASH + ATP --* sorboyl-CoA T AMP T PP,
Principle. This method, based on the observations of Wakil and
Hiibscher, 9 depends on the high extinction of sorboyl-CoA at 300 m/~.
Reagents
Sorbate-MgC12-ATP-buffer mixture. A solution containing the fol-
lowing (final concentrations) is prepared and stored frozen: 20
mM potassium sorbate, 20 mM MgC12, 20 mM neutralized ATP,
0.4M Tris-HC1, pH 8.2. The sorbate may be prepared from
sorbic acid (Eastman Kodak Co., practical grade) crystallized
twice from hot water. The mixture is stable to frequent freezing
and thawing over several months
Acyl-CoA synthetase. This enzyme is prepared from acetone-dried
ox liver mitochondria according to Mahler et al. 1° The enzyme
may be used at the stage described by these authors as fraction
C; (however, if this CoASH assay is to form part of a carnitine
determination, the enzyme must be further purified to remove
carnitine acetyltransferase and acetyl-CoA hydrolase. This is
accomplished by treatment with calcium phosphate gel and
DEAE chromatography at pH 8.7s). Dissolved in 20 mM KHC03
the enzyme is stable for many months when stored frozen.
Procedure. The solution to be tested (containing 2--30 millimicromoles

of CoASH), 0.5 ml of sorbate-MgClrATP-Tris mixture, and sufficient
glass-distilled water to give a final volume of 2.0 ml (after addition of
synthetase) are placed in a silica cuvette with a 1 cm light path. The
optical density at 300 mg is noted, and the reaction is started by addition
of synthetase (0.01-0.05 ml). The enzyme added should be at least 0.05
international units (1 IU = 1 micromole of product per minute at 25°),
or enough to give an initial rate of optical density increase of 0.5 per
minute at saturating CoASH concentration. The increase of optical
density is followed until it ceases, or becomes equal to that of a control
lacking CoASH (this rate should be less than 0.005 per minute). The
optical density increase due to the synthetase itself is subtracted, and
S. J. Wakil and G. Hiibscher, J. Biol. Chem. 235, 1554 (1960).
~oH. R. Mahler, S. J. Wakil, and R. M. Bock, J. Biol. Chem. 204, 453 (1953).
°~ ~ ~
z
7
C9
+
×
f~
¢D I I
¢q
+
+
~9
eD d~ o']
I
T
I
a¢3
c~
¢9
! °~
°~
.¢
v
v
[72] Ass.,,Y OF COA AND ACYL DERIVATIVES OF COA 539
~.~
i
~0 0 u~4 ~ 0
5"q
2 ÷:~ -t-
÷ +
iiPT
÷,. q-
z~ ~'[+o
T ~ /~
+~ -t-
v
~
+
~+
.e ~
~ +~ ÷ Jr
o e$
+ d+
.<
I O
~ °
~~
O ~0.=
540 SEPARATION AND ASSAY METHODS [~
~.~
~.~ x
0
+
v-4
e,
~ ~, ~.~l~ ~+
o
[72] ASSAY OF COA AND ACYL DERIVATIVES OF COA 541
the amount of CoASH is calculated from the net increase. The molar
extinction coefficient for the sorboylation of CoA is 23,500 ± 500 cm-1
(D. J. Pearson and P. K. Tubbs; 5 see footnote 11), so 1 millimicromole of
CoASH gives an optical density increase of 0.0117 in a 2 ml volume. The
assay is performed most conveniently in a recording spectrophotometer;
in this case also the immediate optical density due to the synthetase
itself may be measured directly. To determine this in a manual instru-
ment it is necessary to add a second aliquot of enzyme after the reaction
is complete.
Remarks. The acyl-CoA synthetase also accepts 3'-dephospho-CoA,
although at a considerably slower rate than that found with CoA. It is
unnecessary to add thiols such as glutathione or mercaptoethanol;
indeed, if large amounts of these (more than about 0.5 mM glutathione,
or less with mercaptoethanol) are present interfering acyl-transfer re-
actions may occur, with recycling of CoA. This problem is less acute
with highly purified synthetase. It is necessary that the temperature
remain constant throughout tht assay; warming causes an increase in
optical density at 300 m~ (due to a drop in the pH of the Tris buffer,
giving more undissociated sorbic acid).
The main drawback in this method of CoA assay is the slowing down
of the reaction as the sorboylation approaches completion, due to the
relatively high K,, for CoA (about 5 ~M). For very small amounts of
CoA, therefore, methods based on a-oxoglutarate dehydrogenase are to
be preferred; for larger amounts, or in the absence of a fluorimeter, the
sorbate method is very useful.
Table I, Procedure (8)

Principle. The oxoglutarate dehydrogenase catalyzed reaction
CoASH + 2-oxoglutarate + NAD -~ succinyl-CoA + COs + NADH2
is irreversible [--AFo (pH 7.0) ~ I0 kcal/mole], and the K,~ value for
CoASH is less than 0.1 tLM.7 The reaction is followed by measuring the
appearance of NADH2, either spectrophotometrically at 340 mt~12 or
fluorimetrically. 6 When NAD and 2-oxoglutarate are present in a suf-
ficient excess relative to CoASH, the reaction proceeds to completion
with approximately zero-order kinetics. Oxoglutarate dehydrogcnase from
pig heart is inhibited by both NADH2 and succinyl-CoA, and this
inhibition is abolished if the NAD concentration is greatly in excess of
the concentration of CoASH that is to be assayed. 12 Alternatively the
'1G. Michal and H.-U. Bergmeyer, Biochim. Biophys. Acta 67, 599 (1.963).
'~ F. B. Garland, Biochem. J. 92, 10c (1964).
542 SEPARATION AND ASSAY M E T H O D S [72]
3-acetyl pyridine analog of NAD can be used, giving greater sensitivity
at 366 m/~ and absence of product inhibition? 2
Reagents
Phosphate--MgC13-EDTA buffer mixture. A solution containing
0.1 M KH~P04, 2 mM MgC1._, and 1 mM EDTA is adjusted to
pH 7.0 by adding solid KOH. This solution is stable. Contamina-
tion by growth of microorganisms is prevented by storage at
0-2 ° in the dark
NAD solution. A solution containing approximately 10 mM NAD
is adjusted to pH 6.5 by adding an appropriate amount of 1.0 M
Tris base. The final concentration of NAD is not critical; between
5 and 10 mM is suitable. Storage is at --15 °, and the solution is
stable to freezing and thawing over several weeks
Oxoglutarate solution. A 5--10 mM solution of 2-oxoglutarate at pH
6.5 is prepared and stored in a similar fashion to the :NAD
solution
Oxoglutarate dehydrogenase. This enzyme is prepared from pig
heart by the procedure of Sanadi et al. 13 and assayed under the
same conditions as for CoASH assay, except that a known
amount of CoASH (about 30-50 ~M) is used to initiate the
reaction. The specific activity of the oxoglutarate dehydrogenase
should be about 2-4 units/mg. The enzyme loses activity with
frequent freezing and thawing, and this drawback can be over-
come by dispensing the freshly prepared enzyme solution into
many small tubes {0.1-0.2 ml in each), and keeping each tube at
--15 ° until it is required. Alternatively the enzyme can be
stored unfrozen at --15 ° in 30~ (v/v) glycerol. The enzyme is
stable for several months stored in either manner at a protein
concentration of 2-4 mg/ml. Contaminating enzymes of lower
molecular weight (e.g., malate dehydrogenase, glutamate dehy-
drogenase) can be removed by sedimenting the oxoglutarate
dehydrogenase at 100,000 g for 2 hours. Preparations with higher
purity have also been described 7,1~
Procedure. (a) SPECTROPI-IOTOMETRICASSAY.TM The solution for assay

should have been adjusted previously to pH 6-6.5, and should be fresh.
Even at this pH, losses of CoASH (by oxidation to disulfide) are observed
on storage, and further errors can be caused if the solution also contains
13D. R. Sanadi, J. W. Littlefield, and R. M. Bock, J. Biol. Chem. 197, 851 (1952).
" T . Hayakawa, M. Hirashima, I. Satoshi, M. Hamada, K. Okabe, and M. Koike,
J. Biol. Chem. 241, 4694 (1966).
[72] ASSAY OF COA AND ACYL DERIVATIVES OF COA 54~
CoA thioesters which may slowly hydrolyze. Up to 0.9 ml of the solution

for assay (containing 2-20 millimicromoles of CoASH) is added to 1.0 ml
of phosphate-MgCl2-EDTA buffer mixture in a silica cuvette with a l
cm light path and followed by 0.02 ml of NAD solution, 0.02 ml of
oxoglutarate solution, and sufficient water to give a final volume of 2.0
ml. The optical density at 340 m~ is recorded, and the reaction is started
by mixing in 20 ~g of oxoglutarate dehydrogenase. The increase of optical
density due to the complete utilization of CoASH is over within 30
seconds, and a second addition of enzyme should be made to obtain a
correction value for its own optical density. A very slow increase in
optical density may follow the initial fast increase. The slow increase
results from nonenzymatic hydrolysis of succinyl-CoA with subsequent
recycling of CoASH. The slow increase does not introduce a significant
error provided that sufficient enzyme is added to complete the initial fast
assay in 30 seconds. One millimicromole of CoASH results in an optical
density change at 340 m/~ of 0.0031 in a 2.0 ml final volume.
(b) FLUORIM~rmC ASSAY.e The assay conditions are the same as for
spectrophotometry except that 0.01-2 millimicromoles of CoASH are
assayed. A suitable exciting light is the 366 m~ line of a medium or high
pressure mercury arc lamp, or a combination of the 313, 334, and 366 m/~
lines. Alternatively, a 340 m/~ interference filter can be used in combina-
tion with a 100 W quartz--tungsten-iodine lamp. A secondary filter, such
as the Wratten 2E, protects the photomultiplier against scattered and
reflected exciting light but transmits most of the light emitted by fluoresc-
ing NADtt2. The fluorimeter should be equipped with a suitable zero
suppression for assaying extracts that have a high endogenous fluores-
cence. Calibrations of the fiuorimeter should be made with spectro-
photometrically assayed CoASH or NADtt2.
Remarks. Dephospho-CoA is a substrate for oxoglutarate dehydro-
genase, although the K~ value (60/~M) is 2-3 orders of magnitude greater
than that of CoASH. At low concentrations ( < 5 ~M), dephospho-CoA
is not assayed during the time taken for CoASH assay. The relatively
low pH and absence of added thiols prevent interference by other CoA
species, such as disulfides or thioesters. The assay can also be coupled to
any CoASH releasing system, thereby assaying the activity of the
enzymes involved in CoASH release or the amount of CoASH precursor.
Examples follow:
(i) Phosphotransacetylase* and aeetyl-CoA:
arsenate
CH~COSCoA + OH- , CH3COO- + CoASH
(ii) Citrate synthase* and acetyl-CoA:
CH~COSCoA ~ oxaloacetate --, citrate ~ CoASH
(iii) Carnitine acetyltransferase ~ and short-chain acyl-CoA:
RCOSCoA -}- L-carnitine --* acyl-b-carnitine -}- CoASH
(iv) Carnitine palmitoyltransferase and palmitoyl-CoA:
Palmitoyl-CoA ~ L-carnitine--* pahnitoyl-L-carnitine -]- CoASH
(v) Other acyltransferases, e.g., glycerolphosphate acyltransferase:
L-Glycerol 3-phosphate -}- 2 acyl-CoA --* phosphatidic acid -b 2 CoASH
Fluorimetric assays using "front-face" fluorescence are particularly
suitable for enzymes that are tightly bound to membranes, since the
technique avoids the problems associated with spectrophotometry of
turbid suspensions. The high sensitivity of the fluorimetric assay also
makes it suitable for measuring the CoASH release associated with acyl
transfer from an acyl-CoA to an enzyme (ESH) :
Acyl-SCoA T ESH ~ acyl-SE + CoASH
Assay of CoA Thioesters

For procedures see Table II.
Assay of Acetyl-CoA
Table II, Procedure (1)
Acetyl-CoA -b oxaloacetate --, citrate -b CoASH (i)
CoASH ~- DTNB --* 5-thio-2-nitrobenzoate (~- mixed disulfide) (ii)
[DTNB : 5,5'-dithiobis (2-nitrobenzoate)]
Principle. In the presence of citrate synthase ~ (EC 4.1.3.7) aeetyl-
CoA reacts with oxaloacetate, liberating CoASH [reaction (i) ]. This, like
other thiols, reduces DTNB to give yellow 5-thio-2-nitrobenzoate; this
process [reaction (ii) ; see Table I, Procedure (3) ] gives a molar increase
in optical density of 13,600 cm-1 at 412 m~. 8
Reagents
Tris-HC1 buffer, 1 M, pH 8.0
Oxaloacetic acid
Citrate synthase ~ (crystalline suspension)
DTNB, 10 mM solution, prepared by dissolving DTNB in dilute
(about 50 mM) KHC03, adding HC1 until the color becomes very
pale, and diluting with water to a final concentration of 4 mg/ml.
This solution, which may be stored frozen, should be protected
from light
Procedure. Into a cuvette with a 1 cm light path are placed 0.2 ml

Tris-HC1 buffer, about 1 mg of solid oxaloacetic acid (this need not be
weighed), 25 ~l DTNB solution, and sufficient glass-distilled water to
give a final volume (after acetyl-CoA and synthase addition) of 2.0 ml.
The very low extinction at 412 m~ is recorded, and the sample of acetyl-
CoA (5-100 millimicromoles) is added. The increase in extinction at this
point measures the free thiol (CoASH, etc.) in the sample (increase 0.0068
per millimicromole). Citrate synthase (e.g., 5 ~l suspension) is finally
added, and the further increment in optical density indicates the amount
of acetyl-CoA.
Remarks. This method is the most satisfactory for calibrating stock
solutions of acetyl-CoA; it cannot be used with tissue extracts because
of the large amount of thiols present. Only the acetyl derivative of CoA
reacts at an appreciable speed (short-chain homologs such as propionyl-
CoA do not interfere). Acetyl-3"-dephospho-CoA reacts at about 30% of
the rate, and with increased amounts of citrate synthase may be satisfac-
torily assayed. The simultaneous determination of the free CoASH in
acetyl-CoA solutions is very convenient.
Malate + NAD ¢-* oxaloacetate + NADH~ (i)
Oxaloacetate + acetyl-CoA ---) citrate + CoASH (ii)
Malate + NAD + acetyl-CoA ~ citrate + CoASH + NADH2
Principle. The citrate synthase~-catalyzed reaction (ii) consumes
oxaloacetate, and so displaces the equilibrium of reaction (i), catalyzed
by malate dehydrogenase*; thus acetyl-CoA removal causes the forma-
tion of NADH~, which may be measured fluorimetrically or spectro-
photometrically. However, under the usual conditions acetyl-CoA does
not give rise to a stoichiometric amount of NADH~, for reasons discussed
by Pearson 15 (see also footnote 16), and with low amounts of acetyl-CoA
(as in tissue extracts) a serious underestimate results. This problem can
be overcome either (a) by adding NADH2 [Method (2) below], when a
virtually stoichiometric result is obtained, or (b) by measuring both the
NADH~ formed on addition of malate dehydrogenase and the further
increment formed on addition of citrate synthase; the amount of acetyl-
CoA may then be calculated or obtained graphically [Method (1) below].
Reagents
Tris-HCl buffer, 1 M, pH 7.8
a-Malate (Na or K salt), 1 M (DL-malate may also be used)
NAD, l0 mM
~ D. J. Pearson, Biochem. J. 9,5, 23c (1965).
546 S E P A R A T I O N AND ASSAY METHODS [72]
~._~ ~ o
• ~ ~ ~ , _ N~
2
0
~
~~ . ~~ ' ~~, .~~.~_
"~
~ o ~ ~ - ~ •
o ~ ~'~
¢.O
~ ~ +~
C
0
o~
~l+.~
-4- ~ ~o ~o_
.~.~.
~eL ~
~.
elg
]
m ~
~'~
,¢ r~
+-~
1"+
+~
E
T~ + II ©
,
+~
.~ ;>-.~
~.~
~.~ 0
v
,,-.T
,~
?
NADH2, 10 mM [Method (2) only]
Malate dehydrogenase* (commercial suspension)
Citrate synthase* (crystalline suspension)
Procedure. Into a cuvette (light path 1 em) are placed 0.2 ml of Tris
buffer, 0.02 ml of malate, 0.1 ml of NAD, 0.025 ml of NADH2 [Method
(2) only, see below], the sample to be assayed for acetyl-CoA, and suffi-
cient glass-distilled water to give a final volume of 2.0 ml. The euvette is
placed in a speetrophotometer (preferably a recording instrument), and
the optical density at 340 m~ is measured. Malate dehydrogenase (5 ~l)
is added, bringing reaction (i) to equilibrium, and the increase in optical
density (J~kE1) is recorded. Finally, citrate synthase (5.~I) is added, and
the increment in optical density (~E2) due to acetyl-CoA is noted.
Calculation. METHOD (1), NO NADH2 (OR OXALOACETATE) PRESENT INI-
TIALLY. The molar extinction change for NADH2 formation is 6220 cm-1
at 340 m~ (or 3300 at 366 m~ at 25°). Pearson 15 showed that the amount
of acetyl-CoA present in the 2 ml system is a X (AN1 X 2/6.22) micro-
moles, where a = (f12 ~ 2B)/B ~ 1 and p is given by (AE2/~E1). Thus
a permanent correction graph giving a for different observed values of p
can be prepared, and amounts of acetyl-CoA derived (for example, if
fl = 1 then a = 1.5, or when p = 0.1 then a is about 0.191). An alterna-
tive method of correction has been given. TM
METHOD (2): NADH2 ADDF~. If, before addition of citrate synthase,
the concentration of NADH2 is ten or more times that of oxaloacetate,
then acetyl-CoA does give rise to a virtually equivalent amount of
NADH2 when the synthase is added ;1~ thus addition of NADH~ as above
obviates the need for a correction factor and AN1 need not be measured.
The disadvantage of this method is that the NADH2, besides being
expensive and rather unstable, gives a high initial optical density.
Remarks. Either variant of the coupled assay may be used with tissue
extracts; Method (1) can also be adapted to fluorimetry. Extracts that
have been deproteinized with acid will not contain appreciable NADH2,
and interference by oxaloacetate will not normally occur because of the
low tissue levels of this compound.
As with Procedure (1), this assay is specific for aeetyl-CoA or acetyl-
3P-dephospho-CoA.
Principle. Phosphotransacetylase* catalyzes a rapid arsenolysis of
acetyl-CoA to acetate and CoASH. The CoASH can then be assayed with
oxoglutarate dehydrogenase.
Procedure. The conditions are as described for CoASH assay using
BH.-U. Bergmeyer and H. Moellering, Biochem. Z. 344, 167 (1966).
oxoglutarate dehydrogenase, except that phosphate is replaced by arse-

nate. After CoASH has been assayed by the addition of oxoglutarate
dehydrogenase, 1-5 tLg of phosphotransacetylase is added and the further
increase of optical density due to acetyl-CoA arsenolysis is recorded.
Comment. :Not all preparations of phosphotransacetylase have proved
satisfactory for this assay. As alternatives, CoASH can be released from
acetyl-CoA by the use of oxaloacetate (50 t~M) and citrate synthase (0.2
unit) or L-carnitine (1 raM) and carnitine acetyltransferase (0.2 unit).
However, malate dehydrogenase or short-chain acyl-CoA may interfere
with these respective variants.
Assay of Short-Chain (C2-Clo) Acyl-CoA

Principle
Acyl--CoA + ( - ) -earnitine ~ CoASH + acyl- ( - ) -carnitine (i)
[CoASH is then assayed, e.g., Table I, Procedures (3) or (8)]
Reaction (i) is catalyzed by carnitine acetyltransferase.* For assay of
stock solutions of acyl-CoA this reaction is most conveniently coupled to
estimation of the released CoASH with DTNB [analogous to Procedure
(1) of Table II for acetyl-CoA, described above].
Reagents
Tris-HC1 buffer, 1 M, pH 8.0
DL-Carnitine, 1 M neutral solution
Carnitine acetyltransferase* suspension
DTNB, 10 mM solution, prepared as for Procedure (1) for acetyl-
CoA assay
Procedure. Into a cuvette with a 1 cm light path are placed 0.2 ml of
Tris buffer, 0.02 ml of carnitine solution, 25 ~l of DTNB solution, and
sufficient glass-distilled water to give a final volume (after acyl-CoA and
transferase addition) of 2.0 ml. The optical density at 412 m~ is recorded,
and the acyl-CoA sample (5-100 millimicromoles) added; the increase
in optical density measures the free thiol (CoASH, etc.), in the sample
(increase 0.0068 per millimicromole). Acetyltransferase (e.g., 5-10 t~l
suspension) is then added, and the further increase in optical density
indicates the amount of acyl-CoA.
Remarks. The velocity of the reaction decreases with increasing chain
length of the acyl-CoA, 1~ and with the higher homologs it is necessary to
'~J. F. A. Chase, Biochem. J. 104, 510 (1967).

use more en~.yme. Since DTNB progressively inhibits the transferase,

sufficient enzyme should be used to complete the reaction within 5
minutes, and the activity should then be checked by addition of more
acyl-CoA. A control without acyI-CoA should be run, to allow for the
color yield of enzyme plus DTNB. If oxaloacetate is added to the
system and an addition of citrate synthase, as described above for
Table I, Procedure (1), precedes that of carnitine acetyltransferase, it
is possible to measure both acetyl-CoA and short-chain acyl-CoA (as
well as free thiol) in the same sample, since citrate synthase accepts
only acetyl-CoA.
For measuring short-chain acyl-CoA in tissue extracts the DTNB
method is unsuitable, and oxoglutarate dehydrogenase [Table I, Pro-
cedure (8), described above] should be coupled to the carnitine acetyl-
transferase reaction. Since tissues contain much more acetyl-CoA than
short-chain acyl-CoA, except under very unusual conditionsf the former
must be measured specifically; as with the DTNB procedure above, this
can be done in the same cuvette with the aid of citrate synthase and
oxaloacetate (but beware of interference by malate dehydrogenase).
Assay of Long-Chain (C12 or Longer) Acyl-CoA

These derivatives may be separated from other CoA compounds by
virtue of their insolubility in dilute HCI04 (e.g., 2% w/w). 18-2° Thus,
after grinding powdered frozen tissue with HC104, centrifuging leaves
long-chain acyl-CoA in the sediment while the supernatant contains the
other forms of CoA. Very small amounts of aeyl-CoA, for example,
these formed in in vitro incubations, require the presence of protein (e.g.,
serum albumin) for satisfactory precipitation. After being thoroughly
washed two or three times with dilute HCIO~ and dispersion in water,
the precipitate is extracted with alkali at pH 11-12 for 20-30 minutes at
room temperature; this hydrolyzes the acyl-CoA. A thiol (e.g., 5 mM
glutathione) should be present, to prevent extensive oxidation of CoA
[but see possible interference by thiol in Procedure (7) of Table I]. After
acidification and centrifugation the extract is neutralized and assayed
for CoA [Table I, Procedure (6), (7), or (8)].
Long-chain acyl-CoA can be extracted from HC104-precipitated and
washed material by means of serum albumin; 19 such extracts could
1~O. Wie]and and L. Weiss, Proc. IV Intern. Congr. Biochem. Vienna 15, 158 (1960).
i~p. K. T u b b s and P. B. Garland, Biochem. J. 93, 550 (1964).
~ W. M. Bortz and F. Lynen, Biochem. Z. 339, 77 (1963).
p r e s u m a b l y be a s s a y e d in a c o u p l e d s y s t e m c o n t a i n i n g p u r i f i e d carniLine
p a l m i t o y l t r a n s f e r a s e , excess carniLine, a n d o x o g l u t a r a t e d e h y d r o g e n a s e
( a n a l o g o u s to t h e m e t h o d for s h o r t - c h a i n a c y l - C o A ) . H o w e v e r , the
p a l m i t o y l t r a n s f e r a s c s p e c i f i c i t y is n o t y c t k n o w n in detail. "-"'~
~oaOf the following footnotes, 21-28 are pertinent to Table I; 29-36 to Table II.
21p. D. J. Weitzman, Biochem. J. 99, 1Oc (1966).
=E. R. Stadtman, G. C. Novelli, and F. Lipmann, J. Biol. Chem. 191, 365 (1951).
"Footnote 2, p. 419.
2, R. W. yon Korff, J. Biol. Chem. 200, 401 (1953).
Footnote 2, p. 523.
H. Inoue, K. Adachi, F. Suzuki, F. Fukuniski, and Y. Takeda, Biochem. Biophys
Res. C o m m ~ . 21, 432 (1965).
:7 Footnote 2, p. 513.
2sFootnote 2, p. 517.
P. A. Srere, H. Brazil, and L. Gonen, Acta. Chem. Scand. 17, s129.
W. Pring, W. Schoner, V. Haag, and W. Seubert, Biochem. Z. 346, 206 (1966).
3, p. K. Tubbs and P. B. Garland, unpublished.
3-.C. D. Upper, Ph.D. Thesis, University of Illinois, Urbana, Illinois, 1964.
83S. Cha, C.-J. M. Cha, and R. E. Parks, J. Biol. Chem. 240, PC 3700 (1965).
"Vol. V, p. 446.
"Footnote 2, p. 445.
Footnote 2, p. 425.
[73] REMOVAL OF PHENOLIC COMPOUNDS FROM PLANTS 555
[73] R e m o v a l of P h e n o l i c C o m p o u n d s d u r i n g t h e
I s o l a t i o n of P l a n t E n z y m e s
By W. D. LooMIs
Plants produce a variety of phenolic compounds, which often interfere
seriously in the isolation of plant enzymes or organelles. Some plant tis-
sues contain such high concentrations of "tannins" that all protein is
precipitated during conventional extraction procedures, whereas other
phenols may inactivate or modify enzymes without precipitating them.
Several plant phenolic compounds have been shown to be potent enzyme
inhibitors at concentrations of less than 1 raM.
Since production of phenolics is a function of growing conditions as
well as the species and variety of plant, it cannot be expected that any
one procedure for enzyme extraction will be equally effective with all
plant materials. However, generalizations can be made about the ways in
which phenolic compounds react with proteins and with compounds
related to proteins. These reactions have been reviewed elsewhere1-4 and
will be summarized here. We will also describe some enzyme techniques
which are of value in working with plant tissues rich in phenolic com-
pounds. These techniques, and an understanding of the reactions of
phenols with proteins, will help investigators to develop methods suitable
for their material.
Reactions of Phenolic Compounds

Phenols combine with proteins in two ways: reversibly by hydrogen
bonding, and irreversibly by oxidation to quinones followed by covalent
condensations of the quinones with reactive groups of protein. The
quinones may also oxidize essential groups of proteins.
Hydrogen Bonding of Phenols
The hydrogen bond formed between phenols and N-substituted
amides (e.g., peptide linkages) is very strong, and the equilibrium in
aqueous solution favors the formation of complexes. The evidence indi-
cates that the principal bonding is via the proton of the phenolic hy-
droxyl group, the oxygen of the peptide linkage acting as proton
acceptor. The amount of phenolic material bound to protein by hydrogen
'W. D. Loomis and J. Battaile, Phylochemistry 5, 423 (1966).

2 H. S. Mason and E. W. Peterson, Bioehim. Biophys. Acta 111, 134 (1965).
K. H. Gustavson, J. Soe. Lea~her Trades' Chemisls 50, 144 (1966).
4 W. S. Pierpoint, Biochem. J. 98, 567 (1966).
556 SPECIAL HANDLING OF PLANTS [73]
bonding may be equal to more than one-third the dry weight of the
protein. This itself may precipitate or inactivate enzymes. Moreover, the
hydrogen-bonded complexes tend to become covalent complexes through
oxidation of the bound phenols to quinones.
Quinone Formation and Quinone Reactions

If oxidation of phenols occurs, the resulting quinones may form
permanent covalent linkages to proteins. From the available evidence, re-
active groups of the protein undergo 1,4-addition to the quinones, form-
ing covalent complexes. Sulfhydryl groups are particularly reactive; free
amino and imino groups also react. Quinones with a second reactive ring
position available can react with a second reactive group of proteins,
resulting in rapid cross-linking. The oxidation of phenols may be
catalyzed by either phenol oxidases or peroxidases, or it may occur non-
enzymatically.
Oxidation of phenols commonly results in development of melanin.
Whenever "browning" occurs in a plant extract or homogenate, it may be
assumed that the plant proteins are modified by quinone additions. The
proteins may be extensively "tanned" and converted into dark-colored
insoluble melanoproteins.
If only a few quinone groups add to an enzyme molecule, the damage
may be hardly discernible. Likely effects are: decreased solubility, ab-
normally high UV absorption, fluorescence under UV light, and decreased
stability of the enzyme. However, a single quinone molecule reacting
with a critical -SIt group could cause complete inactivation of the
enzyme.
Quinones may cause damage by oxidation as well as by addition
reactions. However, the same control measures apply to both mecha-
nisms.
Principles of Enzyme Isolation from Phenol-Containing Tissues

Techniques for isolating enzymes or organelles from plants that
contain phenolic compounds should not only separate the phenols from
proteins, but also prevent oxidation of the phenols.
Separation o] Hydrogen-Bonded Complexes

Use o] High pH. Ionized phenols cannot serve as proton donors for
hydrogen bonding. Therefore, the ionized phenols may be removed by
conventional techniques, e.g., dialysis or gel filtration, using media with
a pH of about 8 or higher. Although this method is useful, its applica-
bility is limited because: (1) the high pH may damage enzymes, and (2)
ionized phenols are oxidized more readily to quinones than tile non-
ionized forms.
Use o] Phenol-Complexing Agents. At neutral or acid pH the only
effective means of removing bound phenolics from plant protein is to
supply a phenol-complexing agent that can compete with the peptide
linkages of the plant protein. Many substances with polar groups form
hydrogen-bonded complexes with phenols, but few of them form a strong
enough bond to compete with the peptide linkage. To date, the most
satisfactory agents appear to be various grades of polyvinylpyrrolidone
(PVP). Foreign protein and synthetic polyamides (nylons) have also
been used successfully. Polyethyleneglycols (PEG) have proved useful in
certain methods. ~,~
Phenolic hydroxyl groups act as very strong proton donors in hydro-
gen bonding. The - - C O - - N < group of PVP is a very strong proton ac-
ceptor and cannot act as a proton donor. For this reason PVP appears
to be a stronger and more specific phenol-binding agent than other avail-
able materials. The - - C O ~ N H - - group of proteins and polyamides is a
strong proton acceptor, and a weak proton donor. Proteins and poly-
amides bind phenols tightly, but probably not as tightly as does PVP. In
ordinary nylon, most of the amide groups are tied up by internal hydro-
gen bonding and are not available for binding phenols.
Ordinary PVP, even of high molecular weight, is very water soluble.
Complexes of soluble PVP with phenolics are commonly water soluble if
PVP is present in excess and if there has not been too much oxidative
polymerization of tile phenolics. Thus, soluble PVP is a very effective
agent for removing phenolics in the isolation of plant mitochondria. A
cross-linked, insoluble grade of PVP has been developed and is available
commercially as Polyclar AT. It is very effective in isolating soluble plant
enzymes; since it binds phenols as insoluble complexes, it allows rapid
separation of phenols from soluble proteins.
Polyethyleneglycols, being polyethers, act only as proton acceptors
(except for the terminal hydroxyl groups). They are weaker phenol
adsorbents than PVP, polyamides or proteins, but their solubility in ace-
tone has made them useful in enzyme isolation methods using acetone2
The formation of hydrogen-bonded complexes between phenols and
proteins or other phenol-complexing agents is an equilibrium process.
For this reason a large quantity of the complexing agent must be added
in order to displace the equilibrium and free the proteins.
Buffers of high pH are used commonly in extracting enzymes from
plant tissues, but when an agent such as PVP is used, the pH of the
A. M. Badran and D, E. Jones, Nature 206, 622 (1965).
*D, R. Dilley, Plant Physiol. 41, 214 (1966).
extraction medium should be low enough to suppress ionization of

phenolics. Although media of pH 7.4 or 7.5 have been used successfully, a
pH of 7.2 or lower is probably preferable.
Use o] Organic Solvents. Organic solvents that are capable of hydro-
gen bonding can be used to dissociate hydrogen-bonded protein-phenolic
complexes. 1 Solvents that act as proton acceptors, but not proton donors
(e.g., ketones, esters, dimethylformamide, N-methylpyrrolidone, and di-
methylsulfoxide), interact most specifically with phenols and are least
likely to denature proteins. Thus the conventional acetone-drying tech-
nique removes considerable phenolic material, and if about 20% water is
added to the acetone, the effectiveness in removing phenolics is im-
proved. ~,s A method is described here in which acetone treatment is
combined with the use of a phenol adsorbent. Addition of reducing agents
(e.g., mercaptoethanol) or phenol oxidase inhibitors (e.g., diethyldi-
thiocarbamate) to the acetone might also be advantageous.
Prevention o] Protein Modification by Quinones

There are two approaches to preventing protein modification by
quinones. The best, to prevent the formation of quinones, is difficult. The
alternative is to add reagents that react rapidly with quinones as they
are formed, thereby sparing proteins. Phenol oxidase inhibitors, such as
cyanide or diethyldithiocarbamate (DIECA), may be used if they do not
inactivate the enzyme system one wishes to isolate. Pierpoint 4 has shown
that DIECA not only inhibits phenol oxidase (by chelating copper), but
also reacts with quinones. DIECA (10 mM or 5 raM) has been used in
the isolation of plant enzymes 6 and viruses, 9'1° and it might be a useful
adjunct to the PVP methods described here.
Reducing agents, e.g., ascorbate, do not prevent quinone formation,
but reduce quinones as soon as they are formed. Thiols may also con-
dense with quinones. Reducing agents are useful in isolating plant
enzymes, but in one respect they may be harmful: in the presence of
oxygen, they activate the ortho-hydroxylation of monophenols by phenol
oxidases. In using reducing agents enough must be added to maintain
reducing conditions during the entire time that phenolics and protein
are in contact. The amount of reducing agent per gram of tissue is
important, rather than the concentration of reducing agent in the
medium. Ascorbate appears to be preferable to thiols when used at high
D. S. Bendall and R. P. F. Gregory, in "Enzyme Chemistry of Phenolic Com-

pounds" (J. B. Pridham, ed.), p. 7. Macmillan (Pergamon), New York, 1963.
8G. W. Sanderson and G. R. Roberts, Biochem. J. 93, 419 (1964).
°B. D. Harrison and W. S. Pierpoint, J. Gen. Microbiol. 32, 417 (1963).
~ W. S. Pierpoint and B. D. Harrison, J. Gen. Microbiol. 32, 429 (1963).
levels,' but ascorbate also causes damage to enzymes if extracts are

stored for some time.
For many purposes one can obtain adequate results by taking all
reasonable measures to prevent oxidation of phenols, while adding
quinone-scavcnging reagents. For some plant tissues or enzymes, this
nmy be inadequate, and it may be necessary to work under an inert
atmosphere in a glove box. This was the case in the recent isolation of
cell-free nitrogen-fixing extracts from soybean nodules? ~ Extracts of
high activity could be obtained only by adding both ascorbate and
insoluble PVP under an atmosphere of argon.
Methods of Enzyme Isolation from Phenol-Containing Tissues
Isolation of Soluble Enzymes Using Insoluble PVP (Polyelar AT)

By using insoluble phenol-adsorbents, it has been possible to obtain
active soluble enzymes from peppermint and tea leaves. With conven-
tionaI techniques, peppermint extracts browned badly, and no enzyme
activity other than phenol oxidase could be detected. No soluble protein
could be extracted from tea leaves by conventional methods. 1~
Extracts obtained from peppermint leaves with Polyclar AT 1 con-
tained mevalonic kinase, phosphomevalonic kinase, glutamyl transferase,
and alkaline phosphatase. Extracts obtained from tea leaves with Polyclar
AT 13 contained 5-dehydroshikimate reductase. By a similar technique
using powdered nylon as the phenol-adsorbent, tea leaf catechol oxidase
(formerly thought to be a particulate enzyme) was isolated in completely
soluble form? 4
Desc~iption o] the Method. The method described here is a modifica-
tion of the procedure published previously? Changes from the earlier
procedure involve principally the use of lower pH, and decrease in the
amount of ascorbate.
Polyclar AT is obtained from General Aniline and Film Corporation,
Dyestuff and Chemical Division, 140 West 51st Street, New York, New
York. Before use it is purified by boiling for 10 minutes in 10% HC1 and
washing with glass-distilled water until free of C1-. It can be used moist,
or dried for storage. If it is dried, the lumps should not be removed by
grinding since this may produce traces of soluble PVP.
The amount of Polyclar AT needed for optimal results depends on the
amount of phenolic material in the tissues. With tea 13 and peppermint ~
11B. Koch, H. J. Evans, and S. Russell, Plant Physiol. 42, 466 (1967).
,2 G. W. Sanderson, Tea Quart. 36, 103 (1965).
~ G. W. Sanderson, Biochem. J. 98, 248 (1966).
1, G. W. Sanderson, Biochim. Biophys. Aeta 92, 622 (1964).
leaves, 0.6-1.5 g dry weight of Polyclar per gram fresh weight of tissue
has proved satisfactory.
All operations are carried out in the cold. For each gram of fresh
tissue, 1 g dry weight of purified Polyclar AT is suspended in 10 ml of
0.1 M potassium phosphate buffer, pH 7.2, containing 0.1 M sodium
ascorbate. (The volume of solution can be reduccd, but the bulk of the
Polyclar makes it necessary to use a fairly large volume of liquid.) The
suspension should preferably be allowed to stand for some time before
use to ensure complete wetting of the Polyclar.
The fresh tissue is ground in a mortar with liquid nitrogen. The
frozen powder is stirred gently into the suspension of Polyclar until well
mixed, and the extract is separated from the Polyclar by squeezing
through bolting silk or fine-mesh nylon cloth. The Polyclar may be
resuspended in the buffer solution and extracted a second time. A second
treatment of the extract with Polyclar (0.1 g Polyclar per gram of tissue)
may also prove beneficial. 1~ After the extract has been squeezed through
bolting silk, it is nearly clear, most of the particulate matter remaining
in the Polyclar. The extract is further clarified by centrifugation (20
minutes at 35,000 g). The extract may be used at this stage for some
purposes. If the extract is to be stored for some time, or to be used for
assays of oxidation-reduction systems, or assays which depend on UV
absorption, it will be necessary to remove the ascorbate. This can be done
conveniently and quickly by gel filtration. Sephadex G-50 (Pharmacia,
Uppsala, Sweden) and Bio-Gel P=10 (Bio-Rad Laboratories, Richmond,
California) are satisfactory. As mentioned below, gel filtration may also
be necessary in order to completely remove phenolic compounds.
The use of liquid nitrogen is a very convenient way of macerating
the tissue, while keeping oxidation of phenolic compounds to a minimum.
If another method is used, precautions must be taken to avoid oxidation.
For example, the tissue might be homogenized in cold buffered aseorbate,
possibly in the presence of DIECA or another phenol oxidase inhibitor,
and the Polyclar added to the homogenate as quickly as possible.
Although it would be desirable to have the phenol-binding agent present
during homogenization, we have found that this produces trace amounts
of soluble PYP from Polyclar AT.
Isolation o] Mitochondria Using Soluble PVP
By adding soluble PVP to bind phenolic compounds, Hulme and co-
workers 15-17 have obtained active mitochondrial preparations from apple
I~A. C. Hulme, J. D. Jones, and L. S. C. Wooltorton, Phytochemistry 3, 173 (1964).
~A. C. Hulme, J. D. Jones, and L. S. C. Wooltorton, Nature 201, 795 (1964).
i~j. D. Jones, A. C. Hulme, and L. S. C. Wooltorton, Phytochemistry 4, 659 (1965).
fruit and rose petals, and even from apple peel, a tissue notoriously rich
in phenolic compounds. Conventional techniques for isolating mito-
ehondria had been ineffective with these tissues.
The mitochondrial fraction isolated from apples by the new technique
catalyzed reactions of the Krebs cycle and of electron transport, 1~,1~ and
also demonstrated respiratory control (A. C. Hulme, personal communi-
cation). Electron micrographs 15 rerealed the presence of intact mito-
chondria. Contaminants included a small amount of phenolic material,
and presumably chloroplast fragments (the preparations were green).
Wiskich TM has reported a similar procedure for isolating apple mito-
chondria. His mitochondrial preparation, which was also green, showed
respiratory control in the oxidation of succinate.
Description o] the Method. ~ Pharmaceutical grade PVP (Kollidon
25, molecular weight approximately 28,000) was obtained from Badische
Anilin und Sodafabrik (Ludwigshafen, Germany). The pharmaceutical
grade was essential, as ordinary grades were not sufficiently pure.
All operations were performed in the cold. Apples were peeled with
a stainless steel potato peeler, and 25 g of the peel was added immedi-
ately to 120 ml of cold extraction medium. The tissue was held below
the surface of the liquid by means of a polyethylene disk while vacuum
was applied and maintained for 10 minutes. The desiccator was stmken
gently during this time to allow all the air to escape from the tissue and
be replaced by extraction medium. The vessel containing the tissue was
placed on a magnetic stirrer, running at slow speed, under a special
stainless steel mill. The tissue was macerated by passing it between the
two knurled stainless steel rollers of the mill, as through a wringer, and
was returned to the original medium. During this procedure, the tissue
was bathed with further extraction medium (80 ml) from a wash bottle.
Thus a total volume of 200 ml of extraction medium was used for each
25 g of tissue.
The extraction medium contained: sucrose, 0.4M; tris{hydroxy-
methyl)aminomethane, 0.2M; citrate, 0.02M; KH2P04, 10 mM; ethyl-
enediaminetetraacetic acid (EDTA), 10 mM; and PVP. The pH of the
medium was made to 7.7-7.8 with H3PO4, resulting in a final pH of 7.3
to 7.5 in the homogenate. (A final pH of 7.0-7.2 would probably be
preferable.) Cysteine (10 mM or 30 raM) appeared to be beneficial):
PVP was added at the optimal level, as determined by trials. A PVP
concentration of 0.75% to 1% was suitable for mature Cox's Orange
Pippin apples. More PVP was required for immature fruit, and concen-
trations up to 5% were sometimes used. With very young apples, 5%
PVP was optimal? ~
l~j. T. Wiskich, Nature 212, 641 (1966).
The macerated peel was squeezed through fine cloth and then
centrifuged at 1000 g for 10 minutes. The supernatant was further
centrifuged at 15,000 g for 20 minutes. The mitochondrial pellet was re-
suspended in 20 ml of a solution containing 0.4M sucrose and 10 mM
EDTA, pH 7.5 with KOH, followed by centrifugation at 15,000 g for l0
minutes. (It might be beneficial to add PVP to this washing solution
and to use a lower pH.)
Isolation o] Enzymes by Use o/a Phenol-Binding Agent with Acetone

Badran and Jones 5 tested several phenol-binding agents in combina-
tion with acetone treatment to obtain active soluble phenol oxidase from
green bananas. The best results were obtained with polyethyleneglycols
of high molecular weight (4000 or 20,000). The best procedure was as
follows.
Banana tissue was cut into thin slices in 0.1 M potassium phosphate
buffer, pH 6.5, containing 1% PEG. The tissue slices were vacuum in-
filtrated with the PEG-buffer solution for 30 minutes and then homog-
enized in additional PEG-buffer mixture, and tested with tannic acid to
ensure that there was an excess of PEG (--->tannic acid-PEG precipitate).
The homogenate was poured into two volumes of cold acetone (--10 °)
and centrifuged at 1200 g for 10 minutes at --10% Phenolics and PEG
remained in the supernatant aqueous acetone, while the enzyme was
precipitated. The soluble enzyme was obtained from the precipitate by
several extractions with phosphate buffer, pH 6.5, and was clarified by
centrifugation at 18,000 g for 10 minutes. Addition of a wetting agent
(Triton X~114) to the extracting buffer increased the yield of soluble
enzyme.
Discussion
These methods yield better results than conventional techniques for
isolating active enzymes from plant tissues rich in phenolic compounds.
They are, however, still not perfected; improvements are needed, par-
ticularly for the prevention of oxidation.
Although PVP is useful in plant enzyme work, there may be
complications in its use. For example, there are suggestions 17 that PVP
may inhibit flavoproteins, possibly by binding the flavin coenzymes.
PVP has also been reported to inhibit apple phenol oxidase, ling
There are probably some phenolic compounds that do not form strong
hydrogen-bonded complexes with proteins or PVP, but which are readily
oxidized to quinones. This appears to be the case with tyrosine, and one
i~j. R. L. Walker and A. C. Hulme, Phytochen,istry 4, 677 (1965).

would expect it also in polyphenols in which intramolecular hydrogen

bonding is possible (e.g., eatechol derivatives). Such compounds would
not be removed effectively by PVP and would remain in the soluble
extract as latent inhibitors. Gel filtration or dialysis should remove them
if oxidation is prevented.
PREVIOUSLY PUBLISHED ARTICLES FROM METHODS IN ENZYMOLOGY
~ELATED TO SECTION VI
Vol. III [65]. Preparation of a-Keto Acids. Alton Meister.

Vo|. III [67]. Preparation and Assay of Oxalacetic Acid. Samuel P. Bessman.
Vo|. HI [68]. Preparation of Tricarboxylic Acids. Daniel H. Deutsch and Robert E.
Phillips.
Vol. HI [72]. Itaconic Acid and Related Compounds. Helge Larsen.
Vol. IV [25]. Synthesis and Degradation of Isotopically Labeled Glycolic, Glyoxylic,
and Oxalic Acids. Katherine F. Lewis and Sidney Weinhouse.
Vol. VI [77]. Methylmalonyl Coenzyme A. Martin Flavin.
Vol. VI [121]. Preparation of Tritium-Labeled Substrates. John M. Lowenstein.
[74] STEREOSPECIFICALLY LABELED INTERMEDIATES 567
[74] Stereospecifically Labeled Citric Acid

Cycle Intermediates
By SASHAENGLARDand KENNETHR. HANSON
List of Labeled Compounds 567

Problems in Nomenclature 569
General Procedures for Separation, Purification, Isolation, and Analysis 573
Chemical and Enzymatic Syntheses 577
1. Chemical Synthesis of Fumaric-d2 Acid 577
2. (3R)-L-Aspartate-3-d from Fumarate and (3S)-L-Aspartate-~,8-d2 from
Fumarate-d~ 578
3. (3R)-L-Malate-2,-d from Fumarate and (3S)-L-Malate-~,~-d, from
Fumarate-d~ 580
4. Chemical Synthesis of (3R)-L-Malic-$-d Acid from (3R)-L-Aspartic-8-d
Acid 581
5. (3S)-L-Malate-3-d from (3S)-L-Aspartate-2,8-d2 582
6. Chemical Synthesis of (3R)-D- and (3S)-L-Malic-2-d Acids 583
7. (3R)-Citrate-2fl-d2 from Oxaloacetate-d2 and (3S)-Citrate-~-d2 from
Acetate-d3 _ 584
8. (2R,3R)-Citrate-2-d from (3R)-L-Malate-3-d" and (2S,3R)-Citrate-2-d
from (3S)-L-Malate-2,8-d~ . 586
9. Chemical Synthesis of (3R)-Citric-22-d2 and (3S)-Citric-~-d~ Acids 589
10. Chemical Synthesis of (3S)-Citric-5-14C Acid 591
11. (2S)-Succinate-2-t and (3S)-2-Ketoglutarate-3-t from threo-D,-Isocitrate 592
12. (2R)-Succinate-$-t and (3R)-2-Ketoglutarate-3-t from 2-Ketoglutarate 595
13. Chemical Synthesis of (2R)-Succinic-$-d Acid from (3R)-L-Malic-8-d
Acid or (3R)-L-Aspartic-3-d Acid 596
14. Other Labeled Succinic Acids 598
L i s t of L a b e l e d C o m p o u n d s
Priority order
1. Alphabetical listing according to first letter of chemical name (note that
threo is a configurational prefix and is not considered).
2. Alphabetical listing according to isotope labeling: 1'C > d > t
3. Listing according to extent of labeling : d > d~ :~ d~, etc.
4. Achiral compounds before chiral isomers.
5. Listing according to chirality symbols: D > L, then (R) > (S)
6. Resolved compounds before racemates.
Section numbers have been given to assist in locating methods for preparing the
labeled compounds (see Chapter Contents).
Section
Acetic-d3 acid 7
Acetyl-d8 phosphate 7
(3R)-L-Aspartic-$-d acid 2, 4, 13
(3S)-L-Aspartic-~,8-d2 acid 2, 4, 5
568 PREPARATION OF COMPOUNDS [74]
(3,.g)=L-2-Bromosuccinic-3-d acid 6, 13
( 3S)-D-2-Chlorosuccinic-3--d acid 13
(3S)-r.-2-Chlorosuccinic-3-d acid 13
(3S)-Citric-5-"C acid 10
(2R,3R )-Citric-Z-d acid 8
(2S,3R)-Citric-2-d acid 8
(3R)-( T )-Citric-2,~-d~ acid 7,9
(3S)-(--)-Citric-~,~-d2 acid 7,9
(2R.3R )-Citric-~-t acid 8
Fumaric-d2 acid 1,2,3
Fumaric-t~ acid 1
(4R)-L-Glutamic-4:-d acid 13
(4S)-threo-D.-Isocitric-$-d acid 14
threo-D..L.-Isocitric-3~-d~ acid 14
(38)-2-Ketoglutaric-~-d acid 11
2-Ket oglut aric-~4,4-d~ acid 7, 14
(3R)-2-Ketoglutaric-$A~,~-d, acid 14
(3R )-2-Ketoglutaric-3-t acid 12
(3S)-2-Ketoglutaric-3-t acid 11
2-Ketoglutaric-3,3-t~ acid 11
L-Malic-~-d acid 5
(3R)oL-Malic-3-d acid, dimethyl ester 2, 3, 4, 13
(3R)-D-Malic-3-d acid 6,13
(38)-r.-Malic-2-d acid 5,6
(3RS)-V,L-Malic.2-d acid 6
(3S)-L-Mallc-~,3od2 acid 3,4,8
threo-3-Methyl-L-aspartic-3-d acid 13
(2RS)-2o(Methyl-d~)-malonyl-CoA 14
(3RS)-Mevalonic-~-~C-5,5-da acid 14
(3R )-Oxaloacetic-3-d acid 8
(3S)-Oxaloacetic-3-d acid 5,8
Oxaloacetic-3,3-d.. acid 7
(4R)-Oxalocitramalic-3,3-dt acid 7-lactone 9
(4S)-Oxalocitramalic-$,3-d~ acid T-lactone 9
(3R,4R)-2-Oxoglutaraldehydate-3,~-d~ 14
(6S)-Quinic-6-t acid 8
(2R)-Succinic-~-d acid 13
(2S)-Succinic-~-d acid 11, 14
Succinic-~,2-d~ acid 14
( 2R,3R)-Succinic-~$-d~ acid 14
(2R,3~)-Succinic-~,3-d2 acid 14
( 2S,38 )-Suc cinic-~,$-d~ acid 14
(2RS,3RS)-Succinic-~$-d2 acid 14
(3R)-Succinic-~,~,$-d, acid 14
(3S)-Succinic-~2,3-ds acid 14
Succinic-d4 acid 14
(2R)=Succinic-~-t acid 14, 12
(28)-Succinic-£-t acid 11, 14
(2S,38) -Succinic-$,Sot2 acid 14
(3R)-Succinic-~,~fl-ts acid 14
Succinic-t~ acid 14
[74] STEREOSPECIFICALLYLABELED INTERMEDIATES 560
Problems in Nomenclature*
In describing the structure of a stereospecifically labeled compound,
decisions have to be made as to (l) the way in which labeling is to be
indicated in the chemical name, (2) the symbolism to be employed to
designate absolute and relative configurations, and (3) the way in which
the stereochemical symbolism is to be incorporated into the name. The
older biochemical literature in naming deuterated and tritiated com-
pounds employs deuterio- and tritio- as alphabetically ordered substitu-
tional prefixes (analogous to hydroxy-, chloro-, etc.) and extends carbo-
hydrate and amino acid nomenclature to give such names as
erythro-3-deuterio-L-malate (formula I). ~ This method is particularly
reasonable for deuterated compounds in which protium, the normal iso-
tope, is replaced by deuterium to the extent of 90% or more, and it is
readily understood. Unfortunately the approach has certain real and
potential disadvantages which limits its usefulness. To bring biochemical
practice into line with general chemical practice a different approach
must be made.
COOH COOH
I I
HO--C--H H~C--OH
L }
D-- C~H HOOC - - C ~ H
I )
COOH H--C--D
)
COOH
(1) Cn)
Two systems for naming labeled compounds have come into extensive
use: the Chemical Abstracts system and the "square bracket" system.
In the Chemical Abstracts system s' s the italic symbols d~, tn, ~C~, etc.
are added to the chemical name or to part of a name. (The subscript n
has the value 1, 2, 3, etc., and indicates the number of positions labeled;
the 1 is usually omitted.) Numbers indicating the position of labeling are
italicized. The numbers are omitted if all positions are labeled.
* There is a growing practice to use the terms 8yn and anti in place of cis and
tran, when describing additions to double bonds [see D. J. McLennan, Quart. Rev.
21, 490 (1967) ; and W. Klyne and V. Prelog, Experientia 16, 521 (1969)].
~Frojection formulas are shown according to the convention proposed by Emil
Fischer: horizontal bonds above the plane of the paper and vertical bonds below.
' Chem. Abstr. 63, 4R (1965).
*E. J. Crane, Ind. Eng. Chem. News Ed. 13, 200 (1935); reprints available from
Chemical Abstracts, Columbus, Ohio.
The "square bracket" system4,5 was developed by the British Chem-

ical and Biochemical Societies and adopted by the journal Biochemistry.
In this approach the Roman type symbols 2H., 3H,, ~*C., etc. are placed
in square brackets immediately before the appropriate part of the name,
there being no hyphen linking the square bracket to the name.
In both cases the positions of labeling are indicated by numbers in the
usual way, but in the Chemical Abstracts system the number is italicized.
The use of prior superscripts to show mass numbers has been adopted by
international agreement. In formulas, Chemical Abstracts employs D and
T rather than d and t or 2H and 3H. There appears to be no objection
to the use of the symbols D and T in formulas when the "square bracket"
system is used for the name. The two systems are equivalent, and both
are based on the principle that for satisfactory indexing and ready under-
standing the name o] a labeled compound must correspond as closely as
possible to that of its parent compound. The prefixes deuterio- and tritio-
are not employed. In this contribution we have chosen to use the
Chemical A, bstracts approach.
In specifying absolute configurations (chirality) the italic R,S sym-
bols of Cahn, Ingold, and Prelog 6,7 are used to supplement the small
Roman capital D,L symbols of the extended amino acid and carbohydrate
nomenclature,s-t° The use of the R / S system to supplement the D,L
symbols is specifically authorized by Cahn et al. 6 The R,S symbols are
employed as prefixes to the name or part of the name, and they are placed
in curved brackets with the number designating the specified chiral center
adjacent to the symbol thus: (2R,3S)-. Where only one chiral center is
present the number may be omitted. In specifying isotopic chirality the
R,S symbols employed are those appropriate to the molecular model "in
which a labeling isotope (2H, SH, ~4C, is0, etc.) is substituted for a
conventional normal isotope (1H, 12C, ~eO, etc.) at all the positions in the
compound that are labeled". 1~ The R / S system can readily be used in
conjunction with either method for indicating labeling: (3R)-L-malic-3-d
4j. Chem. Soe. p. 4203 (1953).

sS. L. Thomas and H. S. Turner, Quart. Rev. London, 7, 407 (1953).
"R. S. Cahn, Sir C. Ingold, and V. Prelog, Angew. Chem. 78, 413 (1966); ibid.
Intern. Ed. Engl. 5, 385, errata 511 (1966).
*R. S. Cahn, J. Chem. Educ. 41, 116, errata 508 (1964).
sj. Org. Chem. 28, 281, 291 (1963). American-British Rules of Carbohydrate
Chemistry. Addendum to Amino Acid Rules9
oj. Am. Chem. Soc. 82, 5575 (1960). IUPAC Definitive Rules of Amino Acid
Nomenclature.
~oH. B. Vickery, J. Biol. Chem. 237, 1739 (1962).
i~K. R. Hanson, J. Am. Chem. Soc. 88, 2731 (1966).
[74] STEREOSPECIFICALLY
LABELED INTERMEDIATES 571
acid is equivalent to (3R)-L-[3-2H]malic acid. (Note that the deuterium

is attached to the malie portion of the name: L-malic acid-d2 is L-malic
acid in which the protium of the carboxyl groups is replaced by deu-
terium.)
One problem remains: the use of the prefixes erythro-, threo-, etc. We
have chosen to retain the earlier usage to the extent that we shall speak
of the erythro- and threo-enantiomers of malic-8-d acid. The principle
that the name of a labeled compound should correspond as closely as
possible to that of its parent compound would seem to require, however,
that the relative configurational prefixes like the symbols D and L, should
not be changed or introduced as a consequence of labeling. We would
name the compound shown in formula II therefore (4S)-threo-D~- or
(2R,3S,4S)-isocitric-4-d acid but not xylo-Ds-isoeitrie-4-d acid.
In discussing the chemistry of the enzymatic reactions we shall have
occasion to use an extension of the R/S system which permits the chem-
ically identical atoms or groups (ligands) at a center Xaabc to be
named. 11,12 Such centers are termed prochiral, and the "paired" like
substituents or atoms are named pro-R and pro-S according to a pro-
chirality rule which is closely related to the chirality rule of the R/S
system. In most experiments with stereospecifically labeled compounds
the biochemist is concerned to establish which of the paired substituents
at a prochiral center is substituted or otherwise changed in an enzymatic
reaction, and istotopic labeling is a means to that end. The pro-R/pro-S
system readily permits the results of experiments involving isotopic
chirality to be restated in terms of prochirality. For all the compounds
discussed here, and for all but a few of the compounds of interest to the
biochemist, labeling by a heavy istotope in a pro-R substituent leads to
R chirality and labeling in a pro-S substituent to S chiralty.
Formulas (III) to (V) illustrate the above usage. In formula (III),
C-2 is a chiral center with S chirality, but tradition permits the center to
be designated as L. C-3 is a prochiral center and the paired substituents
(hydrogen) are designated as pro-R at C-3 and pro-S at C-3 (indicated
by subscripts R and S). A word of caution about the subscript usage is
necessary: reference to an R substituent should never be made when a
pro-R substituent is implied. In succinic acid (IV) C-2 and C-3 are
sterically equivalent prochiral centers and there are therefore two equiv-
alent pro-R hydrogen atoms, pro-R at C-2 and pro-R at C-3 (arbitrary
numbering), and two equivalent pro-S hydrogen atoms. Citric acid (V)
has three prochial centers: C-2, C-3, and C-4. There are two like
--CH2COOH substituents at C-3 (the pro-S substituent is derived
tt H. Hirschmann, J. Biol. Chem. 235, 2762 (1~0).
1 COOH 1, 4 COOH
J I
2 HO--C--H 2, 3 Hs--C--H R
i I
3
3, 2 HR--C--H s
I I
4 COOH 4, 1 COOH
(HI) (Iv)
1 COOH t
I pro-R
2 HS - - C - - H R
I
3 HOOC--C--OH
$
4 Ha--C--H
I
s } pro-S
5 COOH
(v)
biosynthetically in the vast majority of organisms from acetate).13-~6 As
in the case of succinate, arbitrary numbering for the carbon chain may be
employed. The two ends of the chain are, however, stereochemically dis-
tinct, and there are advantages to employing stereospecific numbering for
citric acid and its labeled derivatives. After consultation with Dr. Hirsch-
mann and others, we have adopted the numbering shown on the general
principle that, when stereospecifie numbering is used, the R or pro-R
substituent should be given the lower numbers. This reverses an earlier
practice,/,, is which has, however, not been universally adopted. The con-
vention offers the important advantage that it is in keeping with the
general structure of the R / S and pro-R/pro-S systems and the accidental
advantage that the carbon chains of the three substrates of aconitate
hydratase17,18 are numbered in a parallel manner. ~9 In view of the present
confusion in this matter we strongly urge that in future abstracts and
summaries all stereochemical information about citric acid should be
"K. R. Hanson and I. A. Rose, Proc. Natl. Acad. ~ci. U~. 50, 981 (1963).
"H. Hirschmann, in "Comprehensive Biochemistry" (M. Florkin and E. H. Stotz,
eels.), Vol. 12, p. 236. Elsevier, Amsterdam, 1964.
wO. Gotteschalk and H. A. Barker, Biochemistry 5, 1125 (1966); ibid. 6, 1027 (1967).
~J. R. Stern, C. S. Hegre, and G. Bambers, Biochemistry 5, 1119 (1966).
"Aconitase, EC 4.2.1.3.
The names of enzymes given in the text are the recommended trivial names as-
signed by the International Union of Biochemistry (Recommendations, 1964,
Elsevier, Amsterdam, 1965). In footnotes, where desirable, more familiar names are
given together with the Enzyme Commission numbers.
wI. A. Rose and E. L. O'Connell, J. Biol. Chem. 242, 1870 (1967).
explicitly stated in terms of the R / S and p r o - R / p r o - S systems and that

when stereospecific numbering is used, whether according to the former
or the present proposal, it should be regarded as a supplementary means
for communicating stereochemical information.
General Procedures for Separation, Purification, Isolation, and Analysis

The separation of citric acid cycle intermediates and other organic
acids has been achieved by partition chromatography on silica gel or
similar media 2°,21 and by elution chromatography on anion exchange
resins such as Dowex 1-formate or Dowex 1-acetate. 22,23 Except for the
separation of citrate and isocitrate, ion-exchange chromatography is the
method of choice. The resin columns have greater capacity, are more
reproducible in their behavior, and there are no serious difficulties in
applying samples. For many purposes the procedures using a constant
volume gradient device will be found to be perfectly satisfactory. The
general procedure briefly described below, which incorporates the modi-
fications introduced by Hoberman, 24 has been used extensively and found
to be highly reproducible.
The deproteinized and neutralized reaction mixtures are passed
through columns of Dowex 1-formate {XS, 200-400 mesh). Elution is
accomplished with the use of a gradient of formic acid approaching 6 N.
The gradient is developed with the use of three chambers of an Autograd
(Technicon Corporation): the exit chamber and that adjacent to it ini-
tially contain distilled water, and the third chamber, 6 N formic acid.
After this solution has passed through the column, 6 N formic acid is
added to elute such strongly acidic anions as fumarate and 2-keto-
glutarate. Elution of such compounds as hexose diphosphate, nucleoside
diphosphates, and phosphoenolpyruvate requires the further addition of
1 N sodium or ammonium formate, while 2 N sodium or ammonium
formate is needed to elute nucleoside triphosphates. Fractions are col-
lected at a rate of 10-12 drops per minute. The rate is regulated by
means of a peristaltic pump connected between the exit chamber and
column. The following order of elution has been routinely obtained with
the continuously increasing formic acid gradient: glutamate, fl-hydroxy-
butyrate, aspartate, lactate, succinate, and malate. Proceeding with 6 N
formic acid, citrate and isocitrate (unresolved), glucose 6-phosphate,
a-glycerolphosphate, fumarate, and 2-ketoglutarate emerge in that order.
Fructose 1,6-diphosphate followed by phosphoenolpyruvate are subse-
~ F. A. Isherwood, Biochem. J. 40, 688 (1946).

"See Vol. III [64].
..2H. Busch, It. B. Hurlbert, and V. R. Potter, J. Biol. Chem. 196, 717 (1952).
~ J. K. Palmer, Conn. Agr. Ezpl. 8ia. New Haven Bull. 589, 1956.
~H. D. Itoberman, Ann. N. Y. Acad. Sci. 119, 1070 (1965).
quently eluted with 1 M ammonium formate. With ~4C- or 3H-labeled

compounds, radioactivity in the effluent may be monitored by adding an
aliquot from each test tube to a scintillator fluid such as Bray's solution ~5
and counting the samples in a liquid scintillation spectrometer. Alterna-
tively, it has been found convenient to pass the effluent stream through
an anthracene-con+.ai~ing cell located in the well of a scintillation
spectrometer. Pulses from the spectrometer are counted by a scaler set for
1-minute counts. The output of the scaler leads to a digital recorder
which, in addition to printing the 1-minute counts on tape, yields an
analog output which is recorded. To locate radioactive peaks on the chart
of the recorder, a solenoid-activated pen attached to the recorder signals
each tube change.
Formic acid and water arc removed from each tube by vacuum desic-
cation at room temperature in the presence of soda-lime -~2,2, or by evapo-
ration at 46 ° in a current of air delivered through a glass manifold?3 The
identity of each peak can be determined by its position of emergence
from the Dowex 1-formate column (titration with IWaOH) or by paper
chromatography. The individual acids are isolated and purified as
described below.
Fumaric acid is purified by repeated recrystallizations from boiling N
HC1. L-Aspartic acid is isolated as the copper salt which is then de-
composed by treatment with H2S. The free aspartie acid is isolated from
the CuS filtrate by addition of ethanol and recrystallized several times
from water-ethanol. L-Malic acid can be isolated as the cinchonine
salt2~, 2~ which can be recrystallized from methanol-acetone or from water-
acetone. It can also be isolated as the diphenacyl ester which is recrystal-
lized from benzene-petroleum ether. 28 The free acid can be isolated and
purified by repeated recrystallizations from ethylacetate-ligroin.2~ Citric
acid can be isolated as the quinidine salt, ~°,31 or as the free acid by re-
peated recrystallizations from ethylacetate and hexane or from hot nitro-
ethane22 Succinic acid is isolated and purified by recrystallizations from
water or by repeated sublimations in an oil pump vacuum at 120-135 ° .
Succinic acid can also be recrystallized by dissolving the solid acid in a
minimum volume of hot tetrahydrofuran (purified by refluxing for 48
hours over sodium and distilled from sodium) and adding dropwise, at
intervals, 4 to 5 volumes of benzene23
25G. A. Bray, Anal. Biochem. 1, 279 (1960).
~ H. D. Dakin, J. Biol. Chem. 59, 7 (1924).
~TS. 1Ratner and A. Pappas, J. Biol. Chem. 179, 1183 (1949).
~ F. A. Loewus, T. T. Tchen, and B. Vennesland, J. Biol. Chem. 212, 787 (1955).
~*A. I. Krasna, J. Biol. Chem. 233, 1010 (1958).
a°F. G. Breusch and H. Keskin, Enzymologla II, 243 (1944).
~S. Weinhouse, G. Medes, and N. F. Floyd, J. Biol. Chem. 166, 691 (1946).
~S. Englard, J. Biol. Chem. 235, 1510 (1960).
Prior to isotope dilution by the addition of carrier, or for any other

required purpose, each acid can be quantitatively determined as follows:
fumarate by determining the absorbance at 240 m~; 3~ aspartate by the
ninhydrin method as modified by Moore and Stein 35 or by Rosen; '~6
succinate by measuring the total oxygen uptake upon incubation with a
succinic oxidase preparation from heart muscle; 37 L-matate by the
fluoromctric method of Speck as described by Loewus et al., ~ by the
spectrophotometric method of Goodban and Stark, ~s or by the enzymatic
procedure of Holzer and Solig; ~9 citrate by the procedure of Natelson
et al. ~° as described by Stern ~ (see also this volume [66]). References to
additional methods have been listed by Swim and Utter. ~2
In preparation for isotope analysis, samples of deuterated compounds
are generally diluted with their protonated counterparts to contain
between 0.5 and 1.0 atom percent excess deuterium. For deuterium
analysis, the samples are oxidized in liquid-air oxygen, and the water is
converted to hydrogen by passage through a uranium converter connected
in series with a mass spectrometer. 43,~4 Samples of deuterated succinate
can be analyzed for their content of normal, mono-, di-, tri-, and tetra-
deuteratcd succinate molecules. The isolated succinic acid is converted to
succinic anhydride by refluxing with acetyl chloride, evaporating the
reagent and washing the crystals with ether25 Mass analysis of the
product is then carried out to determine the peaks at m / c 56, 57, 58,
59, and 60.
Frequently in handling tritiated water it is necessary to remove water
from a sample by sublimation. This operation is conveniently performed
with a sublimation vessel ("soldier") of the type shown in Fig. 1.~ It is
most important that the stopcock of such a vessel be capable of holding
a high vacuum for at least an hour. The sample to be sublimed is intro-
duced into the bulb, and the sample is shell frozen. The vessel is then
~3M. Spreeher, R. Berger, and D. B. Sprinson, J. Biol. Chem. 239, 4268 (1964).
at E. Racker, Biochim. Biophys. Acla 4, 211 (1950).
35S. Moore and W, H. Stein, J. Biol. Chem. 211, 907 (1954).
~eH. Rosen, Arch. Biochem. Biophys. 67, 10 (1957).
~*H. A. Krebs, Biochem. J. 31, 2095 (1937).
'~A. E. Goodban and J. B. Stark, Anal. Chem. 29, 283 (1957).
H. Holzer and H. D. Solig, Bioehem. Z. 336, 201 (1962).
~°S. Natelson, J. B. Pine, us, and J. K. Lugovoy, J. Biol. Chem. 175, 745 (1948).
4~See Vol. III [69].
'~ See Vol. IV [24].
,s G. Nief and R. Botter, in "Advances in Mass Spectrometry" (J. D. Waldron, ed.).
p. 515. Macmillan (Pergamon), New York, 1959.
*~See Vol. IV [21].
*~G. Popj~k, D. S. Goodman, J. W. Cornforth, R. H. Coruforth, and R. Ryhage,
J. Biol. Chem. 236, 1934 (1961).
,e Obtainable from Birkett Glass Inc., Millville, New Jersey.
576 P~F.P,iRATmN OF COMPOUNDS [74]
attached to a high-vacuum line and evacuated for 2 minutes. The stop-

cock is closed, the vessel is renmved, and the straight leg of the soldier
is placed in a dry ice-acetone bath. When sublimation is complete, the
recovered tritiated water m a y be returned to the storage vessel with the
aid of a disposable Pasteur pipette. This operation should be pcrformed
in a fume hood. A short length of AWG 15 Teflon tubing (Penn Fluoro-
carbon Co., Clifton Heights, Pennsylvania) attached to the end of the
~ ~2o/5l5
l
Fro. 1. Sublimation vessel convenient for handling tritiated wager.
pipette is useful as it permits scavenging for any remaining drops of
wat~er. The traps of the vacuum line should be rinsed out after use. 4~a
Correlations and critical assignments of configurations m a y be
achieved with the aid of nuclear magnetic resonance and optical rotatory
dispersion measurements. For example, with the (?-2 and C-3 carboxyl
groups of malate in a trans configuration, the C-2 and C-3 protons of
(3R)-L-malate-3-d are trans to each other, whereas the same two protons
are gauche to one another in (3S)-L-malate-8-d. 47 The relative proximity
of these two protons can be determined by measuring the spin-spin inter-
actions of the protons by nuclear magnetic resonance. *s-~° Thus, (3R)-L-
Recently we have used sublimation vessels analogous to those described in Yol.
IV, p. 712, but having O-ring seals (Type M, size 116) to attach the U-portion
to the tubes in place of ground glass joints. (Obtainable from the Kontes Glass
Co., Vineland, N.J., as part of a hydrolysis tube assembly.) The apparatus holds
a vacuum for days if necessary, the contents of the tubes are readily withdrawn,
and contamination with grease is avoided.
4~S. Englard, Ann. N.Y. Acad. Sci. 84, 695 (1960).
R. U. Lemieux, R. K. Kullnig, H. J. Bernstein, and W. G. Schneider, Jr., J. Am.
Chem. Soc. 80, 6098 (1958).
~F. A, L. Anet, J. Am. Chem. Soc. 82, 994 (1960).
O. Gawron, A. J. Glaid, III, and T. P. Fondy, J. Am. Chem. Soc. 83, 3634 (1961).
[74] 8TEREOSPECIFICALLY LABELED INTERMEDIATES 577
malate-3-d has been reported to have a coupling constant (referring to

the spin-spin interaction of the two hydrogens on the adjacent C-2 and
C-3) of 6.3, 6.0, 7.1, and 6.7 cps? 9,'°-52 On the other hand, coupling con-
stants around 4.0-4.2 have been reported for (3R)-n-malate-3-d and
for a synthetic racemic mixture of (3R)-D-malate-3-d and (3S)-L-malate-
8-d. 29,49,s°' 52 The two isomeric forms of the monodeuterated succinates
can be distinguished by application of ultraviolet optical rotatory disper-
sion data23 The low negative rotations of (2R)-succinate-2-d which are
measurable in the near visible region decrease to a minimum near 223 m~
and subsequently increase toward the positive direction crossing zero near
210 m~. '~ The trough is the first extrema of a Cotton effect which is asso-
ciated with the absorption of the carboxyl group in this region. The curve
obtained for (2S)-succinate-~-d is almost an exact mirror image of the
curve obtained for (2R)-succinate-2-d22
Chemical and Enzymatic Syntheses

The majority of the following preparations are described for deuterium
labeling. Although the manipulations would have to be changed for the
preparation of tritiated compounds the transposition should present no
serious problems. Sections 11 and 12, which deal with tritiated com-
pounds, illustrate some of the differences in experimental approach
required in handling this isotope.
1. Chemical Synthesis of Fumaric-d2 Acid

Principle. The synthesis of fumaric-d: acid is accomplished by reduc-
ing acetylenedicarboxylic acid dimethyl ester with 1 mole of deuterium
gas per mole of ester.
Method. The following procedure has been adopted after a number
of trials employing various solvents, pressures of deuterium, and cata-
lysts2" Acetylenedicarboxylic acid dimethyl ester, 54.4 g (0.26 mole) is
reduced with 99.8~ D2 at an initial pressure of 37.5 pounds per square
inch in a Parr hydrogenation apparatus. Pd0, 270 rag, is added to provide
Pd as a catalyst. The theoretical uptake of deuterium generally requires
2.8 hours. When this point is reached, the reaction mixture is filtered to
remove Pd, ether being used to effect transfer of the ester and to wash
the catalyst. After removal of the ether by evaporation under reduced
pressure, the product is distilled, also under reduced pressure. The distil-
late is treated overnight with a 5% excess of N NaOH at room tempera-
~' R. A. Alberty and P. Bender, J. Am. Chem. Soc. 81, 542 (1959).
~S. Englard, J. S. Britten, and I. Listowsky, J. Biol. Chem. 242, 2255 (1967).
~J. W. Cornforth, G. Ryback, G. Popj~k, C. Donninger, and G. Schroepfer, Jr.,
Biochem. Biophys. Res. Commun. 9, 371 (1962).
HH. D. Hoberman and A. F. D'Adamo, Jr., J. Biol. Chem. 235, 519 (1960).
578 PREPARATION O F COMPOUNDS [74]
ture, and the hydrolyzate is evaporated to dryness. The residue is refluxed

with 8 N HC1 for 30 minutes to transform maleie to fumaric acid. This
treatment does not cause loss of carbon-bound deuterium from the mole-
cule. The highly insoluble fumaric acid is separated by filtration and re-
crystallized three times from boiling N HC1. The purity of the deuterated
fumaric acid may be ascertained by chromatography on Dowex 1-for-
mate. In an actual run, 18 g (0.16 mole) of the three times recrystallized
material was obtained (62% overall yield). The observed deuterium
concentration in the 2 and 3 positions was 89 atom %. The dilution of
deuterium in the fumaric acid is believed to be due to a Pd-catalyzed
exchange of deuterium gas with the protium of the methyl groups of the
esters. Fumaric-t2 acid has been synthesized by an analogous procedure. 55
2. ( 3R )-L-Aspartate-3-d from Fumarate and ( 3S)-L-Aspartate-2,3-d2

from Fumarate-d~
COOH COOH
J f
H--C O20 H~N--C--H
II + NHs ~ l
C--H D--C~H
J I
COOH COOH
(r) (H)
COOH COOH
[ [
D--C H~O I~N--C--D
Jl + NH S r~ f
C--D H--C--D
I I
COOH COOH
(m) (iv)
Principle. Aspartate ammonia-lyase 5~ catalyzes reversibly the stereo-
specific trans addition of ammonia to fumarate with the formation of
L-aspartate. 29,5~ In D20, (3R)-L-aspartate-3-d (II) is formed from
fumarate (I). The enzymatic amination of fumarate-d2 (III) in normal
water results in the formation of (3S)-L-aspartate-2,3-d2 (IV). Only the
method for the preparation of (3R)-L-aspartate-3-d is given in detail.
Method. Whole cells of Proteus vulgaris are grown as described by
Krasna and Rittenberg28 The harvested cells from 2 liters of media are
H. D. Hoberman, E. A. Havir, O. Rochovansky, and S. Rather, J. Biol. Chem. 239,
3818 (1964).
Aspartase, EC 4.3.1.1; see also this volume [54].
~' S. Englard, J. Biol. Che.m. 233, 1003 (1958).
UA. I. Krasna and D. Rittenberg, J. Am. Chem. Soc. 76, 3015 (1954).
washed twice with distilled water to remove any culture media and are
then washed four times with 99.8% D oO and finally suspended in 5 ml
of D~O. Fumaric acid, 4.64 g (40 millimoles), is dissolved in 30 ml of
99.8% D20 containing 3.2 g of NaOH. The pH of this solution is 6.0. To
this solution is then added 1.34 g of NH~CI (25 millimoles), 77.6 mg of
KH2PO4 and 344 mg of Na2HP04. This quantity of phosphate salts
results in a final buffer concentration of 50 mM. By careful addition of
approximately 1 ml of 10% NaOH in D20 the pH of the solution is
adjusted to 7.4 (the optimal pH for aspartate ammonia-lyase activity).
The Proteus vulgaris cell suspension is added, and the volume is adjusted
to 60 ml with D20. After addition of 6 ml of toluene, the reaction mixture
is flushed with prepurified N2 for 5 minutes and then placed in a shaking
incubator at 37 ° . Samples are removed at intervals and analyzed for
ammonia to measure the approach to equilibrium. The reaction is usually
complete within a half hour. Extended incubations should be avoided to
preclude the further introduction of deuterium in the 2 position of the
(3R)-L-aspartate-3-d by reversible transamination.59 The reaction is
ternfinated by heating the mixture in a bath of boiling water for half an
hour. The solution is then centrifuged to remove dead cells, and the
slightly turbid supernatant is filtered through Celite to give a clear
solution. The filtrate is treated with an excess of a saturated solution of
cupric sulfate and placed in an ice bath until all the copper aspartate
crystallizes out. The copper aspartate is then decomposed with H2S, the
solution is filtered to remove CuS, and free aspartic acid is precipitated
from the filtrate by the addition of ethanol. The aspartic acid is recrystal-
lized three times from water-ethanol in order to remove all exchangeable
deuterium.
As fumarate hydratase ~° occurs in Proteus vulgaris, (3R)-L-malate-
8-d is also formed in tile course of the reaction. In order to isolate both
compounds, the deproteinized and clarified reaction mixture is brought to
pH 1.5 with concentrated HC1 and the solution is placed on a Dowex
50-H ÷ column, 4.2 X 10 cm. The column is washed with water to remove
the L-malic and residual fumarie acids, and the b-aspartic acid adsorbed
on the column is eluted with 1.5 N HC1. After repeatedly evaporating
this eluate to dryness in a vacuum in order to remove the HC], the
aspartate is isolated as its copper salt by treatment with C u C Q and the
copper aspartate is then decomposed as above to yield aspartic acid. The
fumaric and malic acids in the water eluate from the Dowex 50 column
are separated by chromatography on a Dowex 1-formate column.
In a typical experiment, ~9 using the above procedure but an extremely
long period of incubation (to allow for complete equilibration of the
~D. B. Sprinsoa and D. Rittenberg, Nature 167, 484 (1951).
e°Fumarase, EC 4.2,1.2; see also this volume [17].
deuterium between the aspartic and fumaric acids), the deproteinized
reaction mixture was reported to contain 14.8, 8.5, and 2.1 millimoles of
aspartic, malic, and fumaric acids, respectively. In this experiment, an
additional introduction of deuterium into the 2 position of aspartic acid
took place by reversible transamination, and the aspartic and residual
fumaric acids were found to contain 1.24 and 0.16 atoms of deuterium per
molecule, respectively. By terminating the reaction shortly after the
aspartate ammonia-lyase equilibrium is achieved, however, one obtains
aspartie and malic acids labeled exclusively and stereospecifically in the
3 position.
3. (3R)-L-Malate-3-d from Fumarate and (3S)-L-Malate-2,3-d2
from Fumarate-d2
COOH COOH COOH COOH

( [ I I
H--C D~O H O - - C - - H D--C H20 HO--C--D
(( ~-- ( ; [I > J
C--H D--C--H C--D H--C~D
COOH COOH COOH COOH
(ii ) (w)
Principle. Fumarate hydratase catalyzes the reversible stereospecific
trans addition of water to fumarate with the formation of L-malate2 ~'48'5°
In D20, (3R)-L-malate-3-d (II) is obtained from fumarate (I). The
enzymatic hydration of fumarate-d~ (III) in normal water results in the
formation of (3S)-L-malate-$,3-d2. The preparation of (3R)-L-malate-
3-d is described.
Method. A mixture of 2.5 millimoles of fumaric acid and 1.25 milli-
moles of K2HPO~ is dissolved in water, adjusted to pH 7.4, and lyophi-
lized. The dried residue is redissolved in 10 ml of 99.8~ D~O and re-
lyophilized. This procedure is repeated twice more to assure maximum
exchange of protium atoms by deuterium atoms. The dried material is
dissolved in D20 and adiusted to a final volume of 25 mh This yields a
solution whose pH, as measured with a glass electrode, is 7.7.
To this solution is added approximately 100 units of crystalline
fumarate hydratase ~° (obtainable from Boehringer-Mannheim C o r p . ,
New York) and the reaction mixture ia incubated at a defined tempera-
ture (e.g., 28°). A small aliquot of the reaction mixture is transferred to
a silica euvette with a 1 cm light path provided with an 8 mm quartz
insert; the progress of the reaction is measured at the same temperature
by following the decrease in optical density at 300 m~. When the reaction
has reached equilibrium it is stopped by heat inactivation. After cooling,
the solution is titrated with dilute N a 0 H to the phenolphthalein end

point, and the acids are separated by chromatography on Dowex 1-for-
mate.
A preparation of (3S)-L-malate-$,g-d2 may be obtained in an analo-
gous manner. In actual runs/2 using the above procedure, 1.95 millimoles
each of (3R)-L-malate-8-d and (3S)-L-malate-~,3-d2 have been obtained.
4. Chemical Synthesis of (3R)-L-Malic-3-d Acid from

( 3R )-L-Aspartic-3-d Acid
COOH COOH
I I
I-I2N~C--H NaNO2 HO--C--H
D--C--H H2SO4 D--C--H
I l
COOH COOH
(I) (II)
Principle. (3R)-L-Aspartic-3-d acid (I) has been converted to (3R)-

L-malic-8-d acid (II) 29 by treatment with nitric oxide in nitric acid
according to the method of Walden 8~ for the conversion of L-asparagine
to L-malic acid. The use of NaNO~ in H2S04 to achieve this conversion
has been reported and is described below. The action of nitric oxide in
nitrous acid on L-aspartate does not appear to significantly labilize the
hydrogen atom at C-2. ~9 It is therefore likely that (3S)-L-malic-~,8-d2
acid can be prepared in an anologous manner from (3S)-L-aspartic-~,8-d2
acid.
Method. To 0.133 g (1 millimole) of (3R)-L-aspartic-3-d acid in 5.0
ml of 1 N H2S04 is added 1.5 ml of a freshly prepared 30% NAN02 solu-
tion; the addition is made over a period of 20 minutes with continuous
agitation. This solution is stirred for an additional 1 hour and 10 minutes,
after which time a quantitative ninhydrin test ~5 should fail to detect
aspartic acid. The solution is further acidified with dilute H2S0, to pH
1.5, mixed with Celite, and continuously extracted with ether for 9 hours.
The ether-extracted material is dissolved in water and adjusted to pH 8.0
with KOH; the malic acid is isolated by chromatography on Dowex
1-formate.
In a typical experiment ~7 using the above procedure, L-malic acid was
obtained in 88% yield with a quantitative retention of the deuterium
initially present in the starting material. Furthermore, the product of
deamination when subjected to tile action of fumarate hydratase yielded
ul~labeled fum'~r'tte. It i~ therefore evident that lfitrous ~tcid deamitmtioll
6~p. Walden, Bet. Deul. Keram. Ges. 28, 2771 (1895).

does not affect the stereochemistry of carbon atom 3 of aspartic acid and
that the product of deamination is (3R)-L-malic-3-d acid.
5. (3S)-L-Malate-3-d from (3S)-L-Aspartate-2,3-d2

COOH COOH
I 9..ketoglutarate L-glutamate J
H2N--C--D C=O
H--C--D aspartate H-- C--D
I aminotransferase I
COOH COOH
k
I
HO--C--H
[
H--C--D
I
COOH
(nI)
In the absence of a satisfactory alternative procedure, the cnzymatic
synthesis of (3S)-L-malate-3-d (III) from (3S)-L-aspartate-2,3-d2 is a
distinct possibility. The enzymatic conversion 57 is carried out in the
presence of 2-ketoglutarate, aspartate aminotransferase, 62 NADH, and
malate dehydrogenase. 63 Some racemization (and loss of deuterium) will
probably occur by way of the reversible nonenzymatic keto-enol tauto-
merization of the (3S)-oxaloacetate-3-d intermediate (II). The extent of
racemization (or loss of deuterium), however, will depend on the relative
rates of this tautomerism and the subsequent reductive step as catalyzed
by malate dehydrogenase. It is therefore necessary for the purpose of
obtaining (3S)-L-malate-3-d, to generate slowly the (3S)-oxaloacetate-
3-d by limiting the amount of aminotransferase, and to maintain the
high rates of L-malate formation with the use of an excess of dehydro-
genase. The inclusion of a system for the rapid regeneration of NADH
will also minimize the formation of (3S)-oxaloacetate-3-d from (3S)-
L-malate-3-d by reversal of the malate dehydrogenase reaction. The
preparations of aspartate aminotransferase and malate dehydrogenase
*~G l u t a m a t e - o x a l o a c e t a t c transaminas~, E C 2.6.1.1.
,3 E C 1.1.1.37.
used should be free of fumarate hydratase activity. In the pre~encc of

the latter enzyme, (3S)-L-malate-3-d would be dehydrated to yield mono-
deuterated fumarate which on rehydration in normal water would yield
an equimolar mixture of (3S)-L-malate-3-d and L-malate-2-d.
6. Chemical Synthesis of (3R)-D- and (3S)-L-Malic-3-d Acids
MeO.~--] ./OMe HOC1
H "O" "H
(i)
C1 OH OH C1
MeO~>[.. /~/OMe + MeO% .~OMe KOH

H "01 "H H" "0" "H
(n)
H. .O .H
LiAID4
M e O ~ O M e r
H" "O" "H
(III)
D OH OH D
MeO% ~/OMe + MeO% J</OMe

H ~ "0 ~ "H H ~ "01 \H
(IV)
I ttNOs
or Br2, CaCO s
COOH COOH
I I
H--C--OH HO -- C--H
I
D--C--H H--C--D
i I
COOH COOH
(va) (Vb)
584 PREPAnAWlON OF COMPOUNDS [74]
The above sequence of reactions 49.~°,(~4 gives rise to a raeemic mixture

of the threo enantiomers (V): (3R)-D-malic-3-d acid (Va) and (3S)-
L-malic-3-d acid (Vb) ; this mixture may be named (3RS)-D,b-malic-3-d
acid. The configurational assignment rests on the known stcreospecificity
of epoxide ring opening. 2,5-Dimethoxy-2,5-dihydrofuran (I) is com-
mercially available, and the yields for all steps except the last are good
(I--) II, 58%; I I o III, 70%; III o IV, 83%). The reported yield for
the nitric acid oxidation 44 is only 4.6%. It is probable that the yields with
bromine oxidation 49 are better, but the experimental details have not
been published. The enantiomers of malic acid may readily be resolved; ~
the cinchonine salt of the L-acid is sparingly soluble in methanol and less
so in acetone, whereas the salt of the D acid is readily soluble in both,
but sparingly soluble in water.
(3R)-D-Malate-3-d has been obtained by a combination of enzymatic
and chemical procedures. (3S)-L-2-Bromosuccinic-3-d acid is obtained
from enzymatically synthesized (3R)-L-aspartic-3-d acid by the com-
bined action of HBr and NaN0~. (The change in the chirality symbol
applied to C-3 reflects the priority of Br in the sequence rule and does
not imply inversion.) Treatment of (3S)-L-2-bromosuccinic-3-d acid
with KOH in methanol gives (3R)-D-malic-3-d acid? 9 The most direct
method, however, for the synthesis of (3R)-D-malate-3-d involves the
trans-hydration of maleate in D20 as catalyzed by the maleate hydratase
from rabbit kidney. ~2
7. (3R)-Citrate-2,2-d2, from Oxaloacetate-d2 and
(3S)-Citrate-4,J-d2, from Acetate-ds e5
Principle. When citric acid is formed from acetyl-CoA and oxalo-
acetate in the presence of the commonly occurring citrate synthase, ~6 the
pro-R -CH2COOH substituent at C-3 of citrate is derived from oxalo-
acetate. ~,~3,~5,~ The synthesis with this enzyme of stereospecifically
labeled citric acids is described in this and the following section. Reaction
sequence A is initiated by equilibration of oxaloacetate (I) in D~O in
order to achieve complete exchange of the methylene hydrogen atoms
with deuterium. Oxaloacetate-d2 (II) is then treated with acetyl-CoA
in the presence of citrate synthase and an acetyl-CoA regenerating system
(phosphate acetyltransferase, ~7 acetylphosphate, and CoA) to yield (3R)-
citrate-g,2-d~ (III). Oxaloacetate (I) in normal water with acetyl-d~
6,j. (3. Sheehan and B. M. Bloom, J. Am. Chem. Soc. 74, 3825 (1952).
"~For stereospecific numbering and the use of the pro-R/pro-S terminology, see sec-
tion on nomenclature.
Citrate condensing enzyme, EC 4.1.3,7.
6~Phosphotransacetylase, EC 2.3.1.8.
0
~ ~ o ° 8 ~
~ - - r..)-- ~ - - ~ - - r..)
~ 0 0
I ---
I "-"
0
0 8
ffl m
0- r..) ~
0 e~
0 ~
0 0 0
~ 0 ~
phosphate ~s in the presence of the components necessary to achieve citratc

synthesis gives (3S)-citrate-$,4-d2 (V). The combined actions of aconitatc
hydratase and isocitrate dehydrogenase {NADP) 69 on compound (III)
yields unlabcled 2-ketoglutarate (IV) and on (V) yields 2-ketoglutarate-
~,~-d2 (VI).
Method. The procedure is essentially similar to that described below
for the synthesis of (2R,3R)-citrate-2-d and (2S,3R)-eitrate-2-d from
(3R)-L-malate-3-d and (3S)-L-malate-2,3-d2, respectively, except that
NAD +, pyruvate, malate dehydrogenase, and lactate dehydrogenase 7° are
omitted from the reaction mixture. The isolation of (3R)-citrate-2,2-d2
containing 1.68 atoms of D per molecule has been reported from a reac-
tion mixture in which oxaloacetate was equilibrated in a medium con-
taining 91% D20 prior to the addition of the enzymes necessary for
citrate synthesis. ~1
8. (2R,3R)-Citrate-2-d from (3R)-L-Malate-3-d and (2S,3R)-Citrate-

2-d from (3S)-L-Malate-2,3-d2 ~
PT"inciple. Oxaloacetate has been shown to be reduced in the keto form
by the action of malate dehydrogenase 28,7~ and therefore arises in this
form in the reverse reaction. In the malate dehydrogenase-catalyzed
oxidation of L-malate, therefore, the stereochemistry at C-3 is unaffected
so that (3R)-L-malate-3-d (I) and (3S)-T.-malate-2,3-d~ (IV) are con-
verted to (3R)-oxaloacetate-3-d (II) and (3S)-oxaloacetate-3-d (V),
respectively. Since the keto form of oxaloacetate is also the substrate of
citrate synthase, ~1 the stereochemistry at the carbon atom of citrate
derived from the C-3 of oxaloacetate (or from the C-3 of the initial
L-malate), remains unchanged. In view of the known steric course of the
citrate synthase reaction '13 it is evident that by the sequence of reactions
initiated by malate dehydrogenase, (2R,3R)-citrate-2-d and (2S,3R)-
citrate-g-d are obtained from (3R)-L-malate-3-d and (3S)-L-malate-
~,3-d2, respectively. ~ Some racemization (and loss of deuterium) may
occur by way of the reversible nonenzymatic keto-enol tautomerism of the
stereospecifically labeled monodeuterated oxaloacetate intermediates. The
Prepared by the acetylation of orthophosphate with acetic anhydride-d6 (see Vol.
I I I [39] procedure B).
m EC 1.1.1.42.
'*EC 1.1.1.27.
,1S. Englard, J. Biol. Chem. 234, 1004 (1959).
"J. B. Graves, B. Vennesland, M. F. Utter, and R. J. Pennington, J. Biol. Chem
223, 551 (1956).
"In terms of the pro-R/pro-S nomenclature, the pro-R and pro-S hydrogens at
C-3 of t,-malate become, respectively, the pro-R and pro-S hydrogens at C-2
(pro-R to C-3) of citrate.
o~O ~ ~ o
n t~ ° -- 81 i~
8 8
+l ° o
r.)
°T~8
o
v v
I I
-f-
O
~z
8?Oo
I o 8118
v ¢ 9 - - ~9 - ¢ 9 - - ~
I I I I
extent of racemization (or loss of deuterium) will depend, however, on

the relative rates of this tautomerism and the condensation reaction with
acetyl-CoA. In order to minimize the extent of racemization, it is neces-
sary to generate slowly the monodeuterated oxaloacetate isomers by
limiting the amount of malate dehydrogenase, and maintain a high rate
of citrate synthesis with the use of excess citrate synthase and a large
amount of phosphate acetyltransferase for the rapid regeneration of
acetyl-CoA from acetylphosphate and free CoA.
The deuterium atom of (2R,3R)-eitrate-2-d is removed stereospecifi-
cally in the conversion of citrate to c/s-aconitate, whereas the deuterium
atom of (2S,3R)-citrate-2-d is retained and is ultimately transferred to
NADP ÷ by the further action of isocitrate dehydrogenase (NADP).82 In
other words, the stereochemical interrelationships in the citric acid cycle
are such that the hydrogen atom added by fumarate hydratase is re-
moved by aconitate hydratase.
Method. (3R)-L-Malate-3-d and (3S)-L-malate-2,3-d2, 1.65 milli-
moles of each, are neutralized to pH 7.4 and are incubated in separate
flasks with 5.50 millimoles of Tris buffer (pH 8.1), 2.75 millimoles of
potassium pyruvate, 1.38 millimoles of dilithium acetyl phosphate, 37
micromoles NAD ÷, 3.4 mg of CoA (1180 units), 263 units of lactate de-
hydrogenase, 1670 units of malate dehydrogenase, ~4 26 units of phosphate
acetyltransferase, and 500 units of citrate synthase in a total volume of
50.0 ml. The reactions are initiated by the addition of malate dehydro-
genase and are carried out at 28 °. At various times, 0.1 ml aliquots are
withdrawn for the determination of acetyl phosphate by the hydroxyl-
amine method of Lipmann and Tuttle. ~s Under the above conditions, the
reactions are essentially complete within 10 minutes. The reactions are
terminated by acidification to pH 1.1 with concentrated H2S04 and the
solutions are then extracted continuously for 96 hours with ether. The
ethereal extract is evaporated almost to dryness, dissolved in H20 and
neutralized to pH 8.0 with NaOH. Citric acid is isolated by chromatog-
raphy on Dowex 1-formate.
In typical experiments of the above type, 0.84 to 0.85 millimoles of
each isomer of monodeuterated citrate has been obtained. (3R)-L-Malate-
3-d containing 1 atom D per molecule yielded (2R,3R)-citrate-~-d with
0.82 atom D per molecule. (3S)-L-Malate-2,3-d2 with an isotope content
equivalent to 1.73 atoms D per molecule (equally distributed between
C-2 and C-3) gave rise to (2S,3R)-citrate-2-d containing 0.85 atom D
'~Assayed for the reaction: oxaloacetate W NADH -~- H ÷--* L-malate ~ NAD÷. At
pH 8.1 in the direction of malate oxidation the rates are at least 15- to 20-fold less,
so that this enzyme is essentially the rate-limiting step in the overall reaction.
T~F. Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945).
per molecule. (The C-2 deuterium atom of L-malate is lost through the
action of malate dehydrogenase).
(2R,3R)-Citrate-$-d or ~-t can be synthesized by the action of
aconitate hydratase on c/s-aconitate in D20 and THO, respectively.19,;6
(2R,3R)-Citrate-~-t has also been prepared I~ by the oxidation with HI04
and Br2/H20 of (6S)-quinate-6-t (obtained from 5-dehydroshikimate or
quinate and T H 0 in the presence of quinate dehydrogenase77 and 5-de-
hydroquinate dehydrataseTS). This is clearly not the most convenient
method for obtaining the tritiated citric acid but (6S)-quinate-64 could
be a most useful starting point for the synthesis of other stereospecifically
tritiated compounds of biological interest.
9. Chemical Synthesis of (3R)-Citric-2,2-d2 and
(3S)-Citric-4,4-d2 Acids ~5
COOH
C----Of [' C=O, O = C l , ,,

CI~ C--O O--C
O I J O
+ ~ I ~C +
O--C--COOH ( C--COOH HOOC--C---J
) I I
ci% ~c c,%
COOH HOOC COOH
Resolved
(I) (~)
I D20 D20
D202 D~02
5 HOOC COOH 1
I I
4 D~C CD2 2
t J
3 HO--C--COOH HOOC-- C - - O H 3
i L
CH~ 4
1 ttOOC COOH 5
(m) (iv)
~'S. Englard and S. P. Colowick, J. Biol. Chem. 226, 1047 (1957).
,, EC 1.1.1.24.
,s EC 42.1.10.
590 PREPARATION OF COMPOUNDS [74J
Principle. The aldol condensation of oxaloacetate and pyruvate gives

a mixture of products from which the racemic ~,-lactone of (±)-oxalo-
eitramalic acid (I and II) can be isolated. 79 This mixture may be re-
solved with the aid of brucine. The hydrogens at C-3 adjacent to the
C-2 carbonyl groups of the lactones are labile. Stereospecifically deuter-
ated citric acids may thus be obtained by equilibrating the appropriate
lactone with D20 and then oxidizing the product in D~O with D202. s°
The assigned configurations rest on the known stereochemistry of citric
acid biosynthesis, 1~ and the enzyme experiments of Martius and Schorre. si
A provisional assignment-was made by Martius and Schorre on the basis
of Hudson's lactone rule. sl,82
Method. (__--+-)-OXALOCITanMALICACID y-LACTONE. A solution of equi-
molar amounts (0.1 moles) of oxaloacetic acid (13.5 g) and pyruvic acid
(8.8 g) is adjusted to pH 9 with Na~_C03, and the mixture (100 ml) is
left at room temperature for 8 hours. The solution is brought to pH 4
with phosphoric acid and continuously extracted with peroxide free ether
for 3 hours, then strongly acidified and extracted with fresh ether for 40
hours. The second ether extract on slow evaporation in a vacuum desic-
cator gives crystals which are pressed out on a clay plate ("Sie wurden
auf Ton abgepresst!") and recrystallized from ethyl acetate-petroleum
ether: yield, 2.7 g (13.3%), m.p. (decomp) 178 ° with browning at 170 °.
Martius assigned a lactone structure to the compound on the basis of its
C, H analysis (drying conditions not given) and the fact that although
it was neutralized by 3 equivalents of alkali (bromothymol blue) the
end point of the titration was not sharp.
RESOLUTION OF THE LACTONES. A hot solution of brucine (9.7 g) in
water (200 ml) is added to a solution of the racemic lactone (8.3 g) in
50 ml, and the mixture is left at 0 ° for 8 hours. The crude salt correspond-
ing to the (--)-enantiomer (I) is collected by filtration and the mother
liquor is concentrated to 50 ml and left at 0 ° for 8 days. The crude salt
corresponding to the (-f-)-enantiomer (II) is then collected. Each salt is
dissolved in water (5 ml), and the pH is adjusted to 8.4 with Na2CO:~.
After removal of the precipitated brucine, the solutions are extracted 3
times with chloroform and adjusted to pH 1.4 with 2 N H2SO4. The
lactones are obtained by ether extraction and purified to constant specific
rotation by recrystallizations from ethyl acetate-petroleum ether. After
five or six recrystallizations the following melting points and rotations
were found (546 m~, Hg line).
(4R)-(--)-Oxalocitramalic acid ~-lactone (I) : [~]~6s°: _ 2 1 5 °
~C. Martius, Z. Physiol. Chem. 279, 96 (1943).
C. Martius and G. Schorre, Ann. Chem. 570, 140 (1950).
8, C. Martius and G. Schorre, Ann. Chem. 570, 1943 (1950).
P. Schwartz and H. E. Carter, Proc. Natl. Acad. Sci. U.S. 40, 499 (1954).
(water, concentration not given), m.p. I62-163 ° (decomp.), 1.2 g ( 2 9 3 ) .

(4S)-(-~)-Oxalocitramalic acid 7-1actone (II): [a]~°e-----~-213 °
(water, cone. not given), m.p. 161.5-162.5 ° (decomp.), 1.5 g (36%).
PREPARATION OF DEUTERIUMPEROXIDE. P20~ (8 g) is dissolved in D~O
(6 ml, 96.3%), the solution is briefly warmed, and then cooled to --20 °.
Finely powdered Na202 is added to the D3P04 solution with stirring until
the pH is 7.6. The mixture is then acidified with D3PO~ (2 ml from a
solution of 5 g of PzO~ dissolved in 2.3 ml of D20) and is sublimed or
distilled i n v a c u o , s3 Yield 3.8 ml of 29% D202.
PREPARATIONOF LABELEDCITRIC ACIDS.The lactone (1.23 g) is dissolved
in D20 (1.5 ml, 96.3% D) and left at room temperature for 24 hours.
The D20 is removed i n v a c u o and the procedure is repeated 4 more times.
The solid is then dissolved in a small amount of D20, and Dz02 solution
is added (4 ml. 10%). After 24 hours, the D~O and any remaining D:Oz
are removed i n v a c u o and the residue is evaporated with water 5 times
and then recrystallized from ethyl acetate-petrol ether. The following
properties have been reported for the enantiomers.
(3S)-(--)-Citric-~,~,-d2 acid (III) (from (4R)-(--)-lactone): [a]~°6
= ca. --1 ° (in water) and --33.6 ° (in saturated ammonium molybdate
solution with acetic acid added, concentration not given); 2 X 0.961
atoms D per molecule, m.p. 148-149 °, 420 mg (35% yield).
(3R)-(-~)-Citrie-2,~-d~ acid (IV) (from (4S)-(-~)-lactone): [a]~%
~- ca. ~ 1 ° (in water) and ~-31.9 ° (in saturated ammonium molybdate
solution with acetic acid added, c = 2.730 g/100 ml) ; 2 X 0.959 atoms D
per molecule, m.p. 149-150.5 °, 55% yield.
10. Chemical Synthesis of (3S)-Citric-5-~4C Acid 65

Sodium (3S)-citrate-5-~4C (salt of IV) was prepared by Wilcox et El. 8~
by treating a concentrated mildly alkaline solution of (3R)-3-carboxy-
4-chloro-3-hydroxybutyric acid (III) with :Na~'CN and hydrolyzing the
resultant nitrile. The absolute configurations of the above compounds
follow from the observation that on enzymatic degradation of (IV) all
the detectable radioactivity Was recovered in the 2-ketoglutarate (V)
and from the known stereochemistry of the aconitate hydratase ~3 reaction.
Although the conversion of (III) to (IV) is straightforward, the treat-
ment of ethyl 4-chloroacetoacetate (I) with liquid anhydrous H C N at
--5 ° in the presence of a catalytic amount of NaCN should be attempted
only in a laboratory with adequate facilities. The racemic chloro acid
(II), obtained by hydrolysis of the cyanohydrin, was resolved with the
~3H20= in concentrations greater than 50% when mixed with small quantities of
organic materials may become dangerously explosive. 8'
E. S. Shanley and J. R. Perrin, Jet Propulsion p. 382 (1958).
~sp. E. Wilcox, C. Heidelberger, and V. R. Potter, J. Am. Chem. Soc. 72~ 5019 (1950).
COOH COOH COOH

I I I
I HCN, N a C N I Brucine I
C:O COHCOOH ~ HOOC--C--OH
I H+ I I
) I I
C1 C1 C1
(I) (II) (ni)
COOH COOH
I r
C--O C~
I I
CI~ ~ HOOC--C--OH
I I
I
I~COOH 14CIO O H
(v) (IV)
aid of brueine. After l l recrystallizations, decomposition of the brucine
salt gave a low yield of the pure noncrystalline R-enantiomer ( I I I ) ,
[a]~ - 4 4 . 9 ° ( 1 5 ~ , in water)25a
11. (2S)-Succinate-2-t and (3S)-2-Ketoglutarate-3-t from
t/u'eO-D,- Isocitrate
Pr&ciple. When threo-D~-isocitrate (I) is oxidized by N A D P + in the
presence of the NADP-specifie isocitrate dehydrogenase from pig heart,
the hydrogen pro-S at C-3 of the 2-ketoglutarate formed is derived from
the medium; i.e., the overall reaction proceeds with retention of con-
figuration. 86,s7 Thus in tritiated water, (3S)-2-ketoglutarate-3-t is
obtained ( I I ) ; and in D20, (3S)-2-ketoglutarate-8-d. The hydrogen
adjacent to the keto group undergoes a slow nonstereospecific exchange
*~ More recently a satisfactory procedure has been established for resolving inter-
mediate III as its (--)-menthol diester. The absolute configuration of the
(R)-cnantiomer was determined by reference to quinic acid by way of citramalic
acid and, hence, the absolute configuration of the labeled citric acid was estab-
lished (H. Weber, Dissertation 3591, EidgenSssischen Technischen ttochschule,
Ziirich, 1965).
~S. Englard and I. Listowsky, Biochem. Biophys. Rea. Commun. 12, 356 (1963).
s7G. E. Lienhard and I. A. Rose, Biochemistry 3, 185 (.~9S4).
CIO O H NADP NADPH + H+ COOH

H--C--OH C=O
HOOC-- C - - H . ~ T--C--H
I isocitrate L
C I-~ dehydrogenase C H2
COOH COOH
(i) (ii)
COOH
1%O2
T--C--H
t
ci%
I
COOH
(III)
with the hydrogen of the medium. Since this rate of exchange is small
(ca. 1% per hour at 30 ° and pH 7.6),as it is not essential that the 2-keto-
glutarate should be converted as soon as possible after its formation.
An excess of NADP ÷ has been used for the preparation of (3S)-2-
ketoglutarate-8-d; H~02 was added at the end of the reaction to give
(2S)-succinate-2-d. s6 Yeast glutathione reductases9 and oxidized gluta-
thione were used as an NADP ÷ regenerating system for the preparation
of (3S)-2-ketoglutarate-3-t, which was isolated by chromatography prior
to its enzymatic conversion to glutamate.8T
In the procedure described here, however, the nonenzymatic NADP*
regenerating system also produces an equimolar amount of H202 and the
2-ketoglutarate is oxidized as it is formed?° If it is desired to stop at the
2-ketoglutarate stage, an excess of catalase 91 must be added. A kinetic
isotope effect is associated with the isocitrate dehydrogenase reaction ;87.9o
the tritium content of the reaction product was found to be 60-75% of the
expected value.
The NADP + regenerating system consists of the redox dye N-methyl-
phenazonium methosulfate ("phenazine methosulfate") and oxygen. If
P* stands for the dye cation, then the stoichiometry of the system may be
represented as follows:
Z. B. Rose, J. Biol. Chem. 235, 928 (1960).

'~ E C 1.6.4.2.
,o K. R. Hanson, Federation Proc. 24, 229 (1965).
~1EC 1.11.1.6.
NADPH+H + +P+ = N A D P + + PH2+
PH~+ + O 2 = P+ +H202
NADPH+H + +O2 = NADP + + H 2 0 2
In the presence of catalase the overall reaction becomes
NADPH+H + +½02--NADP ++H20
As early users of the system employed crude enzyme preparations con-
taining catalase, the fact that the initial oxidation produced H202 was
not recognized. The mechanisms of these oxidations do not appear to
have been studied, but the dye is analogous in some respects to FAD2 -~
Method. Triethanolamine-HC1 buffer, pH 7.5 (100 mieromoles), a
neutralized solution of KH2 threo-D~-isocitrate (10 micromoles) and
MnC12 (5 micromoles) are added to the bulb leg of a sublimation vessel
(Fig. 1). The solution is then shell frozen and lyophylized using the
high-vacuum line traps to collect the water. When the tube is thoroughly
dry, NADP + (ca. 0.5 mg, 0.7 micromole) and N-methylphenazonium
methosulfate (ca. 1 mg, 3 micromoles) are added as solids. Working in a
fume hood, tritiated water (0.5 ml) is transferred to the sublimation tube
bulb by means of a disposable Pasteur pipette fitted with a Teflon exten-
sion. When the solids have dissolved, 50 ~l of a solution of isocitrate
dehydrogenase is added (in 50% glycerol; Boehringer-Mannheim Corp.,
New York; 18-20 units/ml). The system is briefly flushed with oxygen,
the stopper is inserted but the side arm is left open, and the reaction
mixture is gently agitated at 30 ° for 1.5 hours. At the end of this time,
the reaction mixture is shell frozen, the tube is evacuated, and the triti-
ated water is transferred to the straight leg of the vessel by sublimation.
The residue is not a dry solid, as the glycerol added with the enzyme does
not sublime. The vacuum is then released and the water is allowed to
melt. Working in a fume hood, aliquots of water are removed for radio-
activity measurements. The remaining water is transferred to a tritiated
water storage vessel. Succinic and 2-ketoglutaric acids are isolated from
the residue by Dowex 1-formate ion-exchange chromatography. If (3S)-
2-ketoglutarate-3-t is the desired product, a suspension of crystalline
beef liver catalase (0.1 ml, 0.6 mg) must be added to the reaction mixture
to destroy H202. The reaction between H202 and ketoglutarate is very
rapid, and some succinate is therefore obtained along with the keto-
glutarate.
The above reaction has been performed in a Warburg vessel with
KOH in the center well and enzyme added to the side arm. The time
course of oxygen uptake was found to be linear for 80% of the reaction
F. Dickens and H. McIlwain, Biochem. J. 32, 1615 (1938).

time. The system appears to be saturated with respect to substrate,

cofactors, and dye. In an experiment without added catalase, the oxygen
consumption was 1.04 moles per mole of isocitrate added, and the ratio
of succinate to 2-ketoglutarate in the product was 30:1. No detectable
isocitrate remained.
12. (2R)-Succinate-2-t and (3R)-2-Ketoglutarate-3-t
from 2-Ketoglutarate
COOH COOH COOH

I I I
C --O C =O C --O C OOH
I THO ' H20 I H~O2 I
CI'I~ m CT 2 ~ H--C--T ~ H--C--T
, i Mga+ NADPH I L
CH. CH. CH~ CH~
I I isocitrate I t
COOH COOH dehydrogenase COOH COOH
(t) (n) (ill) (iv)

Principle. The NADP-specific isocitrate dehydrogenase from pig
heart catalyzes the exchange of the hydrogen pro-R at C-3 of 2-keto-
glutarate with the hydrogen of the medium. The reaction requires the
presence of both Mg ÷÷ and NADPH, but not C02. NADP ÷ and N A D H
will not substitute for N A D P H 2 s
Method. PREPARATIONOF 2-KETOGLUTARATE-3,3-t2.A solution of Na2
2-ketoglutarate (100 micromoles) is lyophylized in a glass tube. An
ampule of the type used to freeze dry bacteria is suitable. Tritiated water
(50 ~l) is added to the tube, the solution is frozen, and the tube is sealed
under high vacuum. For additional safety, the tube may be placed in a
metal jacket. The tube is then autoclaved at 115 pounds pressure for
30 minutes. The 2-ketoglutarate is then recovered by chromatography on
Dowex 1-formate. The recovery of 2-ketoglutarate is not quantitative.
A small leading radioactivity peak corresponding to succinic acid is
observed. With the above quantities of 2-ketoglutarate and tritiated
water the hydrogens at C-3 will have approximately 93% of the specific
activity of the hydrogen of the tritiated water employed.
REPLACEMENT OF THE pro-S TRITIUM OF 2-KETOGLUTARATE-3,3-t2 BY
PROTIUM. The following procedure requires approximately 15 columns
(1 X 0.5 cm) of Dowex 1-formate (XS, 2(D-400 mesh). Such columns are
conveniently prepared in disposable Pasteur pipettes using glass wool to
support the resin bed. In order to measure tritiated water in the presence
of tritiated 2-ketoglutarate, a column in which water has drained to the
resin surface is placed in an empty test tube and a sample (e.g., 5 ~I)
is added to the column followed by 4 washes of water (each of 0.5 ml).
When the last portion of water has drained to the resin surface, the
column is removed and an aliquot of the efltuent is analyzed for radio-
activity.
The enzyme catalyzed exchange is performed by maintaining 2-keto-
glutarate-3,S-t2 (16 micromoles) at 30 ° in the presence of triethanol-
amine-HC1 buffer, pH 7.6 (300 micromoles), MgCl~ (1 mieromole),
NADPH (1.5 micromoles) and purified isoeitrate dehydrogenase from
pig heart (Boehringer-Mannheim Corp., New York, 5 units, 0.25 ml, 2.5
mg) in a total volume of 3 ml. The release of tritium into the medium is
followed by analyzing as described above small aliquots withdrawn for
assay at 0.5 or 1.0 hour intervals. Additional enzyme is added after 2
hours. By 5 hours the enzyme-catalyzed replacement is complete and the
extent of elimination is about 51~. 87,9° A further addition of enzyme has
no significant effect, but a very slow spontaneous exchange of tritium is
observed. It is possible to isolate the 2-ketoglutarate by chromatography
without extensive loss of tritium. The compound is, however, rapidly
converted to (2R)-succinate-~-t by the addition of H202 (2 ml of a 3%
solution) to the reaction mixture. The labeled succinie acid is then
isolated by chromatography on Dowex 1-formate. The succinate so
produced is unlikely to contain more than 2 or 3 ~ of labeling in the
pro-S position2 °
13. Chemical Synthesis of (2R)-Succinic-2-d Acid from (3R)-L-Malic-
3-d Acid or (3R)-L-Aspartic-3-d Acid
COOH COOH COOH
i I l
HO--C--H t h i o ~ l chloride H--C--C1 ~ H--C--H
D--C--H pyridine D--C--H Pd D--C--H
I t I
COOH COOH COOH
(i) (ii) (m)

Principle. As a result of the elegant work of Cornforth et al28 the two
enantiomers of monodeuterated succinate have been distinguished by
means of optical rotatory dispersion measurements. In their study, (3R)-
r~-malie-3-d acid (I), obtained by enzymatic trans hydration of fumarate
in D20, was esterified to the dimethyl ester. This ester with thionyl
chloride and pyridine gave the dimethyl ester of (3S)-D-2-chlorosuccinie-
3-d acid (II), which was then reduced in acetic acid by zinc-copper
couple. Hydrolysis by acid and recrystallization from water yielded
(2R)-succinic-Z-d acid (III) containing 0.93 atom D per molecule and
having a plain negative optical rotatory dispersion curve in the 350-250
rn~ region. The procedure described below eliminates the esterification of

the stereospeeifically labeled monodeutrated malate. It has been success-
fully used in the reduction of (3R)-D-malic-3-d acid 93 to (2R)-succinie-
2-d acid ~ and should work equally well for the reduction of the cor-
responding epimer (3R)-L-malic-3-d acid to this compound.
Method. To 0.180 g (1.34 millimoles) of (3R)-L-malic-3-d acid in an
oven-dried flask is added dropwise 0.5 ml (6.9 millimoles) of thionyl
chloride previously distilled from quinoline, linseed oil, and then over zinc
dust. The reaction mixture is cooled to --10 ° and kept in the dark.
Purified pyridine (0.21 ml, 2.6 millimoles) is then added dropwise at a
very slow rate so as to maintain the temperature below --5 ° . The reaction
mixture is allowed gradually to warm up to room temperature, and after
8 hours a saturated solution of KC1 is added. Extraction is carried out
with several portions of ether, and the combined ethereal solution is
evaporated under reduced pressure. The yellow residue is dissolved in
water, treated with charcoal, and filtered. The aqueous solution is taken
to dryness, and the product is recrystallized from chloroform. (3S)-D-2-
Chlorosuccinic-8-d acid is obtained (the reaction proceeds by inversion of
configurationg~).
To 0.090 g (0.6 millimoles) of (3S)-n-2-chlorosuccinic-3-d acid dis-
solved in water is added 70 mg of palladium catalyst on charcoal and the
reaction flask is evacuated. Hydrogen gas is bubbled in, and the reaction
mixture is allowed to stand in an atmosphere of hydrogen at 10 pounds
pressure. The catalyst flocculates after taking up hydrogen for 10 min-
utes. The reaction is allowed to proceed for an additional 4 hours. The
solution is then filtered to remove the catalyst and evaporated to dryness
under reduced pressure. (2R)-Succinic-$-d acid is purified either by re-
crystallization from water or by repeated sublimations.
In a typical experiment, starting with (3R)-D-malic-3-d acid, (3S)-L-
2-chlorosuccinic-8-d acid was obtained in a 61~ yield. Hydrogenation of
the latter compound resulted in an 82~ yield of (2R)-succinic-~-d acid.
The overall procedure resulted in an isotope recovery in the final product
of 91% of the deuterium initially present in the starting (3R)-v-malic-
3-d acid.
(2R)-Succinic-~-d acid can also be obtained (1) by reduction of
~3R)-L-aspartic-3-d acid prepared enzymatically by trans-stereospecific
amination of fumarate in D20 and (2) by chloramine T degradation of
(4R)-L-glutamic-~-d acid obtained by the enzymatically catalyzed re-
oaObtained by enzymatic ~ra~-hydratlon of maleate in ]:)20 as catalyzed by maleate

hydratase.
A. McKenzle and F, Barrow, J. Chem. Soc., p. 99 (1911).
arrangement of threo-3-mcthyl-L-aspartate-3-d. 95 The reduction of

(3R)-L-aspartic-3-d acid is by far the best alternative method for the
production of (2R)-suceinic-2-d acid and is herewith briefly described. 05
(3R)-L-Aspartate-3-d is prepared by incubation of fumarate and
ammonia with whole cells of Proteus vulgaris in D2026 The stereospecif-
ically labeled monodeuterated L-aspartic acid is converted to (3S)-L-2-
bromosuccinic-3-d acid by treatment with NAN02 and HBr according to
the method of HolmbergY A solution of 253 mg (1.3 millimoles) of this
compound in 5 ml of water is added to a suspension of catalyst prepared
by the hydrogenation of 160 mg of PtO~'H20 in 10 ml of water. Hydro-
genation is continued at atmospheric pressure at 23 ° for 2 hours, until
hydrogen uptake (31 ml) has ceased. The catalyst is removed by filtra-
tion and washed with water, and the combined filtrate and washings are
extracted continuously overnight with ether. The ether extract is dried
over MgS04 and evaporated to dryness. The (2R)-succinic-2-d acid is
further purified as above. An 85-90% yield of succinic acid has been
reported by carrying out the hydrogenation of the 2-bromosuccinic acid
as outlined here. In" addition, the overall reduction of (3R)-L-aspartic-3-d
acid to (2R)-suecinic-2-d acid proceeded with almost complete retention
of the initial deuterium in the final product. 95
14. Other Labeled Succinic Acids

Although our primary concern is with individual stereospecifically
labeled compounds, nonresolvable racemic mixtures of labeled succinic
acids and aehiral labeled succinic acids may be required as reference
compounds or as starting materials for syntheses.
Succinic-d4 acid (I) and succinic-2,2-d2 acid (II) may conveniently
be prepared by heating the commercially available compounds tetraethyl
ethane-l,l,2,2-tetracarboxylate and triethyl ethane-l,l,2-tricarboxylate,
respectively, in D20 at 160 ° for 3 hours. 45 The same procedure could also
be used with tritiated water; however, succinic-t4 acid is commercially
available. Compound I may also be obtained by the reduction of di-
methyl acetylenedicarboxylate, as for example with deuterium gas in the
presence of platinum oxide 9s or with sodium-mercury amalgam in the
presence of D20. 09
(2R,3S)-Succinic-$,3-d~ acid (III) may be obtained by the reduction
tSM. Sprecher, R. L. Switzer, and D. B. Sprinson, J. Biol. Chem. 241, 864 (1966).
See this chapter, Section 2.
9'B. Holmberg, Ber. Deut. Keram. Ges. 60, 2205 (1927).
"S. Borcic, Croat. Chem. Acta 35, 87 (1963); Chem. Abstr. 59, 6224d (1963).
mG. Adembri and F. Desio, Ric. ~qci. Rend. A3, 907 (1963) ; Chem. Abstr. 60, 7902d
(1964).
COOH COOH COOH

i i i
C~ CD 2 H--C--D
I t I
C~ C~ H--C--D
I I I
COOH COOH COOH
(t} (III)
COOH COOH
I I
H--C--D D--C--H
t I
D--C~H H--C--D
f I
COOH COOH
(IV) (v)
of maleate with deuterium in the presence of palladium-charcoal lo° or by

diimide generated in D20.1°~-1°3 Either of these methods when applied to
fumarie acid give rise to the racemate (2RS,3RS)-suecinic-~,3-d2 acid
(IV and V). The stereospecificity of the reductions are greater than 95%.
When threo-as-isocitrate was treated with isocitrate lyase T M in D20
in the presence of semicarbazide, recycling took place despite the presence
of the glyoxylate trapping agent. (A result which suggests that the re-
lease of glyoxylate from the enzyme limits the overall rate of the reac-
tion.) Some of the (2S)-succinate-2-d formed by the initial cleavage con-
densed with glyoxylate to give (4S)-threo-Ds-isocitrate-$-d, which on
cleavage took up a second deuterium atom. The isolated product (2S,3S)-
succinic-~,3-d2 acid (V) had a labeling ratio of ca. 1.0:0.423 The prepara-
tion of (2R,3R)-succinic-2,8-d2 acid (IV), with the deuterium equally
distributed between the two methylene carbon atoms, has recently been
reported, l°s The compound was obtained by treating (4R)-4,5-dihydroxy-
2-oxovalerate (i.e., 3-deoxy-L-pentulosonate, not 3-deoxy-2-oxo-L-ara-
bonate) 8 in D20 with the dehydratase for this compound from
Pseudomonas saccharophila, and oxidizing the product (3R,4R)-2-oxo-
glutaraldehydate-8,~-d2 with H202 and KMnO,.
'°°T. T. Tchen and H. Van Milligan, J. Am. Chem. Soc. 82, 4115 (1960).
,o~E. J. Corey, D. J. Pasto, and W. L. Mock, J. Am. Chem. Soc. 83, 2957 (1961).
"2 E. J. Corey and W. L. Mock, J. Am. Chem. Soc. 84, 685 (1962).
~o~S. Hunig, H. R. Muller and W. Thiev, Angew. Chem. Intern. Ed. 4, 271 (1965).
~o~EC 4.1.3.1. See this volume [29].
I~D. Portsmouth, A. C. Stoolmiller, and R. H. Abeles, J. Biol. Chem. 242, 2751
(1967).
COOH COOH COOH

) f I
H--C--D D--C--H H--C--OH
I I I
C~ CD 2 HOOC--C--D
I J I
COOH COOH CD2
COOH
(vD (vm (vIID
(3R)-Succinic-~,~,3-d8 acid (VI) has been obtained by the action of
purified methylmalonyl-CoA mutase 1°6 on chemically synthesized (2RS)-
2-(methyl-d3)-malonyl-CoA. (0nly the (R)-enantiomer is acted upon by
the enzyme.) A similar series of reactions gave the tritiated analog> °7 The
optical rotatory dispersion curve of the product was not determined, but
all the deuterium was retained during the isomerization and the stereo-
chemistry of this reaction has now been established. 1°8
(3S)-Succinic-#,2,3-d3 acid (VII) has been obtained from the ozon-
olysis and further oxidation of a deuterated squalene derived biosyntheti-
cally from (3RS)-mevalonate-#-~'C-5,5-d2. 4",~°~ (0nly the (R)-enanti-
omer of mevalonate corresponding to the (R)-(+)-lactone is converted
into squalene.)
The above compounds could be more readily prepared in most
laboratories by way of threo-D~L,-isocitrate-3,~,$-d3 (VIII and its enan-
tiomer) which is obtainable by chemical synthesis from succinic-d, acid
(I) and chloral> 3,~°,m On treating the threo-racemate in normal water
with isocitrate dehydrogenase, NADP ÷, N-methylphenazonium metho-
sulfate, and oxygen, the threo-D,-enantiomer would be converted into
(3R)-succinate-2,~,3-d3 ~2 (VI). To obtain the enantiomeric (3S)-
succinate-~,$,S-d3 (VII) it would be necessary to isolate (3R)-2-keto-
glutarate-#,~,~-d3 after isocitrate dehydrogenase treatment of the
threo-racemate. Heating this compound in normal water would eliminate
deuterium from the 3-position. Alternatively, 2-ketoglutarate-~,4-d~
could be obhined from (3S)-citrate-~,~-d~? ~a Deuterium could then be
introduced into the alternative position at C-3 to give a center with (S)
*~EC 5.4.99.2. See this volume [34].
'~ J. D. Erfle, J. M. Clark, Jr., R. F. Nystrom, and B. C. Johnson, Y. B£oL Chem.
239, 1920 (1964).
~ M. Sprecher, M. J. Clark, and D. B. Sprinson, J. Biot. Chem. ~41, 872 (1966).
" J . W. Cornforth, R. H. Comforth, C. Donninger, G. Popj~k, G. Ryback, and
G. T, Schroepfer, Jr., Biochem. Biophys. Res. Commun. l l , 129 (1963).
mR. Fittig and H. E. Miller, Ann. Chem. ~ , 43 (1889).
1,, H. B. Vickery, this volume [75].
1, See this chapter, Section 11.
,1~See this chapter, Section 7.
[75] threo-D.-ISOCITRATE 601
chirality by means of isocitrate dehydrogenase. Addition of H20, at the

end of the reaction would give the desired deuterated succinate.114
Although in all cases where it has been necessary to establish the
configuration of a stereospecifically labeled succinic acid the optical
rotatory dispersion of a deuterated compound has been determined, it
may not always be possible to obtain sufficient material for such measure-
ments. An alternative approach is to employ tritium labeling and to
compare the sample compound with reference preparations of (2R)- and
(2S)-succinate-2-t by treating similar amounts of all three compounds
with purified threo-D~-isocitrate lyase in the presence of glyoxylate and
Mg ÷÷. The equilibrium for the lyase favors cleavage so that only a small
portion of the added succinate is present as isocitrate at a given time. The
cycle succinate-isocitrate-succinate ultimately leads to the exchange of
both the pro-S hydrogens of succinate with the hydrogen of the medium;
i.e., tritium is eliminated from (2S)-succinate-2-t, but not from its enan-
tiomer. It should be verified in such experiments that side reactions have
not taken place and that succinate is indeed present at the end of the
reaction. If the isocitrate lyase is contaminated with a trace of aconitate
hydratase, then the pro-R hydrogens of succinate will also be exchanged.
The use of reference compounds in this type of experiment is essential2 °
i,, See this chapter, Section 12.
[ 7 5 ] P r e p a r a t i o n of M o n o p o t a s s i u m threo-m-Isocitrate 1
B y t-I. B. VICKERY
Introduction
Although threo-Da-isocitrate is doubtless present in trace quantities in
all living cells in which the metabolic pathway known as the Krebs tri-
carboxylic acid cycle occurs, so far as is at present known substantial
amounts of this organic acid are found only in the leaves of certain
succulent plants, most of which belong to the family Crassulaceae. Iso-
citric acid was synthesized by Fittig and Milleff in 18892 and later, and
1The nomenclature employed is that recently advocated by H. B. Vickery [J. Biol,
Chem. 237, 1739 (1962)]. The thr¢o prefix indicates that the two asymmetric
centers have opposite configurations, and the l), symbol indicates that the a-carbon
has the D Configuration. The s refers to the type amino acid serine.
The synthesis of threo-DBL~-isocitric acid by a modification of the Fittig and Miller
method has been described by G. W. Pucher and H. B. Vickery [J. Biol. Chem.
163, 169 (1946)]. A further modification in which the acid is isolated as its mono-
potassium salt rather than as the lactone has been described by H. B. Vickery
[Science 132, 892 (1960)].
I R. Fittig and H. E. Miller, Ann. Chem. 255, 43 (1889).
[75] threo-D.-ISOCITRATE 601
chirality by means of isocitrate dehydrogenase. Addition of H20, at the

end of the reaction would give the desired deuterated succinate.114
Although in all cases where it has been necessary to establish the
configuration of a stereospecifically labeled succinic acid the optical
rotatory dispersion of a deuterated compound has been determined, it
may not always be possible to obtain sufficient material for such measure-
ments. An alternative approach is to employ tritium labeling and to
compare the sample compound with reference preparations of (2R)- and
(2S)-succinate-2-t by treating similar amounts of all three compounds
with purified threo-D~-isocitrate lyase in the presence of glyoxylate and
Mg ÷÷. The equilibrium for the lyase favors cleavage so that only a small
portion of the added succinate is present as isocitrate at a given time. The
cycle succinate-isocitrate-succinate ultimately leads to the exchange of
both the pro-S hydrogens of succinate with the hydrogen of the medium;
i.e., tritium is eliminated from (2S)-succinate-2-t, but not from its enan-
tiomer. It should be verified in such experiments that side reactions have
not taken place and that succinate is indeed present at the end of the
reaction. If the isocitrate lyase is contaminated with a trace of aconitate
hydratase, then the pro-R hydrogens of succinate will also be exchanged.
The use of reference compounds in this type of experiment is essential2 °
i,, See this chapter, Section 12.
[ 7 5 ] P r e p a r a t i o n of M o n o p o t a s s i u m threo-m-Isocitrate 1
B y t-I. B. VICKERY
Introduction
Although threo-Da-isocitrate is doubtless present in trace quantities in
all living cells in which the metabolic pathway known as the Krebs tri-
carboxylic acid cycle occurs, so far as is at present known substantial
amounts of this organic acid are found only in the leaves of certain
succulent plants, most of which belong to the family Crassulaceae. Iso-
citric acid was synthesized by Fittig and Milleff in 18892 and later, and
1The nomenclature employed is that recently advocated by H. B. Vickery [J. Biol,
Chem. 237, 1739 (1962)]. The thr¢o prefix indicates that the two asymmetric
centers have opposite configurations, and the l), symbol indicates that the a-carbon
has the D Configuration. The s refers to the type amino acid serine.
The synthesis of threo-DBL~-isocitric acid by a modification of the Fittig and Miller
method has been described by G. W. Pucher and H. B. Vickery [J. Biol. Chem.
163, 169 (1946)]. A further modification in which the acid is isolated as its mono-
potassium salt rather than as the lactone has been described by H. B. Vickery
[Science 132, 892 (1960)].
I R. Fittig and H. E. Miller, Ann. Chem. 255, 43 (1889).
by a different method, by Wislicenus and Nassauer, 4 but its occurrence in

nature was first demonstrated in 1925 by Nelson, s who showed that it is
present in small amounts in the juice of the blackberry. In 1942, Pucher
isolated isocitric acid as its triethyl ester in substantial amounts from
the leaves of B r y o p h y l l u m calycin~m, G and, independently the same year,
the Swedish investigator Nordal; obtained it from the leaves of S e d u m
acre and Echeveria secunda var. glauca. These observations solved the
problem of the identity of the so-called "Crassulacean malic acid" which
had been the subject of a large and puzzling literature since the work
of Mayer in 18782
In 1948, while searching for a convenient derivative for isolation
purposes, Pucher, Abrahams, and Vickery ° prepared the dimethyl ester
of the lactone of threo-D~-isocitric acid, a substance with excellent phys-
ical properties. With the same motive Wilson ~° in 1963 described the
beautifully crystalline monopotassium salt of the lactone. He obtained
it by a procedure which involved the isolation of citric and isocitric acids
from extracts of the dried leaf tissue as a mixed fraction by chromatog-
raphy on Dowex 2-formate followed by lactonization of the isocitric acid.
The lactone was then separated from citric acid by further chroma-
tographic steps and isolated by adjustment of the reaction of the solution
to pH 3.26 with potassium hydroxide and subsequent concentration and
addition of alcohol.
The isolation of monopotassium threo-D~-isocitrate from crassulacean
plants was described by H. B. Vickery and D. G. Wilson in 1958.~1 The
following procedure is based upon their work.
Sou1"ces
A survey of the isocitric acid contcnt of some 58 succulent species of
plants including 39 species belonging to the family Crassulaceae has
been made by Soderstrom. le The table lists the species containing 9%
or more of the dry weight of the leaves as threo-D~-isocitric acid. Choice
W. Wislicenus and M. Nassauer, Ann. Chem. 285, 1 (1895).

~E. K. Nelson, J. Am. Chem. Soc. 47, 568 (1925).
*G. W. Pucher, J. Biol. Chem. 145, 511 (1942).
~A. NordM, Medd. Norslc Farm. Selskap. 5, 129 (1942); Chem. Abstr. 40, 6422
(1946).
~A. Mayer, Versuchs Stat. 21, 277 (1878).
PG. W. Pucher, M. D. Abrahams, and H. B. Vickery, ]. Biol. Chem. 172, 579
(1948).
I°D. G. Wilson, Can. J. Biochem. Physiol. 41, 1571 (1963).
11H. B. Vickery and D. G. Wilson, ]. Biol. Chem. 233, 14 (1958); ibid. B~ochem.
Prep. 7, 72 (1960).
I~T. R. Soderstrom, Am. J. Botany 49, 850 (1962).
[75] threo-D,-ISOClTRA TE 603
SPECIES OF SUCCULENT PLANTS THE LEAVES OF WHICH ARE ESPECIALLY

RICH IN ISOCITRIC ACIDa
Percent
Species of dry weight
Bryophyllum calycinum Salisb. [Kalanchoe pinnata (Lain.) Pers.] 9.4

B. crenalum Baker (K. laxifolia Baker) 15.4
B. fedtschenkoi Hamet and Perrier (K. fedtschenkoi ttamet and 15.5
Perrier)
K. daigremontiana Hamet and Perrier 10.0
A eonium decorum Webb 10.2
Graptopetalum paraguayense (N. E. Brown) E. Walther (Echeveria 10.5
weinbergii Hort.)
Sedum praealtum DC. 10.8
S. spedabile Boreau 10.4
S. montanum L. 12.7
S. gaudinii Christ. 9.0
Crassula falcata Wendl. 9.9
Aloe microstigma Salm-Dyck 13.7
A. saponaria Haw. 19.4
A. spinosissima Hort. 9.1
" The Bryophyllum or Kalanchoe species are common greenhouse plants usually
available from commercial growers. Sedum spectabile is a common garden plant. The
aloes are southern plants. Alternative scientific names are given for some species
since the botanical nomenclature is not uniform.
B. calycinum and K. daigremontiana are easily propagated from the numerous
plantlets that form at the indentations in the margins of mature leaves. Brief
directions for propagation are given by Pucher, Abrahams, and Vickery, J. Biol.
Chem. 172, 579 (1948). Most of the other species such as K. fedtschenkoi, which is
especially rich in isocitric acid, can be propagated from single leaves or small
cuttings the bases of which are dipped in a commercial preparation containing
hormones which promote rootlet formation. The plants are grown in sand with use
of a culture solution until established and then transplanted to rich soil. Plants of
suitable size can usually be produced in four or five months.
a m o n g these for the p r e p a r a t i o n of isoeitric acid is largely a m a t t e r of

availability. M a n y of t h e m are grown c o m m e r c i a l l y as house plants, and
stock material can be obtained from local growers. Biochemists u n f a m i l -
iar with plants m a y require the aid of colleagues in the b o t a n y d e p a r t -
ments of their institutions to obtain facilities for growing the plants in
q u a n t i t y . F r o m 3 to 4 kilograms of fresh leaves are needed to obtain
200-300 g of d r y tissue; this a m o u n t can be obtained f r o m a b o u t l 0
plants of the larger species such as b r y o p h y l l u m s and kalanchoes.
Preparation
T h e leaves are cut from the plants in m i d a f t e r n o o n of a bright s u n n y
day, a time chosen so t h a t the malic acid content is at a low level. P l a n t s
of the family Crassulaceae are characterized by a profound diurnal

variation of the malic and citric acid content, and the isolation of iso-
citric acid is facilitated if leaves of minimal malic acid content are used.
The leaves are dried in a ventilated oven at 80 ° until crisp and are
ground to powder preferably in a Wiley mill. This material can be kept
indefinitely. It is convenient to use 200 g quantities for the subsequent
steps.
Essential equipment for the following procedure is a device for
extraction with ether of a mass of tissue which occupies a volume of
about 1400 ml. In this laboratory the Nolan extractor la is used, but
large-scale equipment of the Soxhlet type can doubtless be substituted.
However, a study of the time required for complete extraction of the
organic acids may be necessary. In addition, equipment for rapid evap-
oration of aqueous solutions at temperatures not exceeding about 40 ° is
required.
A 200 g quantity of the dry powdered leaves is mixed with 330 ml of
6.0 N sulfuric acid and allowed to stand for several hours or overnight
to ensure the complete liberation of all of the organic acids from their
salts. The soft mass is then thoroughly mixed with about 200 g of Celite
545 and transferred to the extraction apparatus.
The technique for the extraction will differ according to the equipment
available. With the Nolan device, the 2-liter beaker may be lined with
glass cloth or even with a closely woven cotton cloth although this will
be severely attacked during the extraction. Alternatively, a circular piece
of stainless steel gauze covered with a circle of glass cloth or even
filter paper may be placed in the bottom of the beaker, and the moist
tissue packed firmly on top of it. The top of the mass is leveled and
covered with a layer of Celite; the extraction is facilitated if a round
piece of stainless steel plate containing numerous small holes is placed
on top. This plate works best if the edge is rolled up slightly so as to
form a very shallow pan. The object is to distribute the ether over the
entire surface of the tissue and avoid channeling in the mass beneath.
Whatever apparatus is used, arrangements must be made so that no
fine particles of tissue are washed through by the ether, and that the
ether runs freely through the mass of tissue. Extraction of the organic
acids is usually complete after the apparatus has been heated in a water
bath for 24 hours. The temperature should be adjusted so that siphoning
with Soxhlet type apparatus occurs at intervals of 10-15 minutes.
The deeply colored ether extract is quantitatively transferred to a
beaker, about 200 ml of water is added, and the ether is evaporated
under a stream of air. The fatty material separates in the cold in soft
~L. S. Nolan, Anal. Chem. '21, 1116 (1949).
[75] threo-Ds-ISOClTRA TE 605
masses that are easily filtered and washed, and the aqueous solution is
treated at room temperature with 3-5 g of deeolorizing carbon. The
filtered solution should be clear and only pale yellow in color. At this
point, if the procedure is to be followed quantitatively, the solution is
made to volume and a small aliquot is removed for the determination of
the isocitric acid content by the isocitric dehydrogenase method of
Ochoa 14 as described by Stern. 1~
The subsequent steps in the procedure will depend on the object in
view. If all that is required is a specimen of potassium isocitrate regard-
less of yield, procedure A may be followed. The solution contains isocitric
acid and its lactone as major components, but a substantial amount of
malic acid and a smaller amount of citric acid are also present. Small
amounts to traces of many other organic acids can be detected by suit-
able methods.
Procedure A. The solution is neutralized to exactly pH 3.50 with the
aid of a glass electrode by the careful addition of 20% potassium hy-
droxide. From 50 to 55 ml are usually required with extracts from B.
calycinum, and attention must be given to the temperature toward the
end of the neutralization, ice being added as necessary. The solution is
then concentrated in an efficient vacuum still operated at a bath tem-
perature of about 40 °. A filtration may be necessary during this opera-
tion as a perfectly clear solution is desirable. Concentration is continued
until a thin sirup, usually at about 60 ml, is obtained, and the solution
is then transferred to a beaker with the minimal amount of water,
allowing ample time for drainage of each small quantity of wash fluid.
This solution is chilled in an ice bath and stirred to induce crystallization,
which with the first preparation attempted may take considerable time.
Once seed material has been obtained, crystallization can usually be
induced promptly. The solution is chilled for at least 24 hours with
occasional stirring so as to prevent deposition of a hard mass of crystals
which adhere strongly to the glass. Addition of a little alcohol favors
complete crystallization but may diminish the purity of the crop. The
crystals are filtered on a sintered glass funnel, the mother liquor being
repeatedly used to aid the transfer; they are sucked fairly dry and
washed with 70% alcohol followed by 95% and absolute alcohol and
finally a little ether. They are then dried in a vacuum desiccator, as the
~' S. Ochoa, J. Biol. Chem. 174, 133 (1948).

~J. R. Stern, Vol. III, p. 428. Inasmuch as one-third or more of the isocitric acid
present in this solution will have been converted to the lactone during the drying
of the leaf tissue and the subsequent treatment, it is necessary, before proceeding
with the analysis of the aliquot, to make it alkaline to phenolphthalein and heat it
on the steam bath for a few minutes in order to open the lactone ring.
moist salt is unstable in the oven at 110 °. Such material is usually 88-
94% pure when analyzed by the Ochoa method, and the yield, if B.
calycinum is the source, is close to 10 g. About one-half of the isocitric
acid in the original extract is obtained. Somewhat more will be obtained
from the richer species. One recrystalli~ation by the technique described
below gives material that is pure within the limits of the analytical
method (about __+2%), with melting point between 179 and 186 ° depend-
ing on the rate of heating, specific rotation [alp 25 : ~-20.4 ° (0.1 M in
water), and nearly theoretical potassium content (KH2C6H50~, K - -
16.99%) and neutralization equivalent (theory, 115). Three recrystalliza-
tions give material suitable for refined physicochemical studies. 1~
Procedure B. The solution is made alkaline to phenolphthalein with
potassium hydroxide and heated on the steam bath for 10-15 minutes.
This operation converts all the isocitric lactone present to the acid. The
cooled solution is then treated with Dowex 50 (H ÷) added in small
quantities with rapid stirring until the pH is brought below 3.50 and
filtered. The resin is thoroughly washed with water at room temperature.
Filtrate and washings are then decolorized and brought to exactly pH
3.50 with potassium hydroxide and concentrated as in procedure A. The
yield of crude potassium isocitrate from an extract from 200 g of B.
fedtschenkoi leaves is about 28 g of purity about 97%.
Procedure C. This procedure depends upon the fact that the barium
salt of isocitrie acid is only sparingly soluble in boiling water although
moderately soluble in cold water. In this respect it resembles the calcium
salt of citric acid. Furthermore, precipitation of barium isocitrate in
hot solution results in substantial purification since much of the malic
acid and most if not all of the acids present in small amounts in the
extract of the leaves remain in the filtrate. The citric acid present will,
however, accompany the isocitric acid.
The solution is heated on the steam bath to a temperature higher
than 90 ° and treated with hot saturated barium hydroxide solution until
strongly alkaline to phenolphthalein. Barium isocitrate separates as a
curdy precipitate and is allowed to digest at 90 ° or higher for a few
minutes to ensure complete saponification of the lactone. The thick sus-
pension is filtered on an 18-cm Biichner funnel through 2 sheets of
Whatman No. 3 filter paper covered with about 1 cm of Celite 545 and
washed with about 1 liter of boiling water. The loss of isocitric acid in
the filtrate at this step is about 10%. After being carefully packed on
the funnel with a spatula and sucked thoroughly, the precipitate is
transferred to a beaker and suspended in about 2 liters of water at room
'~ D. I. Hitehcock, J. Phys. Chem. 62, 1337 (1958).

[75] threo-Ds-ISOCITRATE 607
temperature with the aid of mechanical stirring. The filter paper is

stripped from the mass of precipitate and separately treated with excess
of sulfuric acid. The salt is decomposed by slowly adding 10 N sulfuric
acid in slight excess, 45-70 ml being required. Approach to the end point
is detected with a universal indicator paper. An excess of sulfuric acid can
usually be demonstrated when the solution approaches pH 1.5. After the
solution has been thoroughly stirred, a final test for the presence of
excess sulfuric acid is made by centrifuging a 5 ml sample and dividing
the clear supernatant fluid between two test tubes. To one a drop of 1%
sulfuric acid (v/v) is added, and to the other a drop of cold saturated
barium hydroxide. A precipitate of barium isocitrate will at once form in
the second tube, but this will dissolve if the tube is shaken, leaving a
turbid solution containing barium sulfate if excess of sulfuric acid is
present. Treatment of the main suspension with sulfuric acid must be
continued until this test is positive, but substantial excess of sulfuric acid
is to be avoided. After excess of sulfuric acid has been demonstrated, cold
saturated barium hydroxide solution is added with stirring until the
faintest visible test for the presence of sulfuric acid is obtained. If this
point is overshot, further dilute sulfuric acid must be added. With skill,
persistence and a little luck, a precise balance indicating exact removal
of both acid and barium ion can be obtained. This is desirable although
not essential. The barium sulfate is filtered on a Biichner funnel on
Whatman No. 3 paper covered with a layer of filter aid and, if sufficient
Celite was used for the filtration of the barium isocitrate, the filtrate will
run through fairly freely. Alternatively, the barium sulfate may be
centrifuged. In either case, the barium sulfate must be thoroughly
washed with water at room temperature.
Filtrate and washings are neutralized to pH 3.50 and concentrated as
described in procedure A, but when the volume has been reduced to
about 300 ml a further filtration is usually necessary to remove traces of
barium sulfate. A treatment with a little decolorizing carbon may also be
desirable at this stage. The trace of pale yellow pigment usually present
is not, however, easily removed by the carbon. It gives no further trouble.
The yield of crude crystals if B. calycinum extracts are used may be
expected to be about 12 g of purity near 94%. If several preparations by
procedure A have been carried out, it is worth while to combine all
mother liquors of the crude crystals, concentrate, and carry out procedure
C. In this way nearly 80% of the isocitric acid in the extracts may be
recovered in crude crystalline form. It is not worth while to attempt to
obtain second crops by evaporation of the mother liquors of the crude
crops, as such material is grossly impure.
Recrystallization of Potassium Isocitrate. The crude crystals are
suspended in a little more than twice their weight of hot water and
quickly heated until dissolved; the solution is filtered through a hot
funnel. With large amounts, a steam-jacketed funnel is essential. The
filtrate is received in a beaker or flask chilled in ice, and the solution
should be frequently stirred so that the crystals are prevented from
forming a mass that adheres to the glass. The entire operation must be
carried out with the utmost dispatch as lactonization takes place fairly
rapidly, and serious losses may occur if there is any delay in chilling the
hot solution. The addition of about one-half a volume of alcohol after
crystallization is well under way increases the yield, which shouJd be at
least 80% of the weight of the crude material.
The solubility of monopotassium threo-Ds-isocitrate in water at 0 ° is
about 3.5 g in 100 ml and about 0.5 g in 5 0 ~ alcohol. The salt separates
in short often flattened orthorhombic needles or long prisms which ag-
gregate into radiating masses. The corresponding ammonium and rubid-
ium salts have been prepared and also have good properties, but the
sodium and lithium salts separate as hydrates and are much more soluble
than the potassium salt.
Synthesis of threo-DsLs-Isocitrate
The following procedure is a modification of the Fittig and Miller 3
method and is based on the work of Pucher and Vickery 2 and of
Vickery? Chloral is condensed with sodium succinate in the presence of
acetic anhydride, the lactone of trichloromethylparaconic acid produced
is isolated and hydrolyzed by being boiled with excess of barium hydrox-
ide, and the barium isocitrate is decomposed with sulfuric acid. Mono-
potassium threo-DsLs-isoeitrate is then isolated essentially as described
under procedure C.
COONa COOH COOH
I I I
CC13--CHO + CH~ --~ CC18--CH--CH COOH--CHOH--CH
[ I I
CH2 CH2 H2C--COOH
I I
COONa 0,,, CO
Anhydrous sodium succinate (20 g), 16 ml of chloral (33~ excess
over 1 equivalent), and 12.6 ml of redistilled acetic anhydride (1 equiv-
alent) are heated for 1 hour in a 500 ml flask equipped with a reflux con-
denser and mechanically operated stirrer in an oil bath maintained at
140 ° . The reaction mixture turns black and becomes viscous but remains
fluid. The tarlike product is dissolved in 200 ml of hot water, boiled with
about 15 g of decolorizing carbon, and the filtered dark solution is con-
[7S] threo-D,-ISOCITRATE 609
centrated in a vacuum still until at about 120 ml further concentration

becomes difficult because of the separation of sodium salts. The solution
is then heated to dissolve the salts, and 30 ml of concentrated hydro-
chloric acid is added. The dark red oil which separates is induced to
crystallize by chilling the solution in an ice bath and stirring it with a
rod. After being chilled overnight, the crystals are filtered, pressed down
hard on the funnel, and washed with a little ice water. The filtrate and
washings are extracted with ether to recover a further small quantity
of the lactone, the ether extract being washed with water before being
evaporated to give a few drops of oil. The crystals, after being dried in a
vacuum desiccator, weigh 22-24 g (72-78% of theory).
The crude trichloromethylparaconic acid lactone, together with the
small amount of oil from the ether extract, is slowly added (danger of
frothing) to a hot solution of 120 g of barium hydroxide octahydrate in
150 ml of water (20% excess over 6 equivalents), and the thick suspen-
sion of barium isocitrate is boiled for an hour under a reflux condenser
with mechanical stirring in a flask heated in an oil bath. The boiling
solution is filtered on two sheets of Whatman No. 3 filter paper covered
with a thick layer of Celite 545, and is washed with boiling water. The
subsequent decomposition of the barium salt and isolation of the mono-
potassium salt are carried out as described under procedure C above.
The solution of synthetic isocitric acid obtained contains the threo
and erythro disastereomers in the proportion of approximately 10 to 1,
as shown by chromatographic analysis on Dowex 1 with elution with
3.5 N formic acid by the method of Palmer. 17 A little suceinic acid is
usually also present. The erythro isomer is eluted immediately after the
threo isomer. Crystallization of the monopotassium threo-isocitrate is
favored and the yield is improved by the addition of about one-quarter
volume of alcohol to the mother liquor, but if too much alcohol is added
the product may be contaminated with the erythro isomer. The yield is
14-16 g (49-56% of theory), and the preparations usually contain be-
tween 49 and 50% of potassium threo-Ds-isocitrate as determined by the
method of Ochoa. Once recrystallized from a mother liquor that contains
25% of alcohol, the salt is essentially pure.
Monopotassium threo-DsL~-isocitrate crystallizes in small flattened
rhombic needles aggregated into masses of fascicles. It is soluble in its
own weight of boiling water, and the solubility of recrystallized material
is close to 8 g in 100 ml of water at 0 ° and about 1.5 g in 25% alcohol
at the same temperature. It is thus appreciably more soluble than the
D~ isomer. It decomposes at 175-176 ° with evolution of gas and the
production of the lactone as the major product.
,7j. K. Palmer, Conn. Aor. Expt. Sta. Bull. 589 (1955).
[76] Synthesis of threo- and erythro-Isocitric Acids 1

By HAKUJI KATSURA
CO.C2H~ + H~C--CO2C~H5
CO2C2H~ H2C--CO2C2H 5
Diethyl o x a l a t e / Diethyl succinate
NaOC2H s
CO2C2H5 COzC2H5
I
CO Raney- Ni HC O
HC-- CO2C~H5 + H~ HC--CO~C2H5
]
H2C-- CO2C~H5 I~C
i I
CO
Triethyl Diethyl
oxalosuc cinate isocitric lactone
HCl/
CO2HI / C02HI
0 C--H 0 C--H
I H - - C'I - - C O , H + i HO~C--C--H
'I
oc CH. OC CH.
t h r e o - Isocitric erythro- Isocitric

acid lactone acid lactone
Triethyl Oxalosuccinate2
A 500 ml three-necked flask is fitted with a reflux condenser, a
dropping funnel, and stirrer. The opening of the dropping funnel is fitted
with a drying tube filled with anhydrous calcium chloride. Absolute
ethanol (180 ml) is placed ill the flask, followed by 12.5 g (0.5 g atom)
*lq. Katsura, Nippon Kagaku Zasshi (d. Chem. Soc. Japan Pure Chem. Sect.) 8@-,
91 (1961).
sThis procedure is based upon a private communicationfrom Setsuji Sakura~.
[76] SYNTHESISOF threo- AND erythro-isoCITRIC ACIDS 611
of metallic sodium, added in small portions and dissolved by gentle

heating. Ethyl oxa!ate, 81 g (0.5 mole), is then added dropwise at 60 °,
and the solution is stirred continuously for a few minutes after the
addition is complete. This is followed by 87 g (0.55 mole) of ethyl suc-
cinate, added in one portion. The reaction mixture is set aside at 10-30 °
overnight. After ethanol is removed in vacuo, the reddish residue is dis-
solved in 500 ml of water and extracted to remove impurities with 100 ml
portions of ether until the ether extract is colorless. The aqueous layer is
acidified with 100 ml of concentrated hydrochloric acid, using Congo-
red paper as an indicator.
An oily substance separates, which is extracted with 100 ml portions
of ether, until the aqueous layer is almost colorless. The ether layer is
washed with 100 ml portions of 0.1 N sodium hydroxide until the com-
bined washings are no longer acidic to litmus paper. This is followed by
one extraction with 100 ml of water. The ether layer is then dried with
anhydrous sodium sulfate. The solvent is removed in vacuo. The residue
consists of almost pure ethyl oxalosuccinate.
The yield is 102-113 g (72-83% of calculated value). This substance
may be used in the subsequent steps without further purification.
Diethyl Isocitric Lactones

Preparation o] Catalyst. To a solution of 26 g of sodium hydroxide
in 100 ml of water, 20 g of Raney-alloy (nickel content about 50% by
weight) is added in small portions to permit a continuous, vigorous
evolution of hydrogen gas. After all alloy is added, the reaction mixture
is heated on a boiling water bath until gas evolution stops. The resulting
nickel catalyst is washed repeatedly with distilled water until the
washings are free of alkali.
Chloroplat~nate Solution. One gram of chloroplatinic acid is dissolved
in 10 ml of 1 N hydrochloric acid. It is neutralized with 1 N sodium
hydroxide solution just before use.
Method A. To a solution of 6.3 g (0.023 mole) of ethyl oxalosuccinate
in 120 ml of methanol, add the catalyst (prepared from 20 g of alloy)
and 1.5 ml of the chloroplatinate solution. Shake the mixture in an
atmosphere of hydrogen gas at room temperature. Hydrogen absorption
occurs immediately and 368 ml of hydrogen (about 80% of the theo-
retical amount) is absorbed within 1 hour; thereafter hydrogen absorp-
tion is slow. Continue the reduction another 8 hours until the ferric
chloride test is negative. The hydrogen uptake reaches 448 ml (almost
the theoretical amount). Filter the catalyst from the reaction mixture
and wash it with methanol several times. Combine the filtrate and wash-
ings and concentrate them in vacuo. Distill the residue under reduced
pressure (5 mm Hg), discarding a small amount of the early distillate.

A mixture of the lactones of ethyl isocitrate distills at 145-165 °. The
yield is 4.8 g (90~ of theoretical value).
Method B. To a solution of 60 g (0.22 mole) of ethyl oxalosuecinate
in a 500 ml autoclave, add the catalyst (prepared from 80 g of alloy),
3 ml of the chloroplatinate solution, and 150 ml of methanol. Shake the
mixture with hydrogen at a pressure of about 50 atmospheres at room
temperature. Although the hydrogen absorption ceases within 1 hour,
the reduction must be continued for another 2 hours. The lactones of
ethyl isocitrate are obtained as described in Method A. The yield is 45 g
(90~ of theoretical value; B.P.2 135-140°).
threo- and erythro-Isocitric Lactoncs
Heat diethyl isocitrate (70 g) for 6 hours under reflux with 700 ml of
3 N hydrochloric acid. Concentrate the reaction mixture in vacua and
heat the residue in a boiling water bath under reduced pressure. A mix-
ture of threo- and erythro-isocitric lactones is obtained in a quantitative
yield.
Separation of threo- and erythro-Isocitric Lactones
Dissolve the mixture of laetones (4.3 g, 0.025 mole) in 43 ml of
water. To this solution, add 8.6 ml (8.4 g, 0.106 mole) of pyridine and
43 ml of 94~ ethanol. Refrigerate the solution overnight. Filter off
crystals of the pyridine salt of the erythro isomer; these are recrystallized
from 9 4 ~ ethanol. The yield is 1.4 g (22.4~). The pure salt melts at
139.5-141 °2
Dissolve the above salt (1.8 g) in 1.5 N aqueous sodium hydroxide
solution and concentrate it to dryness in vacua. Acidify the residue with
concentrated hydrochloric acid, approximately 3.2 ml, concentrate the
mixture to dryness in vacua, and heat it at 100 ° for 2 hours in vacua.
The resulting residue is extracted ten times with 30 ml portions of hot
ethyl acetate. Evaporate the combined extracts at 40--50° in vacua to
obtain pure erythro-isocitric lactone. The yield is 1.0 g (80%).
The filtrate obtained from the pyridine salt of the erythro isomer
contains threo-isoeitric lactone. It is made alkaline by adding 25 ml of
1.5 N sodium hydroxide and is then concentrated to dryness in vacua.
The residue is acidified with about 10 ml of concentrated hydrochloric
acid, the mixture is concentrated to dryness in vacua, and is heated in
vacua at 100 ° for 2 hours. The resulting residue is extracted ten times
*A. F. Senear, [ J . Am. Chem. Sac. 77, 2564 (1955)] reported a melting point of
138-140".
[77] ISOLATIONAND PROPERTIES OF HYDROXYCITRIC ACID 613
with 100 ml portions of hot ethyl acetate. The combined extracts are
evaporated to dryness in vacuo at 40-50 ° to yield pure threo-isocitric
lactone. The yield is 2.9 g (67.5~fl; B. P. 159.5-161°).
The threo- and erythro-isocitric lactones can be identified by means
of paper chromatography in the solvent system butanol-formic acid-
water ( 4 : 1 : 2 ) . The spots are detected by spraying with 0.05% thymol-
blue in 94% ethanol. The R I values are 0.55 and 0.60 for the threo and
erythro isomers, respectively.
Addendum
Isocitric acid was synthesized from sodium succinate and chloral, 4 and
by the reduction of ethyl oxalosuccinate with sodium amalgam. 5 threo-n~-
(--)-Isocitric lactone was separated in considerable amounts from leaves
of Bryophyllum calycinum by Pucher and Vickery, 6 and erythro-L,-(~-)-
isocitric lactone was separated from a culture of Penicillium purpulo-
genum var. rubrisclerotium Thom. No. 1148 by Sakaguchi. 7
R. Fittig and H. E. Miller, Ann. Chem. 25~ 43 (1889); H. A. Krebs and L. V.
Eggleston, Biochem. J. 38, 426 (1944); G. W. Pucher and H. B. Vickery, J. Biol.
Chem. 163, 169 (1946); H. P. Kato and S. R. Dickmann, Biochem. Prep. 3, 52
(1953).
~W. Wisleccnus and N. Nassauer, Ann. Chem. 285, 1 (1895); G. W. Pucher and H. B.
Vickery, J. Biol. Chem. 163, 169 (1946).
~G. W. Pucher, J. Biol. Chem. 145, 511 (1942); G. W. Pucher, M. D. Abrahams,
and H. B. Vickery, ibid. 172, 579 (1948).
~T. Beppu, S. Abe, and K. Sakaguchi, Bull. Agr. Chem. Soc. Japan 21, 263 (1957);
K. Sakaguchi and T. Beppu, Arch. Biochem. Biophys. 83, 131 (1959).
[77] Isolation and Properties of Hydroxycitric Acid

B y Y. S. LEWIS
Hydroxycitric acid (1,2-dihydroxypropane-l,2,3-tricarboxylic acid)
has two asymmetric centers, hence two pairs of diastereoisomers or four
different isomers (I, II, III, and IV) are possible. 1 Being a ~,-hydroxy
acid, it cyclizes readily to the corresponding lactone. The relative rates of
cyclization of the different isomers to the lactones are not known.
1The nomenclature for tile stereoisomers of hydroxycitric acid was kindly suggested
by Dr. H. B. Vickery, Connecticut Agricultural Experimental Research Station,
New Haven, Connecticut.
[77] ISOLATIONAND PROPERTIES OF HYDROXYCITRIC ACID 613
with 100 ml portions of hot ethyl acetate. The combined extracts are
evaporated to dryness in vacuo at 40-50 ° to yield pure threo-isocitric
lactone. The yield is 2.9 g (67.5~fl; B. P. 159.5-161°).
The threo- and erythro-isocitric lactones can be identified by means
of paper chromatography in the solvent system butanol-formic acid-
water ( 4 : 1 : 2 ) . The spots are detected by spraying with 0.05% thymol-
blue in 94% ethanol. The R I values are 0.55 and 0.60 for the threo and
erythro isomers, respectively.
Addendum
Isocitric acid was synthesized from sodium succinate and chloral, 4 and
by the reduction of ethyl oxalosuccinate with sodium amalgam. 5 threo-n~-
(--)-Isocitric lactone was separated in considerable amounts from leaves
of Bryophyllum calycinum by Pucher and Vickery, 6 and erythro-L,-(~-)-
isocitric lactone was separated from a culture of Penicillium purpulo-
genum var. rubrisclerotium Thom. No. 1148 by Sakaguchi. 7
R. Fittig and H. E. Miller, Ann. Chem. 25~ 43 (1889); H. A. Krebs and L. V.
Eggleston, Biochem. J. 38, 426 (1944); G. W. Pucher and H. B. Vickery, J. Biol.
Chem. 163, 169 (1946); H. P. Kato and S. R. Dickmann, Biochem. Prep. 3, 52
(1953).
~W. Wisleccnus and N. Nassauer, Ann. Chem. 285, 1 (1895); G. W. Pucher and H. B.
Vickery, J. Biol. Chem. 163, 169 (1946).
~G. W. Pucher, J. Biol. Chem. 145, 511 (1942); G. W. Pucher, M. D. Abrahams,
and H. B. Vickery, ibid. 172, 579 (1948).
~T. Beppu, S. Abe, and K. Sakaguchi, Bull. Agr. Chem. Soc. Japan 21, 263 (1957);
K. Sakaguchi and T. Beppu, Arch. Biochem. Biophys. 83, 131 (1959).
[77] Isolation and Properties of Hydroxycitric Acid

B y Y. S. LEWIS
Hydroxycitric acid (1,2-dihydroxypropane-l,2,3-tricarboxylic acid)
has two asymmetric centers, hence two pairs of diastereoisomers or four
different isomers (I, II, III, and IV) are possible. 1 Being a ~,-hydroxy
acid, it cyclizes readily to the corresponding lactone. The relative rates of
cyclization of the different isomers to the lactones are not known.
1The nomenclature for tile stereoisomers of hydroxycitric acid was kindly suggested
by Dr. H. B. Vickery, Connecticut Agricultural Experimental Research Station,
New Haven, Connecticut.
COOH COOH COOH

I I I
H--C--OH HO--C--H H--C--OH
I I I
H O O C -- C-- O H H O - - C -- C O O H H O -- C -- C O O H
i i I
H-- C - - C O O H H--C-- COOH H - - C -- C O O H
l l l
H H H
(z) (II) (rII)
COOH COOH COOH

I I l
HO--C-- H - - C--H C--H
l [ i f ,
HOOC - - C -- OH O HO--C--COOH O HOOC--C--OH
i I I
H--C--COOH H--C--C--O H--C--C-~-O
I i J
(IV) (v) (Vl)

(i) acid,
Ds Dg - H y d r o x y c i t r i c
erythro- Ds - h y d r o x y c i t r i c acid
(II) L s L g - H y d r o x y c i t r i c acid,
erythro - Ls- h y d r o x y c i t r i c acid
(III) DsLg- H y d r o x y c i t r i c acid,
threo-Ds-hydroxycitric acid
(IV) L s D g - H y d r o x y c i t r i c acid,
threo-Ls-hydroxycitric acid
(V) L a c t o n e f r o m acid (II)
(VI) L a c t o n e f r o m acid (IV)
Hydroxyeitrie acid was synthesized by Martius and Maue 2 from
trans-aconitic acid by a modification of the method of Pawollek. s It
was resolved chemically into its isomers. The natural occurrence of
isomer IV of hydroxyeitric acid in the calyxes of Hibiscus sabdari]~a
(Family Malvaceae) has been reported. '-8 More recently isomer (II)
was isolated from the fruit rind of Garcinia cambogia (Family Gutti-
2C. Martius and R. Maue, Z. Physiol. Chem. 269, 33 (1941).
3A. Pawollek, Ann. Chem. 178, 155 (1875).
' C. Griebel, Z. Lebensm. Untersuch.-Forsch. 77, 560 (1939)-(Chem. Abslr. 33, 7491).
C. Griebel, Z. Lebensm. Unters~ch.-Forsch. 83, 481 (1942)-(Chem. Abstr. 37, 4704).
6M. Bachstez, Ciencia Mex 9, 121 (1048); Chem. Abstr. 43, 7044 (1949).
[77] ISOLATIONAND pROPERTIES OF tIYDROXYCITRIC ACID 615
ferae).7.8 The exact stereochemistry of either the synthetic or naturally

occurring isomers is not known with certainty, and unequivocal methods
are necessary to establish this aspect beyond doubt? The stereochemical
structures (II and IV) suggested for the naturally occurring isomers are
based on the existing chemical evidence.
Hydroxycitric Acid from Garcinia

The acid is present to the extent of 20-30% in the dried fruit rinds 1°
of Garcinia cambogia, Garcinia atroviridis, and Garcinia indica, which
are used for culinary purposes and are available commercially in India. 11
The acid can be isolated from these materials in the form of its lactonc
by either of two methods. Method B is recommended, and Method A was
employed during the original isolation, s
Method A
Principle. Neutralization of concentrated extract of the acid in
alcohol with K O H results in the separation of the potassium salt as a
heavy oily liquid which is freed of impurities by repeated washing with
alcohol. Solutions of the purified potassium salt are converted to the free
acid on a cation exchange column. Evaporation of the aqueous solution of
the acid yields the lactone (V).
Procedure. Fruit rind of Garcinia cambogia (200 g) is autoclaved
with 600 ml of water at 115 ° for 15 minutes. The cooled extract (25-30 °)
is decanted through several folds of cheesecloth and filtered on a Bfichner
funnel (Whatman No. 1 paper) ; the residue is washed with water. The
dark brown filtrate (volume 600 ml) is concentrated to about 100 ml on
a water bath and treated with 200 ml of ethanol with stirring. The
resulting precipitate of pectinous material is removed either by centrif-
ugation or filtration. The acidic filtrate is neutralized (pH paper) by
cautious addition of 40% KOH, with careful stirring. The heavy oily
~Y. S. Lewis and S. Neelakantan, Current Sci. India 33, 82 (1964).
Y. S. Lewis and S. Neelakantan, Phytochemi~try 4, 619 (1965).
X-ray crystallographic studies with this goal are being pursued by collaborators of
the late Dr. Patterson at the Institute for Cancer Research, Philadelphia, Penn-
sylvania.
loThe commercially available fruit rinds of Garcirda cambogia are hard and dark
brown in color. They contain considerable amounts of sodium chloride added during
the curing procedure. Significant variations in the yield of hydroxycitric acid are
not encountered with different batches of the material, ttydroxycitric acid was
found to be the major organic acid present as determined by paper chromatographic
examination.
~1Dried fruit rinds of Garcinia cambogia were purchased through the Pepper Research
Station, Taliparamba, Kerala, India. For a description of Garcinia cambogia see
"The Wealth of India (Raw Materials)," Vol. IV, p. 99. Council Sci. Ind. Res.,
New Delhi, India, 1956.
liquid which is formed is allowed to settle for a few minutes and the
supernatant is decanted and discarded. The oily liquid is washed with
60% ethanol (five portions of 100 ml). It is next washed with absolute
alcohol (two portions of 100 ml), the suspension being left to stand for
60-90 minutes each time. A further portion of 100 ml of absolute ethanol
is added and allowed to stand overnight. Ethanol is decanted, and the
yellow (very hygroscopic) semisolid thus obtained is dried in vacuo at
80 ° to remove traces of ethanol and stored in a desiccator. The yield is
about 40 g. A 10% solution of the salt (ca. 5 g) is passed through a
column (20 X 3 cm) of cation exchange resin (e.g., Zeocarb-215). The
column is washed with water until free of acid and the effluent is evap-
orated to dryness on a water bath. (A considerable amount of colored
material is retained on the resin.) The residue is dried in a vacuum oven
at 100 ° for 8-10 hours. A crude crystalline mass is obtained after the
residue has stood in a desiccator for 7-8 days. Instead of being dried
in vacuo, the residue (a thick sirup) can be seeded with a few crystals of
the lactone to induce crystallization. The crude crystalline material (light
brown in color) is further purified by extraction and recrystallization
from ether.
Method B
Principle. The acid is extracted from the fruit rind with acetone. The
acetone extract is concentrated, and the acid is taken up in water. The
aqueous solution yields the crystalline lactone on evaporation.
Procedure. Fruit rind of Garcinia cambogia (1 kg) is kept immersed
in 1500 ml of acetone in a 4 liter flask and left overnight. It is reextracted
with an equal volume of acetone in a similar manner. Acetone is removed
from the combined extracts by distillation in vacuo. The viscous residue
is stirred with a liter of water (45-50°), and the material is filtered
through several folds of cheesecloth. The precipitated insoluble resinous
material is thus removed. The reddish brown filtrate (80-90 ° ) is treated
with activated charcoal (about 20 g) and concentrated to a thick sirup
(light brown in color) on a water bath. It is seeded with a few crystals
of the lactone and left overnight in a desiccator. A light brown, crystal-
line material is obtained. The yield is about 180 g. The material is
vigorously extracted with 3 liters of ether (ten portions of 300 ml), and
the combined extracts are dried over anhydrous sodium sulfate. A
considerable proportion of the color is ether insoluble. The extract is de-
colorized with activated charcoal if necessary. Ether is then removed by
distillation, and the sirupy mass thus obtained is heated as a thin layer
on a water bath (10-15 minutes) to remove traces of ether. This results
in a white solid. The yield is about 150 g.
[77] ISOLATION AND PROPERTIES OF HYDROXYCITRIC ACID 617
Purification
The lactone obtained by either of the above procedures is further
purified by extraction with ether (1 g/20 ml) in several portions. The
ether-soluble material is concentrated to one fourth its volume and an
equal portion of dry chloroform is added with stirring. Upon standing,
the lactone crystallizes as needles. The crystals are collected on a sintered
glass funnel and are dried in vacuo. The pure material thus obtained has
the properties described in the table.
Hydroxycitric Acid (IV) from Hibiscus

The acid is present in the calyxes of Hibiscus sabdarif]a. It is also
found in the acidic leaves of Hibiscus cannabinus and Hibiscus ]urcatus.
The best source for the isolation of the acid is the dried calyx of Hibiscus
sabdari]Ja. 1~ A sample obtained from a crop grown in India contained
28% (dry weight basis) of the acid. The procedure described for its
isolation is essentially similar to that of Griebel2 '~
Principle. The acid is extracted from the dried calyx powder with
acetone. Water is not recommended as mucilaginous material is also ex-
tracted which makes further processing difficult. The acetone extract is
concentrated, and the acid is taken up in water. It is precipitated as the
lead salt and after removal of lead is isolated as the tactone from aqueous
solution by concentration.
Procedure. Powdered calyxes of Hibiscus sabdarif]a (100 g) were
left immersed in 1 liter acetone at room temperature overnight. The sus-
pension was filtered on a Biichner funnel (Whatman No. 1) and most of
the acetone was removed by distillation in vacuo. The concentrate thus
obtained was stirred with 500 ml of water and filtered on a Biichner
funnel (Whatman No. 1 paper). The filtrate was treated with about 10 g
of activated charcoal and filtered. The solution was neutralized (pH
paper) with a saturated solution of neutral lead acetate. The insoluble
lead salts were collected by eentrifugation and washed thoroughly with
water to remove any excess lead acetate. A fine suspension of this material
in water was treated with hydrogen sulfide. The lead sulfide was filtered
off and the filtrate was evaporated to a sirupy consistency on a water
bath. The semisolid material was kept in a desiccator for 2 or 3 days;
during this time it solidified to a crystalline mass. The yield was about
20 g. Further purification was achieved by preparing a saturated solution
of this material in ether; during this process impurities are eliminated.
~'~For a description of the plant, see J. M. Watt and M. G. Breyer Bradwyic, in
"Medicinal and Poisonous Plants of Southern and Eastern Africa," p. 787. Living-
stone, London, 1962.
Addition of an equal volume of chloroform to the ether solution results in
the crystallization of the product.
Properties
T h e pure lactones (V a n d VI) from acids I I a n d I V are hygroscopic,
b u t are s t a b l e a t room t e m p e r a t u r e when k e p t in a desiccator. T h e i r
general properties are listed in the table. Both the lactones form c r y s t a l -
PROPERTIES OF HYDROXYCITRIC ACID LACTONES
Hydroxycitric acid Hydroxycitric acid

lactone (¥), lactone (VI),
Property Hibiscus lactone Garcinia lactone
Melting point 183° 178°

+122 ° +100 °
For free acidb +31 ° --20 °
After saturation with Boraxc +31 ° - 920
Crystal shape Needles Needles

Hygroscopicity High Slight
Solubility Very soluble in water Very soluble in alcohol
and alcohol; slightly and water; fairly
soluble in ether soluble in ether
Susceptibility to purified threo- Negative Negative
D.-isocitrate: NADP oxido-
reductase (decarboxylating)
(EC 1.1.1.42), pig heart
Paper chromatography
R! valuesd
BFW: Acid 0.15 0.24
Lactone 0.39 0.42
PAW: Acid 0.35 0.36
Lactone 0.26 0.26
Metavandate sprays
Acid spot Yellow Reddish orange
Lactone spot Yellow Yellow
Quinine salt, m.p. 227° decomp. 215° decomp.
Two percent solution in water. The optical rotatory dispersion curves of both
lactones indicate a positive Cotton effect with a maximum at 233 n ~ (W. Klyne,
personal communication).
b The lactones are warmed with sufficient equivalents of aqueous KOH for complete
conversion to the tripotassium salt and passed through Zeocarb-215. Measurements
of rotation and total acid are made on the column effluents.
, Solutions of the acids, obtained as described in footnote b, are saturated by addition
of solid Borax, and the rotations are measured. The values mentioned are not
absolute but give an indication of the configuration.
BFW: n-butanol-90% formic acid-water (4:1:5), Whatman No. 2 paper.
PAW: n-propanol-ammonia-water (6:3:1), Whatman No. 2 paper.
• H. A. Stafford, Am. J. Botany 46, 347 (1959).
[78] HOMOCITRIC~HOMOACONITIC~ AND HOMOISOCITRIC ACIDS 619
line calcium salts (CaC~H~07"4 H20). The lactones give a positive test
with hydroxylamine and can be assayed (0.1-1 /A~/) as their hydroxa-
mates by a slight modification13 of the Lipmann-Tuttle procedure. 14
Hydroxyeitrie acid (IV) can be determined by a modification1~ of the
metavanadate procedure. 15 Some preliminary work on the microbial
metabolism of hydroxycitric acid (II) has recently been reported. 16
The structures predicted for the H i b i s c u s and Garcinia acids s have
now been verified and found correct by X-ray diffraction studies carried
out by Dr. Jenny Glusker, The Institute for Cancer Research, Phila-
delphia, Pennsylvania (private communication).
Acknowledgment
The author is indebted to Dr. P. R. Krishnaswamyand Dr. D. Rajagopala Rao
for their assistance in the preparation of this article.
,3Unpublished procedure of D. R. Rao, 1965.
"F. Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945).
"J. R. Matchett, R. R. Legavit, C. C. Nimmo, and G. K. Notter, Ind. Eng. Chem.
36, 851 (1944).
"D. Rajagopal Rao and M. Ramakrishna, Biochem. Z. 344, 399 (1966).
[78] P r e p a r a t i o n of H o m o c i t r i c , H o m o a c o n i t i c ,
a n d Homoisocitric Acids
B y ANTHONYF. TuccI, Lovls N. CECI,and
JNANENDRA K. BHATTACHARJEE
Principle
The methods for the preparation of homocitrie and homoaconitic acids
are based on the work of Strassman and co-workers?,2 The synthesis of
homoisocitric acid is based on the method of Yamashita2 Homoisocitric
acid (I) is obtained by reduction of triethyl 2-oxaloglutarate, prepared
by condensation of diethyl glutarate with diethyl oxalate in the presence
of sodium ethoxide. The free acid is obtained from the triester of homo-
isocitrie acid by hydrolysis. Homoaconitic acids (II) are prepared by
dehydrating triethyl homoisoeitrate with acetyl chloride, followed by
hydrolysis to the free acids. The cis and t rans isomers of homoaconitic
acid may be separated by anion-exchange chromatography; t r a n s - h o m o -
' M. Maragoudakis and M. Strassman, 3. Biol. Chem. 241, 695 (1966).

' M. Stras~aan and L. N. Ceci, J. Biol. Chem. 241, 5401 (1966).
s M. Yamashita, J. Org. Chem. 23, 835 (1958).
[78] HOMOCITRIC~HOMOACONITIC~ AND HOMOISOCITRIC ACIDS 619
line calcium salts (CaC~H~07"4 H20). The lactones give a positive test
with hydroxylamine and can be assayed (0.1-1 /A~/) as their hydroxa-
mates by a slight modification13 of the Lipmann-Tuttle procedure. 14
Hydroxyeitrie acid (IV) can be determined by a modification1~ of the
metavanadate procedure. 15 Some preliminary work on the microbial
metabolism of hydroxycitric acid (II) has recently been reported. 16
The structures predicted for the H i b i s c u s and Garcinia acids s have
now been verified and found correct by X-ray diffraction studies carried
out by Dr. Jenny Glusker, The Institute for Cancer Research, Phila-
delphia, Pennsylvania (private communication).
Acknowledgment
The author is indebted to Dr. P. R. Krishnaswamyand Dr. D. Rajagopala Rao
for their assistance in the preparation of this article.
,3Unpublished procedure of D. R. Rao, 1965.
"F. Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945).
"J. R. Matchett, R. R. Legavit, C. C. Nimmo, and G. K. Notter, Ind. Eng. Chem.
36, 851 (1944).
"D. Rajagopal Rao and M. Ramakrishna, Biochem. Z. 344, 399 (1966).
[78] P r e p a r a t i o n of H o m o c i t r i c , H o m o a c o n i t i c ,
a n d Homoisocitric Acids
B y ANTHONYF. TuccI, Lovls N. CECI,and
JNANENDRA K. BHATTACHARJEE
Principle
The methods for the preparation of homocitrie and homoaconitic acids
are based on the work of Strassman and co-workers?,2 The synthesis of
homoisocitric acid is based on the method of Yamashita2 Homoisocitric
acid (I) is obtained by reduction of triethyl 2-oxaloglutarate, prepared
by condensation of diethyl glutarate with diethyl oxalate in the presence
of sodium ethoxide. The free acid is obtained from the triester of homo-
isocitrie acid by hydrolysis. Homoaconitic acids (II) are prepared by
dehydrating triethyl homoisoeitrate with acetyl chloride, followed by
hydrolysis to the free acids. The cis and t rans isomers of homoaconitic
acid may be separated by anion-exchange chromatography; t r a n s - h o m o -
' M. Maragoudakis and M. Strassman, 3. Biol. Chem. 241, 695 (1966).

' M. Stras~aan and L. N. Ceci, J. Biol. Chem. 241, 5401 (1966).
s M. Yamashita, J. Org. Chem. 23, 835 (1958).
aconitic acid is eluted first from a column of Dowex 1-formate according

to the method of Busch et al?
Homocitric acid (HI) is prepared by treating diethyl B-ketoadipate
with hydrogen cyanide, followed by hydrolysis of the cyanohydrin to the
free acid. The intermediate diethyl B-ketoadipate is prepared by con-
densing magnesium malonic ester with p-carbethoxypropionyl chloride
followed by thermal decomposition of diethyl p-keto-a-carbethoxyadipate
in the presence of fl-naphthalenesulfonic acid.
HOCH--COOH HC--COOH I-I~7--COOH

I II
CH--COOH C--COOH HOC--COOH
I i i
I t I
CI"I2 CH 2 CH2
f I I
COOH COOH COOH
Homoisocitric Homoaconitic Homocitric

acid acid acid
{I) (~) (III)
Procedure
H omocitric Acid
Preparation o] Diethyl D-Ketoadipate? The preparation of diethyl
fl-keto-a-carbethoxyadipate via the condensation of magnesium malonic
ester with fl-carbethoxypropionyl chloride is described in Vol. III [88].
To 220 g of diethyl fl-keto-a-carbethoxyadipate is added 14 g of p-naph-
thalene sulfonic acid monohydrate. The reaction mixture is heated slowly;
gas evolution occurs at 130-140 °, and heating is continued to 190-200 °,
until effervescence ceases. The mixture is cooled, and ether is added (100
ml). The ether solution is washed with four portions of cold 10~ sodium
carbonate solution. The combined aqueous washes are extracted once
with ether. The combined ether solutions are washed successively with
water, M sulfuric acid, and water, and then dried with anhydrous sodium
sulfate. The carbonate solution is acidified and extracted with ether. This
ether extract contains approximately 50 g of unreacted diethyl fl-keto-a-
carbethoxyadipate, which may be treated again with 5 g of D-naphthalene
' tI. Busch, R. B. Iiurlbert, and V. R. Potter, J, Biol. Chem. 196, 717 (1952); and
It. Busch, Vol. III [70].
BB. ltiegel and W. M. Lilienfeld, ]. Am. Chem. Soe. 67~ 1273 (1945).
[78] HOMOCITRIC,HOMOACONITIC, AND HOMOISOCITRIC ACIDS 621
sulfonic acid and worked up as described above. The ether layers are
combined and fractionated through a 30 cm Vigreux column at a pressure
of 0.5 mm Hg after removal of ether. The forerun, b.p. 80-90 °, consists
of diethyl succinate and diethyl malonate and is discarded. The main
fraction distills at 122-126 ° at 0.5 mm Hg and is relatively pure (95%)
diethyl/~-ketoadipate (86 g, 40% yield).
Preparation of Homocitric Acid. The diethyl fl-ketoadipate obtained
above is dissolved in 300 ml of ether to which is added 30 ml of water
and 45 g of finely powdered potassium cyanide. The mixture is stoppered
and cooled in an ice bath for 10 minutes. Then 60 ml of concentrated
hydrochloric acid is added in 5 ml portions with vigorous shaking over a
period of 2 hours. The reaction mixture is kept at room temperature over-
night, the ether layer is separated, and the aqueous layer extracted 4
times with 50 ml portions of ether. The combined ether solutions are
dried with anhydrous sodium sulfate and evaporated. The residue is
dissolved in 125 ml of concentrated hydrochloric acid and heated on a
steam bath for 6 hours. After cooling, the mixture is filtered to remove
ammonium chloride. The filtrate is evaporated to dryness under reduced
pressure and the residue is dissolved in 100 ml of hot ethyl acetate. The
remaining insoluble ammonium chloride is filtered off. Evaporation of
the ethyl acetate yields a viscous residue of homocitric acid (72 g). To
crystallize, the residue is dissolved in water, the pH is adjusted to 1 with
concentrated sulfuric acid and continuously extracted with ether for 2
days, fresh ether being added after 24 hours. The combined ether extracts
are evaporated to dryness, and the residue is crystallized from hot ethyl
acetate, yielding homocitric lactone, m.p. 160-162 ° (70 g, 93% yield).
The salt of the free tricarboxylic acid is obtained by heating the lactone
in excess base.
Homoisocitric acid
Preparation o] Triethyl ~-Oxaloglutarate. To an ice-cooled stirred
suspension of freshly made sodium ethoxide (34 g) in 370 ml of anhydrous
ether is added 73 g of diethyl oxalate; when nearly all the ethoxide is
dissolved, 94 g of diethyl glutarate is added over 5 minutes with con-
tinued stirring and cooling until the solution becomes clear (the color
turns from yellow to red). The mixture is kept at 0-5 ° for 3 days, then
poured onto ice. The ether solution is separated, and the aqueous layer
is washed with ether. The aqueous solution is acidified with cold M
sulfuric acid, and then extracted with ether. The ether solution is shaken
with 1 g of barium carbonate, dried with anhydrous sodium sulfate, and
filtered. Evaporation of the ether filtrate yields 120 g (81% yield) of a
viscous, light yellow residue of triethyl fl-oxaloglutarate.
622 PREPARATION OF COMPOUNDS [?8]
Preparation o] Homoisocitric Acid. Thirty grams of triethyl/~-oxalo-

glutarate, 500 ml of 95% ethanol, and 180 mg of platinum dioxide are
shaken with hydrogen at 45 psi pressure for 16 hours. The mixture is
filtered, and the filtrate is evaporated under reduced pressure. The residue
is dissolved in 500 ml of ether, washed 3 times with 50 ml portions of 10%
potassium carbonate, dried over anhydrous sodium sulfate, and evapo-
rated under reduced pressure to yield 28 g of a residual oil having a
fruitlike odor. On distillation, the colorless product (b.p. 140-141 ° at
0.005 mm Hg) triethyl homoisocitrate is collected. The triethyl homo-
isocitrate is refluxed for 2 hours with 262 ml of 0.9 N sodium hydroxide
(about an equivalent amount). Then the solution is acidified with
hydrochloric acid to pH 1 and continuously extracted with ether for 5
days; the ether extract is evaporated to dryness under reduced pressure.
The residue is dissolved in water and evaporated to dryness again to
remove traces of hydrochloric acid. The residual sirup, stored over
phosphorus pentoxide in vacuo, solidifies. Homoisocitric acid is crystal-
lized from acetone-benzene (m.p. 127-129 °, 81% yield).
Homoaconitic Acid
Triethyl homoisoeitrate (140 g, 0.48 mole), obtained as described
above, is placed in a flask immersed in ice. One hundred milliliters of
freshly distilled aeetyl chloride is added, the flask is stoppered and
shaken vigorously, a condenser fitted with a drying tube is inserted, and
the flask is heated to reflux for 3 hours. The reaction mixture is rapidly
cooled to 20 °, an additional 75 ml of acetyl chloride is added, and
refluxing resumed for an additional 3 hours. The reaction mixture is
cooled, and volatile material removed by evaporation under reduced
pressure, followed by distillation at a pressure of 0.5 mm Hg at 70 °,
yielding a viscous yellow residue.
The mixture of triesters is hydrolyzed by refluxing with 400 ml of 5 N
sodium hydroxide for 1 hour. The solution is neutralized by the addition
of 6 N hydrochloric acid and treated repeatedly with 5 g portions of
activated charcoal. After removal of the charcoal, the solution is evapo-
rated to dryness, leaving a light yellow glassy residue. The residue is
dissolved in 300 ml of water with warming, the solution adjusted to pH
1 with concentrated hydrochloric acid, and is continuously extracted with
ether for 24 hours. The extraction is interrupted after 3 hours and again
after 10 hours. The 0-3 hour ether extract contains 18 g of homoaconitic
acid (predominantly the trans isomer) and the 3-10 hour extract contains
15 g of a mixture of c/s-homoaconitic acid (approximately 3 g), trans-
homoaconitie acid, and approximately 11 g of homoisocitric acid. The
last ether extract and the aqueous phase contains predominantly un°
reacted homoisoeitric acid.
[79] FLUORO ANALOGS 623
cis-Homoaconitic acid can be obtained in pure form by chromatog-

raphy of the 3-10 hour ether extract on a column of Dowex 1-formate:
Five grams of the residue are dissolved in 10 ml of water and neutralized
to pH 7 by addition of 5 N sodium hydroxide. The solution is placed on a
Dowex 1-formate column (3.5 X 40 cm) and the column is eluted by
gradient elution with 6 N formic acid flowing into a mixing flask con-
taining 400 ml of water. Fractions of 10 ml are collected. Homoisocitric
acid is eluted in fractions 90-130, trans-homoaconitic acid in fractions
225-275, and cis-homoaconitic acid in fractions 350-450. Both c/s- and
trans-homoaconitic acids can be crystallized from ethyl acetate-benzene.
t~'ans-Homoaconitic acid melts at 148-149 °, c/s-homoaconitic acid at
80-81 °. cis-Homoaconitic acid forms an anhydride more readily than
trans-homoaeonitic acid and may be separated from trans-homoaconitic
acid by sublimation of the anhydride at 80 ° at a pressure of 1 t~ Hg.
Alternative Procedures
Optically active homocitric acid has been isolated from the culture
medium of a lysine-rcquiring yeast mutant'; its enantiomorph, ( + ) -
homocitric acid lactone, has been synthesized by oxidation of (--)-qumic
acid to (--)-5-dehydroquinic acid, reduction of the product to (--)-5-
deoxyquinic acid, and oxidation of the latter with periodate followed by
bromine: Natural isomers of all three acids, homocitric, cis-homoaconitic,
and homoisocitric acids have been isolated from the culture medium of a
single lysine-requiring yeast mutant:
~U. Thomas, M. G. Kalyanpur, and C. M. Stevens, Biochemistry 5, 2513 (1966).
~J. K. Bhattacharjee and M. Strassman, J. Biol. Chem. 242, 2542 (1967).
[ 7 9 ] C h e m i c a l P r o p e r t i e s a n d S y n t h e s i s of F l u o r o A n a l o g s of
C o m p o u n d s R e l a t e d t o S u b s t r a t e s of t h e Citric A c i d C y c l e 1
By ERNEST KUN TM a n d ROBERT J. DUMMEL
I. Introduction 624
II. General Chemistry of Aliphatic Fluorocarboxylic Acids 624
A. Stability of the C-F Bond in Fluorocarboxylic Acids 626
1The work reported in this review was supported by research grants from the U.S.
Public Health Service (RO1 HD-01239-10 and R01 CA-07955-03). We are grateful
for the criticism of Professor Warren D. Kumler, and for the skillful assistance of
Miss Karen Louise Karvonen in assembling the compendium and references, and
for her and Miss Tina Moya's help with preparation of the manuscript.
~'Recipient of the Research Career Award of the United States Public Health
Service.
cis-Homoaconitic acid can be obtained in pure form by chromatog-

raphy of the 3-10 hour ether extract on a column of Dowex 1-formate:
Five grams of the residue are dissolved in 10 ml of water and neutralized
to pH 7 by addition of 5 N sodium hydroxide. The solution is placed on a
Dowex 1-formate column (3.5 X 40 cm) and the column is eluted by
gradient elution with 6 N formic acid flowing into a mixing flask con-
taining 400 ml of water. Fractions of 10 ml are collected. Homoisocitric
acid is eluted in fractions 90-130, trans-homoaconitic acid in fractions
225-275, and cis-homoaconitic acid in fractions 350-450. Both c/s- and
trans-homoaconitic acids can be crystallized from ethyl acetate-benzene.
t~'ans-Homoaconitic acid melts at 148-149 °, c/s-homoaconitic acid at
80-81 °. cis-Homoaconitic acid forms an anhydride more readily than
trans-homoaeonitic acid and may be separated from trans-homoaconitic
acid by sublimation of the anhydride at 80 ° at a pressure of 1 t~ Hg.
Alternative Procedures
Optically active homocitric acid has been isolated from the culture
medium of a lysine-rcquiring yeast mutant'; its enantiomorph, ( + ) -
homocitric acid lactone, has been synthesized by oxidation of (--)-qumic
acid to (--)-5-dehydroquinic acid, reduction of the product to (--)-5-
deoxyquinic acid, and oxidation of the latter with periodate followed by
bromine: Natural isomers of all three acids, homocitric, cis-homoaconitic,
and homoisocitric acids have been isolated from the culture medium of a
single lysine-requiring yeast mutant:
~U. Thomas, M. G. Kalyanpur, and C. M. Stevens, Biochemistry 5, 2513 (1966).
~J. K. Bhattacharjee and M. Strassman, J. Biol. Chem. 242, 2542 (1967).
[ 7 9 ] C h e m i c a l P r o p e r t i e s a n d S y n t h e s i s of F l u o r o A n a l o g s of
C o m p o u n d s R e l a t e d t o S u b s t r a t e s of t h e Citric A c i d C y c l e 1
By ERNEST KUN TM a n d ROBERT J. DUMMEL
I. Introduction 624
II. General Chemistry of Aliphatic Fluorocarboxylic Acids 624
A. Stability of the C-F Bond in Fluorocarboxylic Acids 626
1The work reported in this review was supported by research grants from the U.S.
Public Health Service (RO1 HD-01239-10 and R01 CA-07955-03). We are grateful
for the criticism of Professor Warren D. Kumler, and for the skillful assistance of
Miss Karen Louise Karvonen in assembling the compendium and references, and
for her and Miss Tina Moya's help with preparation of the manuscript.
~'Recipient of the Research Career Award of the United States Public Health
Service.
B. The Effect of Fluoro Substitution on Neighboring Functional Groups 628

C. Stereochemistry 633
III. Organic Reactions Applied in the Synthesis of Fluoro Aliphatic Esters
and Acids 635
A. General Methods for the Introduction of Fluorine 635
B. General Methods of Synthesis from Fluoro Aliphatic Esters 635
IV. Specific Procedures 638
A. Saturated Acids 638
B. Unsaturated Acids . 647
C. Hydroxy Acids 649
D. Keto Acids 658
V. Compendium of Fluoro Aliphatic Acids and Esters 672
I. Introduction
This chapter deals primarily with propertie~ and synthesis of certain
fuorine-containing carboxylic acids. Trifluoromethyl and other polyfluoro
acids are not included in this review. I t would be inappropriate to at-
tempt an exhaustive review of fluoro organic compounds, partly because
excellent comprehensive coverage of the general chemistry of fluoro com-
pounds is available in monographsY -4 I t would be equally faulty to
describe merely synthetic procedures because the chemistry of fluoro
organic compounds is a difficult specialty requiring considerable insight
into mechanisms leading to possible side reactions. The use of fluoro in-
hibitors in enzymology and related fields is restricted presently to a few
available substances, but the need for more specific inhibitors is likely
to increase. Evaluation of potential modes of action of untried substances
or attempts to synthesize fluorocarboxylic acids, requires knowledge of
reaction mechanisms in order to prevent failures and erroneous experi-
mental designs. For these reasons, a summary of certain principles of
fluoro chemistry specifically related to carboxylic acids--a subject
presently not available in the literature is considered to be as important
as reliable laboratory procedures. Therefore an abridged survey of or-
ganic chemical mechanisms related both to chemical behavior of fluoro
carboxylic acids and to their syntheses has been included.
II. General Chemistry of Aliphatic Fluorocarboxylic Acids

The effects of surrounding groups on the reactivity of the C-F bond
and the influence of fluoro substitution on neighboring groups such as H,
z F. L. M. Pattison, "Toxic Aliphatic Fluorine Compounds." Elsevier, Amsterdam,

1959.
8M. I-Iudlicky, "Chemistry of Organic Fluorine Compounds." Macmillan, New York,
1962.
' R. E. Banks and It. Goldwhite, in "Handbook of Experimental Pharmacology" (0.
Eichler et al., eds.), Vol. 20, p. 1. Springer, New York, 1966.
[79] FLVORO ANALOGS 625
C=O, OH, 1NH2, or C 0 0 H attached to the same aliphatic chain will be

briefly discussed.
In aliphatic hydrocarbons the covalent C-F bond is comparable to
the C-H bond and is relatively stable, as predicted from the single
electron deficiency in the ls orbital of hydrogen or in the 2p2 orbital of
fluorine. As a first approximation, the effects of fluorine substitution in an
aliphatic molecule are predictable from the well-known electron with-
TABLE I
TAFT CONSTANTS a FOR ALIPHATIC ACIDS
Substituent R a* Substituent R a*
H-- 0.49 HOOC-- 2.08

CH,-- 0.00 F,C-- 2.61
C1CH2-- 1.05 CI3C-- 2.65
FCH2-- 1.10 CF,--CO-- 3.7
C12CH-- 1.95 NO2-- 4.0
CH,O--CO-- 2.0
F2CH-- 2 05
° For substituted formic acids, R--OOOH, acid dissociation can be calculated from
the following equation: pKo = 4 . 6 6 - 1.62a*. For substituted acetic acids,
R--CH2COOH: pK~ = 5.16 - 0.73~*. [G. B. Barlin and D. D. Perrin, Quart.
Rev. 18, 3 (1964); ibid. 20, 75 (1966).]
drawing properties of fluorine, which influence its vicinity by a strong

negative inductive effect of the same order of magnitude as chlorine. The
inductive effect, as measured by the acidity of carboxylic acids, is com-
pared to that of other groups in Table I. It is sufficient to account for
differences in reactivity previously attributed to hyperconjugation2 How-
ever, fluorine can serve as an electron donor also in resonance interaction
with neighboring unsaturated systems, e as shown in Formula (1).
:F--C--O ~ : F - - C - - (+)
"" l "" I
R R
+
:F--C--C--R ~ ~ :F--C--C--R
• " I I "" I l
H H H H
T h e properties of fluorocarboxylic acids are e n t i r e l y d e p e n d e n t u p o n

their specific s t r u c t u r e . A l t h o u g h g e n e r a l i z a t i o n s a b o u t the effects of
A. Streitweiser, Jr. and D. Holtz, J. Am. Chem. Soc. 89, 692 (1967).
• R. D. Chambers and R. H. Mobbs, Advan. Fluorine Chem. 4, 50 (1965).
neighboring groups, based on analogies derived from fluoro hydrocarbons,

are somewhat artificial, they m a y serve as an approximate guide in
predicting properties of substituted fluoro carboxylie acids.
A. Stability of the C - F B o n d in Fluoroearboxylic Acids

The C - F bond in fluoroalkanes is considerably more stable to dis-
p l a c e m e n t reactions than the C-C1 bond. Nucleophilic displacement of
chlorine occurs 10 -° to 106 times faster than t h a t of C - F from a compara-
ble site. 7 As in aliphatic fluorohydrocarbons, electron withdrawing groups
on the same carbon atom (e.g., another F) increase the stability of C - F
bonds (as in the case of geminal dihalo, or geminal halo-fluorohydro-
carbons). Geminal difluoro acids are in general more resistant to de-
fluorination than corresponding monofluoro acids. In the case of fluoro-
malonic acids, nonionized carboxyls stabilize the C - F bond, similarly to
other electrophilic substituents. However, under strongly basic conditions
when carboxylate ions act as electron donors, F in fluoromalonic acids
is likely to be displaced by OH-. Electron attracting groups on a carbon
a t o m adjacent to the C - F bond diminish stability and favor elimination
of H F ; e.g., vicinal difluoro or fluoro-halo acids are much less stable than
TABLE II
DECOMPOSITION OF FLUORO CARBOXYLIC AcIDs BY NUCLEOPHILIC DISPLACEMENT
Compound Reagent Product reported Reference
Ethyl fluoroacetate Hydroxide (Fluoride) a

Ethyl fluoroacetate Hydrazine (Fluoride) b
Ethyl fiuoromalonate Hydrazine (Fluoride) b
Fluoroacetic acid Enzymatic Glycolic acid c
catalysis -[- fluoride
2-Fluorocrotonic h Hydroxide 2-Ketobutyric d
2-Fluoro-2-hexenoic Hydroxide 2-Ketohexanoic d
v-Fluoroacetoacetic acids Acid ~-Dihydroxyethylene cmpd. e
Trifluoromethylacrylic acid h Base Methylenemalonic f
2-Trifluoromethylpropionic acid ^ Base Methylmalonic f
2-Amino-3-fluoroisobutyric acid pH 5-6 2-Amino-3-hydroxy g
isobutyric acid
" B. C. Saunders and G. J. Stacey, J. Chem. Soc. p. 1773 (1948).
b F. I. Abezgauz, S. V. Sokolov, and G. P. Udilov, Zh. Obshch. Khi~n. $4, 2695 (1964).
P. Goldman, J. Biol. Chem. 240, 3434 (1965).
d E. D. Bergmann and I. Shahak, J. Chem. Soc. p. 4003 (1961).
[o H. Machleidt, Ann. Chem. 667, 24, 35 (1963).
f -~I. W. Buxton, M. Stacey, and J. C. Tatlow, J. Chem. Soc. p. 366 (1964).
E. D. Bergmann and A. Shani, J. Chem. Soc. p. 3462 (1963).
h May involve nucleophilic addition followed by elimination.
~R. E. Parker, Advan. Fluorine Chem. 3, 63 (1963).
corresponding monofluoro acids. The critical role of the position of C-F

with respect to stability in a fluorocarboxylic acid is illustrated by
fluoropropionic acids. Owing to induction by F, a-fluoropropionic acid is
a stronger acid than fl-fluoropropionic acid. Consequently carboxyl is
present more in the ionized form at constant pH. The carboxylate ion, by
virtue of its electron donating property, t~nds to labilize C-F toward
nucleophilic displacement. On the other hand, in fl-fluoropropionic acid,
the inductive effect of F results in increased acidity of the a - H , and
elimination of HF even under acidic conditions. A carbonyl group plays
a particularly interesting role when vicinal to a C-F bond. The inductive
effect of the carbonyl group should weaken the C-F bond, but the
inductive effect of the fuorine weakens the carbonyl double bond, which
facilitates earbonyl reactions such as hydration and favors the keto form.
In base, the fluorine is stabilized on the enolate ion by resonance. Ex-
amples of decomposition of fluorocarboxylic acids by nucleophilic dis-
placement are shown in Table II.
TABLE III
REACTIONS ~LLUSTIL~TING ELIMINATION OF FLUORINE
Compound Conditions Products reported Reference
3-Fluoropropionic acid Base Acrylic Cf. a

3,3-Difluorobutyric acid Base 3-Fluorocrotonic b
4-Fluorobutyric ester Base Lactone ~ c
4-Fluoro-5-oxohexanoic ester Base Lactone k d
5-Fluorovaleric ester Base Lactone ~ c
4-Fluoro-2-alkylacetoacetic ester Base Tetronic lactone k d
7-Fluoroheptanoic acid Distillation 6-Heptenoic e
Fluorosuccinic ester NH4OH Maleic, fumaric amide f
2,3-Difluorosuccinic acid Water Acetylenedicarboxylic g
3-Fluoroaspartic acid (enzymatic pH 7.4 Oxaloacetic -t- NH3 h
intermediate, not isolated)
2-Carboxy-3-fluoroaspartic ester Conc. HC1 Inorganic salts i
2-Amino-2-carboxy-4-fluorobutyric acid Conc. H F Unidentified product j
a F. L. M. Pattison and R. A. Peters, in "Handbook of Experimental Pharmacology"

(O. Eichler et al., eds.), Vol. XX, p. 407. Springer, New York, 1966.
b A. L. Henne and W. J. Zimmerscheid, J. Am. Chem. Soc. 69, 281 (1947).
c F. L. M. Pattison, S. B. D. Hunt, and J. B. Stothers, J. Org. Chem. 21, 883 (1956).
d H. Machleit, Ann. Chem. 667, 24, 35 (1963).
• F. J. Buckle, F. L. M. Pattison, and B. C. Saunders, J. Chem. Soc. p. 1471 (1949).
l E. D. Bergmann and S. Szinai, J. Chem. Soc. p. 1521 (1956).
M. S. Kharasch, U.S. Patent 2,426,244 (1947).
h E. Kun, D. W. Fanshier, and D. R. Grassetti, J. Biol. Chem. 235, 416 (1960).
M. Hudlicky, Collection Czech. Chem. Comm~llt. 26, 1414 (1961).
i M. S. Raasch, J. Org. Chem. 23, 1567 (1958).
k May be displacement.
In general, cleavage of the C-F bond by nucleophilic displacement
is much less common than loss of F as HF by elimination (Table III).
Eliminations complicate the synthesis of many 2-fluorocarboxylic acids
and may cause explosions of stored fluoro esters2 Both electron attracting
and electron donating groups on carbon atoms adjacent to the C-F group
favor elimination. This effect may extend to the D-carbon. Examples of
effects of vicinal electron donating groups are less than those of electron-
attracting substitutions. A vicinal amino group (an electron donor),
such as occurs in fluoroaspartate, introduces extreme instability, and this
fluoroamino acid exists only as an enzymatic intermediate2 Another ex-
ample of H F elimination is that which follows the addition of fluoro-
pyruvate to thiol groups, forming a hemi-mercaptal. 1° This type of effect
is probably responsible for the fact that fluoro amino acids containing
F vieinal to the amino group have not thus far been isolated as the free
acids. Elimination is generally favored in fluorocarboxylic acids, but
displacement may occur by way of lactone formation in carboxylic acids
containing F in the 4 or 5-position. 11
The fate of the C-F bond in hydroxyfluorocarboxylie acids is inti-
mately connected with stability of the OH group. For example, under
basic conditions (pit above 10.0) monofluoromalic acid slowly eliminates
HF, yielding epoxysuceinie rather than hydroxymaleic acid. ~ The mech-
anism of the intramolecular displacement is related to the inductive effect
of F, rendering the H on OH more acidic. Intramolecular hydrogen
bonding also contributes to the acidity of the hydroxyl.
2-Fluoro-2-alkenoic acids are converted by hydroxide to 2-keto
acids. ~s The fuorine inductively favors hydroxide addition at the 2-
position, and this unstable structure eliminates F-, giving the enol.
lB. The Effect of Fluoro Substitution on Neighboring Functional Groups

The simplest and best-known effect of the fluoro group on neighboring
groups is due to its electron withdrawal by negative induction. This ren-
ders adjacent H atoms more acidic than in corresponding F-free mole-
cules (Table I). The effect is apparent in both dissociation of the
carboxyl group and adjacent acidic C-tI bonds.
It is of importance to consider the effect of F substitution on adjacent
enolizable earbonyl groups (Table IVa). In a nonaqueous medium esters
s F. L. M. Pattison and R. A. Peters, in "Handbook of Experimental Pharmacology"
(0. Eichler et al., eds.), Vol. 20, p. 407. Springer, New York, 1966.
~E. Kun, D. W. Fanshier, and D. R. Grassetti, J. Biol. Chem. 235~ 416 (1960).
I°E. D. Bergmann and A. Mielewitz, I. Chem. Soc. p. 3736 (1963).
11F. L. M. Pattison, S. B. D. Hunt, and J. B. Stothers, J. Org. Chem. 21, 883 (1956).
~A. I. Krasna, J. Biol. Chem. 236, 749 (1961); ibid. 237, 1418 (1962).
u E. D. Bergmann and I. Shahak, J. Chem. Soc. p. 4003 (1961).
TABLE IVa
ENOLIZ.~TION OF OXALOACETICESTERS
Percent Percent
Ester Enol (NMR) Enol (titration) Reference
Diethyl oxaloacetate 79 a
50 (methanol) 72-79 (hexane) a,b

Diethyl chlorooxaloacetate 24-27 (hexane) b
Diethyl fluorooxaloacetate 0 8-9 (hexane) a,b
W. D. Kumler, E. Kun, and J. D. Shoolery, J. Org. Chem. 27, 1165 (1962).

b I. Blank, J. Mager, and E. D. Bergmann, J. Chem. Soc. p. 2190 (1955).
of fluoroketo acids (as well as a-fluoro-substituted ketones) exist in the

keto form,1' in contrast to nonfluorinated homologs. The reason for the
ketonizing effect of F in fluoroketo acid esters is probably related to a
resonance interaction (Formula 1) of F with the enolate form, resulting
in diminution of acidity of the enolate H and subsequent stabilization of
the keto form. In free a-keto-~-fluorosuccinic acid (monofluorooxalo-
acetic acid), both stability of the C-F bond and predominance of the
keto form appear to be related to the formation of a stable hydrogen-
bonded hydrate, as shown in Formula (2).
o /H-.o.'H'.o o/H'~o-'H~o
l 11 I H20 II I I
o/C H~C,.F 0 0 ..?

C\c/C%
I /\
H" H---F H
Fluoro substituted earbonyl compounds form stable hydrates or hemi-
ketals. 1~ Hydrate formation is characteristic of earbonyl groups with
strong electron-withdrawing groups in the vicinal positions, such as
chloral. Formation of the hydrate is further stabilized by proton donation
and hydrogen bonding by an adjacent carboxylic acid group. Thus both
mono- and difluorooxaloacetates crystallize as stable hydratesJ ~," Pure
diethyl fluorooxaloacetate exists entirely in the keto form,t' but in basic
solution enolization of this compound does occur. The inductive effect of
the fluoro group tends to polarize the double bond, favoring positive
1, W. D. Kumler, E. Kun, and J. D. Shoolery, J. Or{/. Chem. 27, 1165 (1962).
H. P. Braendlin and E. T. McBee, Advan. Fluorine Chem. 3, 1 (1963).
=E. Kun, D. R. Graasetti, D. W. Fanshier, and R. M. Featherstone, Biochem.
Pharmacol. I~ 207 (1958).
" E . Kun, L. K. Gottwald, D. W. Fanshier, and J. E. Ayling, J. Biol. Chem. 238,
1456 (1963).
charge on the carbonyl carbon and negative charge on the fluorine-

substituted carbon, as shown in Formula (3).
o- r
I I
EtOOC - - C : C - - C OOEt
f,a +
R- R+
This polarization directs attack by nucleophilic reagents on the carbony]
carbon and e]ectrophilic reagents on the F-substituted carbon. This
mechanism is probably responsible for the formation of stable hydrates
of free fluoroketo acids.
Decarboxylation of unsubstituted aliphatic carboxylic acids by acids
or bases is increased by a-fluoro substitution as compared to the non-
fluorinated analogs (e.g., fluoroacetate decarboxy]ates on standing).
The influence of F-substitution on ketone cleavage of keto acids
depends on the position of F and on experimental conditions. Acid-
catalyzed decarboxylation of oxa]oacetate under mild conditions (at room
temperature) is retarded by mono or by difluoro substitution in the
fl-carbon because this diminishes the tendency to form carbonium ion
on the same carbon, which is essential for decarboxylation. A similar
diminution in the rate of metal ion-catalyzed decarboxylation of oxalo-
acetate is brought about by mono and difluoro substitution 19 in the
fl-carbon of oxaloacetate. Fluoro substitution apparently inhibits the
mechanism proposed by Steinberger and Westheimer.TM Under conditions
of biochemical experiments (neutral pH, 25-30°), C-F bonds in fluoro-
oxaloacetates are stable and decomposition by ketone cleavage plays no
role. However, such cleavage occurs under strongly acidic conditions
such as boiling in mineral acid. 19 Fluorooxaloacetates (and their esters)
show diminished rates of ketone cleavage2° as compared to oxaloacetate
itself. The products of ketone cleavage of fluorooxaloacetate are oxalate
and fluoroacetates.
In acetoacetates, enolization is influenced by fluoro substitution in a
manner shown in Table IVb. Increasing the number of fluoro substitutions
on the same carbon (i.e., 2,2- or 4,4-difluoro) results in an increase in
stability of fluoroacetoacetates toward decarboxylation in acid, while the
tendency toward ketal formation increases.2'
Decomposition of fluoroacetoacetates in base is more complicated.2-~
'81~. Steinberger and F. W. Westheimer, J. Am. Chem. Soc. 73, 499 (1951).
I'~I. Blank, J. Mager, and E. D. Bergmann,J. Chem. Soc. p. 2190 (1955).
~op. V. Nair and H. Busch, J. Org. Chem. 23, 137 (1958).
"E. T. McBee, O. P. Pierce, H. W. Kilbourne, and E. R. Wilson,J. Am. Chem. Soc.
75, 3152 (1953).
T A B L E IVb
ENOLIZATION OF ACETOACETIC ESTERS (OBTAINED BY N M R ANALYSES)
Ester Percent Enol Reference
Methyl acetoacetate 0 a
Ethyl acetoacetate 8 a
Ethyl 2-chloroacetoacetate 15 a
Ethyl 2-bromoacetoacetate 5 a
Ethyl 2-fluoroacetoacetate 15 a
Ethyl 4-fluoroacetoacetate 7 b
E t h y l 4,4-difluoroacetoacet ate 53 b
Ethyl 4,4,4-trifluoroacetoacetate 89 b
J. L. Burdett and M. I". Rogers, J. Am. Chem. Soc. 86, 2105 (1964).
b R. Filler and S. M. Naqvi, J. Org. Chem. 26, 2571 (1961); Tetrahedron 19, 879
(1963).
2-Fluoro-2-alkylacetoacetates decompose by either of two routes, deter-

mined by experimental conditions. In aqueous 1 N Na0H (boiling for 2
hours), decarboxylation yields the predicted 3-fluoro ketone (72%)
(Formula 4). On the other hand, in the course of synthesis of ethyl 2-
fluoro-2-alkyl acetoacetates in absolute ethanol, ethyl 2-fluoroalkanoate
was isolated as a side product (10%). This side reaction takes place by
ketonic fission involving an intermediate carbanion stabilized by the
fiuoro groups (Formula 5).
I ,-+-, I I f ~ NaOH
CH3--C--C'--CR O- -r[CH3--C--C--R
[ 1 + co,
O F
I%O _ It i
r C H a - - C - - C - - R + OH-
I
H
(72% yield)
OEt
I r~
CH3--C--CF--COOEt ~ CH3COOEt + ( R - - C F - - C O O E t )
I
tt
EtOH I
~---R--CF--COOEt + EtO-
:" H. Machleidt, Ann. Chem. 667, 24, 35 (1963).
In aqueous 1 N NaOH used as above 4-fluoro-2-alkyl acetoacetate
yields the defluorinated tetronie acid (49%). This reaction occurs by way
of displacement of F by the neighboring carboxylate group. The dis-
placement is favored by the allylic structure of the enol (Formula 6).
R O" R 0-
\ / \, /
C-----?~"~a ('~ NaOH C----C
0 = C\o_~.~CI~-- F ~ O--C\o/CI ~ + F-
In the same system the 1-fluoro ketone, resulting from decarboxylation

of 4-fluoro-2-alkyl acetoacetate was also isolated (26%), and the car-
boxylic acid due to ketonic cleavage of the parent compound (at a) was
also detected.
In the derivative 2,4-difluoro-2-alkyl acetoacetate, in which the allylic
structure of Formula (6) is not possible, decarboxylation (18%) and
ketonic cleavage (35%) occur.
Introduction of fluorine markedly increases the acid strength of
acrylic acidsY8 The magnitude of this effect depends primarily on the
position of F, as shown in Table V, and is not simply predictable from
TABLE V
ACIDITY OF FLUOROACRYLIC ACIDSa
Acid Dissociation constant, K X 10a

Acrylic acid 5.6
4,4,4-Trifluorocrotonic acid 45
3,3-Difluoroacrylic acid 68
2-Fhoroacrylic acid 280
2,3,3-Trifluoroacrylic acid 1580
2,3,3-Trichloroacrylic acidb 6200
A. L. Henne and C. J. Fox, J. Am. Chem. Soc. 76, 479 (1954).

bFor comparison.
the known inductive effect of fluorine. Both resonance (of the fluorovinyl
structures) and hydrogen bonding of the carboxyl to the p-fluoro group
contribute to the magnitude of the acidity constant.
The inductive effect of the trifluoromethyl group is similar to that of
fluorine substitution itself. 15 The unexpected instability of F in 2-tri-
fluoromethyl acrylic acid in 2 N NaOH has been attributed to hyper-
conjugation/' although the allylic position of the fluorine is sufficient to
"A. L. Henne and C. J. Fox, Y. Am. Chem. Soc. 76, 479 (1954).
M. W. Buxton, M. Stacey, and J. C. Tatlow, J. Chem. Boc. p. 366 (1954).
predict this instability. The product of the reaction (i.e., displacement of

3 F by OH- ions) is methylene malonic acid. However, the much smaller
degree of instability of the trifluoromethyl group in 2-trifluoromethyl-
propionic acid is probably the result of the inductive effect. After elimina-
tion of HF, addition to the double bond of hydroxide is followed by
fluoride elimination, until all the fuorines are replaced.
The lower reactivity of acyl fluorides compared to acyl chlorides7
may be attributed to resonance stabilization (shown in Formula 1), or
to an increase in the double-bond character of the carbonyl group in
O : C - - F compared to O--C--C1.
C. Stcreochemistry
Alterations in the stereochemistry of carboxylic acids by fluoro
substitution is a field which has received as yet little attention, although
introduction of optically active centers by F substitution has advantages
over other substitutions because no drastic change in molecular dimen-
sions occurs, e.g., bond distance of aliphatic C-F is about 1.41 )L, com-
pared with that of C-C of 1.54 ,~; van der Waals' radius of H is about
TABLE VI
OPTICAL ISOMERS OF FLUOROCITRIC ACIDS
Citric acids Isomers
2-Fluoro d-, l-erythro and d-, l-threo

2,2-Difluoro d and l
2,4-Difluoro d, l, and meso
2,2,4-Trifluoro d-,/-eryLhroand d-, l-threo
Tetrafluoro One
1.2 A, that of F is 1.35 .~.' Recently 3-fluorolactic acid has been resolved
into optical isomers and its absolute configuration determined by optical
rotatory dispersion? 5 Resolution of 2-fluoro-3-hydroxypropionic acid has
also been reported. ~e Cis- and trans-4-fluoro-L-proline have been syn-
thesizedY Fluorocitric acid 28 and fluoromalic acid 12 have been separated
into erythro and threo isomer pairs.
As shown in Table VI, substitution of fluorine in the four available
positions on citric acid results in five compounds with a total of fourteen
isomers.
"J. C. Craig, R. J. Dummel, E. Kun, and S. K. Roy, Biochemistry 4, 2547 (1965).
"P. W. Kent, G. Hebblethwaite, and N. F. Taylor, J. Chem. Soc. p. 106 (1960).
'TA. A. Gottleib, Y. Fujita, S. Udenfriend, and B. Witkop, Biochemistry 4, 2507
(1965).
uI). W. Fanshier, L. K. Gottwald, and E. Kun, J. Biol. Chem. 237, 425 (1964).
T A B L E VII
METHODS FOR INTRODUCING FLUORINE ATOMS AT SPECIFIC POSITIONS
ON A CARBON CHAIN
Identifying
letter Reaction"
Displacement of halide with potassium fluoride, or other metal fluoride

Displacement of sulfonate ester with potassium fluoride
Addition to a triple bond or to an epoxide of HF, or of potassium hydrogen
fluoride as a source of HF
l) Reaction of a terminal olefin with "bromonium fluoride"
E Reaction of a perchloryl fluoride with an enolate
F Reaction of a earbonyl with sulfur tetrafluoride
G Reactions of fluoro olefins or fluorohalo olefins
H Replacement of OH by F using 2-chloro-l,l,2-trifluoroethylamine
Comments on methods A-H:

A. Introduction of F by KF requires high temperatures (150-250 °) and high pres-
sure. Its principal use is restricted to the synthesis of intermediates, e.g., ~-fluoro
alcohols as precursors to acids. [A. K. Barbour, L. J. Belf, and M. W. Buxton,
Advan. Fluorine Chem. 3, 181 (1963).]
B. The advantage of this method lies in the preparation of volatile fluoro esters
separable by distillation from the starting material (e.g., toluene sulfonic ester).
This reaction is stereospecific.
C. HF catalyzes both decomposition and polymerization; therefore the method is
of limited usefulness. Potassium hydrogen fluoride as a source of HF diminishes
the extent of polymerization. [E. D. Bergmann and S. Cohen, J. Chem. Soc.
p. 2259 (1958).]
D. A mixture of HF and N-bromosuccinimideadds to a terminal olefin, yielding the
1-bromo-2-fluoro compound. Conversion to a-fluorocarboxylic acid requires two
further steps. [F. L. M. Pattison, D. A. V. Peters and F. H. Dean, Can. J.
Chem. 45, 1689 (1965).]
E. Perchloryl fluoride reacts with an enolate ion by replacing all available a-hydro-
gens. In this reaction F appears to have a positive charge.
F. Sulfur tetrafluoride under high pressure reacts with carbonyl functions, replacing
oxygen by a geminal difluoro group. Under the same conditions an aldehyde
group is converted to --CHF2, and a free carboxyl group is converted to --CF3.
G. Trifluoroethylene (see text footnote 43), tetrafluoroethylene [D. W. Wiley, U.S.
patent 2 988 537 (1961)], and trifluorochloroethylene [M. S. Raasch, J. Am.
Chem. Soc. 81, 2678 (1959); M. S. Raasch and J. E. Castle, OrE. Syn. 42 (1962)]
react under high pressure with acrylic esters and other unsaturated substances
capable of serving as acceptors in Michael condensation to give cyclobutanes.
Reduction by Zn to cyclobutenes followed by permanganate oxidation results
in fluorodicarboxylic acids. Fluoro olefins react also with formaldehyde (see text
footnote 43), and malonic ester [D. C. England, L. R. Melby, M. A. Dietrich,
R. V. Lindsey, Jr., J. Am. Chem. Soc. 82, 5116 (1960)] to yield fluorocarboxylic
acids by subsequent reactions.
H. The replacement of OH by F takes place under mild conditions, e.g., at 0° in
inert solvents. [E. D. Bergmann and A. Cohen, Israel J. Chem. 3, 71 (1965).]
I I I . Organic Reactions Applied in the Synthesis of

Fluoro Aliphatic Esters and Acids
I n the following s u m m a r y each method carries a letter or number to
identify the procedure yielding the compounds listed in Sections IV and
V: Specific Procedures and Compendium.
A. General Methods for the I n t r o d u c t i o n of Fluorine

The principal methods for introducing one or two fluorine atoms at
specific positions on a carbon chain are listed in Table VII. Equations
describing reactions A - H are shown in Fig. 1.
B. General Methods of Synthesis from Fluoro Aliphatic Esters

Most fluorocarboxylic acids of biochemical interest have been syn-
thesized from ethyl fluoroacctates as starting material. The sequence of
syntheses is illustrated in Fig. 2. Fluoro aliphatic esters react in a pre-
dictable fashion in these syntheses. Reactions 1-7, 10, and 11 (which arc
TABLE VIII
METHODS OF SYNTHESIS FROM FLUORO ALIPHATIC ESTERS
Identifying
number Reaction a
Claisen ester self condensations (including mixed esters)

Claisen condensation of fluoro esters with aprotic esters
Condensation with aldehydes or ketones
Michael condensations
Alkylation with alkyl halides
Acylation with acid halides
Reformatsky and Grignard reactions
Reactions involving fission of C--C bonds (decarboxylation, decarbonyla-
tion, ketonic fission)
Reactions which leave the carbon chain and C--F structure unaltered
(e.g., oxidation, reduction, dehydration, dehalogenation)
10 Condensations or addition reactions of fluoro ketones
11 Wittig reaction
" Comments on methods:

Reactions involving the enolate of ethyl fluoroacetate in general have low yields
because of side reactions. In contrast to nonfluorinated esters, mixed ester con-
densations (Method 1) with fluorine on one or both reactants yield predominantly
one product [E. T. McBee, O. P. Pierce, H. W. Kilbourne, and E. R. Wilson,
J. A m . Chem. Soc. 7§~ 3152 (1953)]. Synthesis of diethylfluorooxaloacetate is the
most important example of Method 2. The Wittig reaction (Method 11) permits
stereospecific synthesis of one isomer of an unsaturated fluoro ester [H. Machleidt,
A r~l~. Chem. 674, 1 (1964)]. This is an advantage over dehydration.
636 rRe.PARAT~ON OF COMPOUNDS [79]
ff 0
!
0
~+ 80
+
0 ff ff 0
0 0 ,"
o 0
80
r,.)
+ o
0
g~ 80 - 0,- 0 8
o ff
o
80 ~r
o
~r
r~ ~ 0 0 o
N 0
+ 0
+ ff +
~ r,.)
Z 0 z
8 o ff .~
0
0 r~ 0
0
+ r.r.] ~ o- r~-o
4-
8=8 e
I +
ff 80
0
0 o r,.)
0 II
~r r,.)
ff
0 o d
4-
=d/.5/
0
0 /\
8r,.)
r,) r~
r,.)
r.) 0
N
r,.)
2M
0
0
+
0
Z
0 ~
o
0
0
+ u
r~ r,.) v
.o
N +
r..)
8 r,.)
0
r,.) 8 0
o
u
0 +
o~
r,.) u
r,.) 0-~
II
8 c,
inoisobu ric
\ ev lonic
Oxa o eetic Citric
A r He
FI~. 2. Synthetic pathways to fluoro aliphatic esters.

type reactions) are described in Fig. 3, and reactions 1-11 are listed in
Table V I I I .
I n certain reactions shown in Fig. 2, fluoroacetic ester m a y be
replaced by difluoroacetic ester yielding the corresponding difluoro deriv-
ative. Self-condensation of fluoroacetic and fluorooxaloacetic esters give
the 2,4-difluoro derivatives.
IV. Specific P r o c e d u r e s 2sa
A. S a t u r a t e d A c i d s
2-Fluoropropionic Acid (Method B, abstract)

Ethyl 2-Fluoropropionate. ~9 E t h y l lactate toluenesulfonate was mixed
with a 50% excess of potassium fluoride in sufficient diethylene glycol to
dissolve the salt at 50 °. The ethyl 2-fluoropropionate (75% yield), b.p.
122 ° , was distilled from the mixture.
2-Fluoropropionic Acid? ° The ester was refluxed with 6 N hydro-
chloric acid for 36 hours. The product was extracted with ether. Distilla-
tion gave 2-fluoropropionic acid (51% yield), b.p. 63-66 ° at 13 mm,
nn 25 1.3837. (2-Fluoropropionic acid or its ester has been synthesized by
,8, Most of the following procedures are quoted directly from the original author's
experimental data. For brevity, a few are abstracted. Elemental analyses, some
references, and some descriptive details are omitted, and a few tables are converted
to text. Comments not in the original article are enclosed in parentheses at the end
of the procedure.
29E. D. Bergmann and I. Shahak, Chem. & Ind. 1, 157 (1958).
F. L. M. Pattison, R. L. Buchanan, and F. H. Dean, Can. J. Chem. 43, 1700 (1965).
five additional methods: A, '~J D, ~9 E, 29 the reaction of diazoethane with

ethyl fluoroformate, '~2 and the oxidation of 2-fluoropropanol2 ~)
Fluoromalonic Acid ~4 (Method 8)

B u t y l E t h y l Fluoramalonate. Potassium persulfate (100 g) was added
with stirring and cooling to a mixture of concentrated sulfuric acid (180
g) and water (80 ml); the stirring was continued at 10-15 ° for 30
minutes, and successively butyl alcohol (100 ml) and diethyl oxalo-
fluoroacetate (61.5 g) in butyl alcohol (100 ml) were added. After 1 hour
at 15 °, 1 hour at 25 °, and 12 hours at room temperature, toluene (150
ml) and ice (200 g) were added, the organic layer was separated, and the
aqueous layer was extracted twice with ether (100 ml). The combined
organic extracts were washed with 5% sodium hydrogen carbonate solu-
tion and water, dried, and freed from ether. The remainder was sub-
]ected to azeotropic distillation, after addition of toluene-p-sulfonic acid
(1 g). When no more water collected in the trap, the solution was washed
with 5% sodium hydrogen carbonate solution and water, dried, and
concentrated in a vacuum of 30 mm and the residue was distilled under
1 mm pressure (column). After a forerun of butyl ethyl oxalate (b.p.
84-86°/1 mm), butyl ethyl fluoromalonate (37 g, 60%), b.p. 93-95 ° at
1 ram, was obtained. I t was identified by transformation into fluoro-
malondiamide, m.p. 190 °.
Dibutgl Fluoromalonate. If in the foregoing procedure, the first treat-
ment with sodium hydrogen carbonate was omitted, acid-catalyzed trans-
esterification took place. Thus, dibutyl oxalate, b.p. 111-112 ° at 1 mm,
130-135 ° at 35 mm, and dibutyl fluoromalonate (48 g, 69%), b.p. 128-
131 ° at 1 mm, 163-165 ° at 35 mm, were the products of the reaction.
Fluoromalo~ic Acid2 ~ Hydrolysis of the ester with slight excess of
alcoholic potassium hydroxide at room temperature, followed by acidifi-
cation, gave the free acid hydrate. Recrystallization from water and
dehydration gave fluoromalonic acid, m.p. 135-136 °.
Fluorosuccinic Acid 36 (Method E)

Potassium Enolate o] Triethyloxalylsuccinate. The potassium enolate
was prepared by the standard procedure ~ with two modifications: (a)
3~B. C. Saunders and G. J. Stacey, J. Chem. Soc. p. 1773 (1948).
:~'-E.
' D. Bergmann and I. Shahak, Israel ,l. Chem. 3, 73 (1965).
~:E. Gryszkiewicz-Trochimowski and O. Gryszkiewicz-Trochimowski, Bull. Soc.
Chim. France, p. 928 (1949).
~ E. D. Bergmann and I. Shahak, Chem. & Ind. p. 591 (1961).
*~E. D. Bergmann, S. Cohen, and I. Shahak, J. Chem. Soe. p. 4033 (1961).
~eF. H. Dean and F. L. M. Pattison, Can. J. Chem. 41, 1833 (1963).
~" L. Friedman and E. Kosower, Org. Syn. Coll. III, 510 (1955).
81 ff +
r..) ~- r..)-~ oN
~ r..)
II ~ = ~ ~ +
o+ +
g
8 I
8c? o
O 8
I I ~-?~7 ~ O O I
o--r~ O _O~,O
I I O'-rJ CD- ~ - - ~
O'-~ I G
8
8° l
t O
O
O
O I
O~rD + ff 4"
I
I O---~ ~ +
~r
O
4~
8 - 8G)
~ O
+ I O I
+
0 O--~ ~- -~
N
0
o
~ 8 I
O
,=
o
8 ~ 8 8
:~ r.,.., F.r.1
[79] VLUORO ANALOGS {7)41
0 0
0~ ~ 0 ~ +
0 o 0 o
Z--O -0
ff
8
o
0=0
0 r..)
0
I 0
I
0 0
ff 0 ~rzff
0
ff
r..)
8 o
ff + m
© 0
Z +~
0"-0 N ~
0
+ +CS + +
o
t-- M o
0 0
o 8 ,,0 &--8 ~1
o
X
the work was carried out in a nitrogen atmosphere, and (b) the freshly
cut potassium was not washed with ether. By this means, the formation
of potassium hydroxide and carbonate was reduced.
Diethyl succinate was redistilled freshly through a 23-plate spinning
band column (b.p. 97 ° at 8 mm, n~25 1.4185, d42° 1.035). Diethyl oxalate
was similarly purified (b.p. 76 ° at 10 mm, 7Zv25 1.4090, d~-°° 1.075). The
anhydrous ethanol was stored over Linde molecular sieves, Type 5A.
The enolate was washed with dry ether and placed in a P_o05 vacuum
desiccator for 15 hours at 0.5 mm. Yield: 91-97%, as a light yellow,
friable solid.
Diethyl Fluorosuccb~ate. A 500 ml three-necked flask was equipped
with a stirring magnet, a gas-inlet tube with a fritted-glass gas disperser,
a drying tube filled with indicating Drierite, and a --50 ° to +50 °
thermometer secured by gum-rubber tubing to a ground-glass holder.
After the flask had been flushed with dry nitrogen, dry potassium enolate
(23.6 g, 75.5 millimoles) was introduced, followed by anhydrous ethanol
(300 ml). To the mixture was added 2 ml of bromothymol blue (0.05%
in absolute ethanol) and dry, reagent grade potassium bicarbonate (1.75
g, 17.5 millimoles). Dry nitrogen was passed through the mixture, which
was stirred and cooled in an ice bath. When the temperature was steady
at 4 °, the perchloryl fluoride cylinder was attached to the gas disperser
by gum-rubber tubing and wired on securely. (Rubber containing carbon
black may explode in contact with perchloryl fluoride.) Perchloryl fluoride
gas was then introduced until the temperature rose to 8-10 ° . The gas flow
was then stopped until the temperature had again fallen to 4 °, when it
was resumed. In this manner, 10.7 g (104.3 millimoles) of perchloryl
fluoride were added over a 1 hour period, the actual duration of gas
inflow being 21 minutes. As the perchloryl fluoride is being added, the
original deep-green color lightens due to the formation of potassium
chlorate; the end of the reaction is determined readily by a color change
from green to yellow and by a fall in temperature on the addition of
more perchloryl fluoride. The gas cylinder was then detached, and an
additional quantity (10.2 g, 102 millimoles) of potassium bicarbonate
was added.
The ice bath was removed, and the mixture was stirred at room tem-
perature for 20 hours. The flask was flushed with nitrogen to remove any
unreacted perchloryl fluoride, water (800 ml) was added, and the solu-
tion was extracted thoroughly with ether. The combined ether extracts
were washed with water and dried over magnesium sulfate. The ether
was removed on a water bath, and the alcohol at aspirator pressure. The
residue was then distilled through a 6 inch Vigreux colmnn under oil-
pump pressure. Two main fractions were obtained: (a) diethyl oxalate
[79] FLVORO ANALOGS 643
(4.15 g), b.p. 72 ° at 9 mm to 62 ° at 2.75 ram, n~ 2~ 1.4092-1.4090; and (b)

diethyl monofluorosuccinate (15.0 g), b.p. 75 ° at 2.75 mm to 72 ° at 0.25
mm, nD25 1.4135--1.4133. The first fraction, on redistillation through a spin-
ningband column, gave 3.23 g of pure diethyl oxalate, b.p. 72 ° at 8 ram, n~25
1.4087, d42° 1.076, the infrared spectrum of which was identical in all
respects with that of a sample of the pure ester. Redistillation of the
second fraction, which contained about 8% of diethyl maleatc (ultra-
violet analysis), through the spinning band column gave pure diethyl
monofluorosuccinate (10.6 g, 73%) as a stable, colorless liquid, b.p. 72 °
at 1 mm, nD25 1.4125, d42° 1.142, M R , 41.98 cc/mole; estimated 42.19
cc/mole. (Gitter, Blank and Bergmann, s6b 1953, report b.p. 70--71° at 0.8
mm. nD2~ 1.4240, d42s 1.1090, MRD 44.23 cc/mole.) Principal infrared
bands (3% solution in CCI4), em-l: 1769.5 (C = O) s, 1746.5 (C = O) s,
1372.8 (CH3) s, 1286.0 ( C - O) s, 1264.0 m, 1179.5 (C w O) s, 1097.0
( C - F) s, 1051.2 m, 1028.5 s.
Nuclear magnetic resonance spectrum (27.7% w/v in CC14 using
tetramethylsilane as internal standard): Chemical shifts (T), coupling
constants (c/s). Two CH3CH.~O: 8.69 T, 8.73 T (t) ; 5.78 T, 5.85 T (q) ; Jm~
7.0 C/S. CH2:7.17 r; JHH 5.9 C/S; JH~ 23.3 C/S. CHF: 4.85 T; Jm~ 5.9 c/s;
JHv 47.6 c/s. The ester showed only weak end-absorption in the ultra-
violet, ~22o100.5 in 95% ethanol, indicating the absence of diethyl maleate
(C22o6380); cf. diethyl succinate, ~22o11.
M o n o f l u o r o s u c c i n i c A c id . Pure diethyl monofluorosuccinate (3.3 g,
17.1 millimoles) and 5 9 sulfuric acid (20 ml) were heated under reflux
at 110 ° for 30 minutes, that is, until the mixture appeared to be homo-
geneous. The cooled solution was extracted once with 35-60 ° petroleum
ether, to remove any unchanged ester, and then was subjected to con-
tinuous ether extraction for 38 hours; no further acid was obtained after
a further 24 hours of extraction. The extract was dried with magnesium
sulfate, filtered, and concentrated using a Rinco rotary evaporator. The
addition of benzene (5 ml) and removal of residual ether by boiling
induced the acid to crystallize (1.33 g), m.p. 140-147 °. Concentration of
the mother liquors gave a further quantity (193 mg) of the crude acid.
The petroleum ether extract gave 90 mg of unchanged ester. Yield of
crude acid (1.523 g) based on ester consumed: 67%. It is important that
any residual ester be removed and that the ether solution be dry to avoid
difficulties in the initial crystallization of the acid. The acid may be
recrystallized from benzene-ether, or, better, from hot ethyl acetate. An
analytical sample is conveniently obtained by sublimation at 105 ° (0.3
mm) as a colorless, crystalline solid, m.p. 144-145°; positive fluorine test.
~bS. Gitter, I. Blank, and E. D. Bergmann, Koninkl. Ned. A~ad. Wetenschap. Proc.
C_~6, 427 (1953).
The infrared spectrum in acetonitrile showed a band at 1747 cm-1
(C = O) with a shoulder on the high-frequency side; cf. succinic acid,
1744 cm-1. Ultraviolet absorption spectrum in 95% ethanol (1-.ram cells) ;
~tm,x 198 m~, ~m~x130 (cf. maleic acid: A~,. 203 m#, ~ x 14,900).
P,,e-Difluorosuccinic Acid 37 (Method G)
Step 1. 1,1,~-Trichloro-$,3,3-trifluorocyclobutane ]rom Vinylidene
Chloride and Chlorotrifluoroethylene. A stainless steel bomb is charged
with 350 parts of vinylidene chloride, 1 part of hydroquinone, and 300
parts of chlorotrifluoroethylene. The mixture is heated for 10 hours at
180 ° under autogenous pressure. The product is filtered from polymer
and then distilled to give 266 parts (48% yield) of 1,1,2-trichloro-2,3,3-
trifluorocyclobutane boiling at 120--121°, n925 1.4139.
Step 2. 1-Chloro-~,,3,3-trifluorocyclobutene ]rom 1,I ,P,-Trichloro-2,3,3-
trifluorocyclobutane. In a glass reaction vessel fitted with an efficient
reflux condenser, sealed stirrer, and addition funnel are placed 150 parts
of 95~ zinc dust and 243 parts of absolute ethanol. The alcohol is
refluxed and 400 parts of 1,1,2-trichloro-2,3,3-trifluorocyclobutane is
added slowly. The mixture is refluxed for 2 hours after the end of the
addition. The mixture is cooled and 1500 parts of water containing 25
parts of hydrochloric acid is added. The organic layer is separated, dried
over calcium chloride, and distilled. The yield of 1-chloro-2,3,3-trifluoro-
cyclobutene is 177 parts (66~), b.p. 51.5-52 °, n~ 25 1.3614.
Step 3. ~,~-Difluorosuccinic Acid ]rom 1-Chloro-~,3,3-trifluorocyclo-
butene. In a glass reaction vessel fitted with stirrer, thermometer, addition
funnel, and ice-salt bath are placed 4000 parts of water, 160 parts of
sodium hydroxide, and 316 parts of potassium permanganate. 1-Chloro-
2,3,3-trifluorocyclobutene (214 parts) is added slowly at 15-20 °. The
reaction is complete in 3 hours. The manganese dioxide is filtered off and
washed with water; the filtrate is reduced in volume on a steam bath.
After addition of 312 parts of sulfuric acid, the filtrate is extracted with
ether. After the ether solution has been dried with magnesium sulfate
it is evaporated to give 182 parts (79~ yield) of 2,2-difluorosuccinic
acid. The product is recrystallized from acetone-benzene in 88~ return
(2 crops) and melts at 144-145 ° .
$-Fluoroglutaric Acid ~° (Method E)

Dimethyl a-Fluoro-a-Carbomethoxyglutarate. In a 500 ml three-
necked flask carrying a drying tube, a dropping funnel, and a gas inlet
tube was placed redistilled dimethylformamide (75 ml). Dry nitrogen
M. S. Raasch, U~. Patent 2~24,888 (1958).

[79] FL~ORO ANALOGS 645
was passed through and a 56.5% dispersion of sodium hydride in mineral

oil (10.0 g, 235 millimoles) was introduced. The glutarate ester (50.0 g,
229 millimoles) was added dropwise over 1.5 hours with stirring and
cooling (ice bath). The dark mixture was stirred for a further 2.75 hours
at room temperature. Anhydrous methanol (20 ml), to destroy the excess
sodium hydride, and then dioxane (50 ml) were added. The flask was
cooled in an ice bath, and, when the temperature had fallen to 3.5 °,
perchloryl fluoride was introduced intermittently with stirring; the tem-
perature was held in the range 3.5-16 ° . The addition required about 1.75
hours. The end of the reaction was indicated by a fall in temperature on
passing in perchloryl fluoride. After standing overnight, the mixture was
diluted with water (800 ml) and the product was extracted with ether.
The extract was washed with water and the ether was removed. Benzene
was used to azeotrope any residual water. The resulting residue was
heterogeneous because of the presence of mineral oil. The latter (upper
layer) was removed with a dropper prior to fractionation on the spin-
ning-band column. After a forerun (6-7 g), the required fluoro ester
(23.85 g, 44%) was obtained as a slightly turbid liquid. To obtain a clear,
analytical sample, the ester was extracted three times with 60-80 °
petroleum ether and redistilled through a 23-plate platinum-plated spin-
ning band column.
a-Fluoroglutaric Acid. The above ester (ll.0 g, 46.5 millimoles) was
hydrolyzed and decarboxylated by heating under reflux with 6 N hydro-
chloric acid (40 ml) at 115 ° for 6 hours. The cooled hydrolyzate was
extracted with petroleum ether to remove any residual mineral oil or
ester. The hydrochloric acid was distilled off under aspirator pressure.
The crude acid was then distilled directly, using a simple distillation head
attached to a vacuum adapter; the solid thus obtained (4.64 g, 66.6%)
of b.p. 115-155 ° at 0.1-0.22 ram, was shown by infrared analysis to be
the acid and not the anhydride (acetonitrile as solvent). The solid was
recrystallized four times from 50:50 benzene-ether and dried at the
temperature of refluxing chloroform at 0.5 mm for 3 hours. No hydrate
formation could be detected.
2-Fluoroglutaric Acid 3s (Method 4)

Dicthyl fluoromalonate (10.74 g, 0.065 mole) was added in one por-
tion to a solution of sodium (0.14 g, 0.006 mole) in absolute ethanol (20
ml). Ethyl acrylate (7.4 g, 0.074 mole), dissolved in ethanol (10 ml),
was added dropwise over a period of 15 minutes to the rapidly stirred
~olution of diethyl sodiofluoromalonate. Heat was evolved, and the
~ L. K. Gottwald, J. E. Ayling, and E. Kun, J. Biol. Chem. "239, 435 (1964).

reaction mixture turned brown. The mixture was left at room temperature
for 20 hours and then heated under reflux for 30 minutes. After cooling,
the mixture was treated with glacial acetic acid to bring the pH to 7.0 and
then the solvents were evaporated at 50-60 ° under vacuum. The residue
was taken up in 50 ml of anhydrous ether and filtered in order to remove
salts. The solvent was removed under vacuum and the residue distilled,
yielding diethyl a-fluoroglutarate (4.3 g, 37%), b.p. 120-128 ° at 5-8
mm. A sample, redistilled for analysis, had a boiling point of 110-112°/5
mm. A second, higher-boiling fraction was identified as triethyl a-fluoro-
a-carboxyglutarate (6.1 g, 37%), b.p. 158-160°/5-8 mm. A sample of
this fraction was distilled for analysis and had a boiling point of 148--
149°/1 mm.
a-Fluoroglutaric Diamide. Diethyl a-fluoroglutarate (2.06 g, 0.01
mole) was mixed with ammonium hydroxide (7 ml, 15 N) while cooling
in an ice bath. On standing overnight at 0 ° white crystals deposited and
were filtered, washed with ethanol, and dried, yielding 1.0 g of diamide,
m.p. 165-167 °. A sample recrystallized from ethanol had a melting point
of 169-169.5 ° .
a-Fluoroglutaric Acid. A mixture of diethyl a-fluoroglutarate (4.3 g,
0.022 mole) and 10% hydrochloric acid (30 ml) was heated under reflux
for 10 hours. The solvent was removed under vacuum, leaving 3.4 g of
white crystalline product. After one recrystallization from trifluoroacetic
acid, colorless crystals were obtained (2.0 g, 61%), m.p. 110-113 °. One
subsequent recrystallization from trifluoroacetic acid raised the melting
point to 113% Infrared spectrum of the free acid (0.5% in KBr pellet) :
~.,..~x -= 3.4 (broad), 5.8, 7.0, 7.45, 7.8, 8.15, 9.1, 9.2, 10.8, 11.35, 12.43, 13.5,
14.4, and 15.3. Alternatively, a-fluoroglutaric acid can be prepared from
triethyl a-fluoro-a-carboxyglutarate with a 40% yield by heating the
ester under reflux with 37% HCI for 12 hours. On evaporation of the
solvent the crude acid is obtained, which upon recrystallization from
trifluoroacetic acid yields the pure product, m.p. 113 °.
(The overall yield of 2-fluoroglutaric acid was 29% by method E,
and 45% by method 4. However, the low yield of the intermediate diethyl
malonate, 23.6%, see footnote 30, actually reduces method 4 to 11%
overall yield based on ethyl fluoroacetate as the starting material.)
Diethyl 3,3-Difluoroglutarate 39 (Method F)

General Procedure. Reactions were carried out under pressure in
stain,less steel-lined shaker tubes of 80-1000 ml capacity. Liquid or solid
39W. R. Hasek, W. C. Smith, and V. A. Englehardt, J. Am. Chem. Soc. 82, 543
(1960).
contents were introduced under a nitrogen atmosphere. After evacuation

and chilling with dry ice, sulfur tetrafluoride was introduced. After the
reaction, the gases were vented and the products treated with water or a
suspension of sodium fluoride in inert solvent to remove hydrogen fluoride.
The product was purified by distillation. Diethyl acetonedicarboxylate
(0.25 mole) was reacted with sulfur tetrafluoride (0.50 mole) at 80 ° for
6 hours. Diethyl 3,3-difluoroglutarate (29% yield) was obtained, b.p.
63-65 ° at 2 mm, no 2~ 1.4038. [Hydrolysis to the frec acid was not reported
but should be possible.]
B. Unsaturated Acids
2-Fluoroacrylic Acid
Butyl 2-Fluoroacrylate 4° (Method 3, Abstract). A mixture of butyl
fluorooxaloacetate (190 g), formaldehyde trimer (22 g) and pyridine (1
ml) was heated at 90 ° for 4 hours to give butyl 2-oxo-3-fluoro-3-carboxy-
butyrolactone. The latter was dissolved in benzene and heated with
aqueous 30% potassium carbonate. Evaporation and fractionation of the
benzene layer gave butyl 2-fluoroaerylate (50% yield), b.p. 145-148 °,
2-Fluoroacrylic Acid. This compound, prepared from 2,3-dibromo-2-
fluoropropionic acid, was obtained as a sublimable solid, m.p. 51.5-52 °,
which slowly polymerized on standing at room temperature. 41 (Acid
hydrolysis of the ester appears to be possible.)
2-Fluorocrotonic Acid 13 (Method 3)

Ethyl a-Fluorocrotonate. In this case, because of the low boiling point
of the product, tetrahydrofuran or ethanol is used as reaction medium.
Ethyl oxalate (32 g) and ethyl fluoroacetate (1 g) were added with
stirring to a suspension of sodium hydride (4.8 g) in tetrahydrofuran (100
ml). When the reaction had been initiated by refluxing, the balance of
the fluorinated ester (20.2 g) was slowly added at 40-45 ° and the whole
was heated for 3 hours at 60 °. Then freshly distilled acetaldehyde (9 g)
was added at 0 °, and the mixture was brought slowly to the boiling point,
at which it was maintained for 1 hour; then it was poured into water
(400 ml) and extracted with methylene chloride (100 ml). The organic
layer was washed with 5% sodium carbonate solution (100 ml) and water
(100 ml), dried, and distilled. The ester (15 g, 61%) boiled at 134-136 °.
Hydrolysis of the ester gave a a-fluoroerotonie acid, which, sublimed
at 80--90°/5 mm and reerystallized from light petroleum (b.p. 40-60°),
"H. Gault, D. Rouge, and G. Gordon, Fr. Pat. Addn. 79,944 (1963); Fr. Pat.
1,255,459 (1961).
" A. L. Henne and W. J. Ziramerseheid, J. Am, Chem. Soc. 69, 281 (1947).
had m.p. 111-112 °, no selective absorption above 210 m/~ (in ethanol),
V~az (in KBr) 3030-2857 (associated OH), 1668 (CO), 1162 (:CF),
1075 ( C - - F ) cm -~.
Methyl 4-Fluoroerotonate 42 (Method A)

Methyl ~,-Fluorocrotonate. 5lethyl ~-bromocrotonate (10 g. 0.056
mole) and silver fluoride (10 g, 0.08 mole) were mixed thoroughly and
then heated gently under reflux until a vigorous reaction occurred with
evolution of hydrogen fluoride. The product was cooled and extracted with
ether, and the ethereal extracts were washed with water and dried
(Na2S04). After removal of the ether, the residue yielded a small frac-
tion of crude methyl ~,-fluorocrotonate, b.p. 47 ° at 16 ram, and then
unchanged bromo ester. This fluorination was repeated several times, the
recovered bromo ester being used in each case. Yield: 2.5 g of crude
fluoro ester from 18.5 g of pure methyl ~/-bromocrotonate. The fluoro
ester was redistilled, and the colorless, pleasant-smelling liquid of b.p.
46 ° at 16 mm was collected, (2.1 g, 17%). Care is needed in handling this
material because of its great toxicity. (The free acid has not been
reported.)
Fluoro]umaric Acid 43 (Method 9)

To a solution of 97 g (2.42 moles) of sodium hydroxide in 300 ml of
water were added 100 g of ice and 125 g (0.81 mole) of 2,2-difluorosuc-
cinic acid. The solution was heated on a steam bath for 16 hours, cooled,
"and 100 ml of water and 205 ml (2.44 moles) of hydrochloric acid were
added. The solution was extracted with ether and the fluorofumaric acid
obtained by evaporating the dried ether solution was recrystallized from
acetone-benzene to give 83.5 g (77% yield) in three crops, m.p. 236-237 °.
From the mother liquors 4 more grams of fluorofumaric acid plus 1 g of
sirup was obtained. Therefore, little or no fluoromaleic acid is formed in
the reaction.
DifluoroJumaric Acid 43 (Method 9)

A mixture of 10 ml of water, 2.4 g (0.06 mole) of sodium hydroxide
and 3.44 g (0.02 mole) of pure trifluorosuccinic acid '~1 was heated on a
steam bath for 15 hours. The mixture was cooled and 8 ml of sulfuric
acid in 5 ml of water was added. It is necessary to use a high concentra-
tion of acid, otherwise the difluorobutenedioic acids cannot be extracted
easily. The solution was extracted with ether and the ether solution was
~F. L. M. Pattison and B. C. Saunders, J. Chem. Soc. p. 2745 (1949).
u i . L. Knunyants, B. L. Dyatkin, L. S. German, and E. P. Machalina, Isv. Akad.
N a u k S S S R Otd. K h i m . Nauk. p. 1678 (1962).
deeolorized with carbon, dried, and evaporated to give 3.02 g (99%

yield) of a mixture of difluorofumaric and difluoromaleic acids. Recrystal-
lization of the mixed acids from 6 ml of water gave 1.05 g (34% yield)
of difluorofumaric acid, m.p. 267 °. After a second recrystallization it
melted at 268-270 ° .
C. Hydroxy Acids
3-Fluorolactic Acid
4-F luorometh yl-2,2-dimeth yl- I ,8-dioxolane T oluenesul] onate 44 (Meth-
od B). In a three-necked l-liter flask, fitted with stirrer, condenser, drop-
ping funnel, gas-inlet, and thermometer, 4-1~ydroxymethyl-2,2-dimethyl-
1,3-dioxolane p-toluenesulfonate (300 g) was added to a mixture of
carefully dried potassium fluoride (90 g) and anhydrous diethylene
glycol (300 ml), and the mass was heated quickly until reaction set in,
and then brought slowly to 150 °. After 5 minutes at this temperature, a
current of air was passed through the mass for l0 minutes. The distillate
was dissolved in ether and washed successively with 10% hydrochloric
acid, sodium hydrogen carbonate solution, and water. Distillation of the
dried product gave at 146-147 ° at 760 mm the fluorine compound (123 g,
84%; larger batches gave somewhat lower yields).
I-Fluoropropane-2,3-diol2 ~ A mixture of 4-fluoromethyl-2,2-dimethyl-
1,3-dioxolane (29.8 g) in water (36 ml) and 37% hydrochloric acid (11
ml) was heated under reflux for 0.5 hour. The solvents were removed
under vacuum and water was added to the residue. This water was again
removed under vacuum and the remaining sirup was taken up in ethanol
(50 ml). The ethanol solution was treated with solid sodium bicarbonate
and filtered. Distillation gave pure l-fluoropropane-2,3-diol: 14.3 g, 68%,
b.p. 100-102 ° at 10 mm or 117 ° at 30 mm, n~ 25 1.4225; lit. b.p. 117 ° at
30 ram, n. 25 1.4230.
E t h y l Fluorolactate. 45 A mixture of 1-fluoropropane-2,3-diol (11.0 g),
water (36 ml), and 70% nitric acid (50 ml) was heated on a steam bath.
When the temperature of the mixture reached 80-90 °, a spontaneous
reaction set in and the reaction mixture was removed from the steam
bath. (Caution: the reaction can become violent.) When the reaction
subsided, the mixture was maintained at 55 ° for 5 hours and then left
at room temperature for 12 hours, heated an additional hour at 65 °, and
concentrated at 65 ° (20-30 mm). After addition of water (25 ml), the
distillation was repeated and continued to dryness, leaving as a sirup
'4 E. D. Bergmann, S. Cohen, and I. Shahak, J. Chem. Soc. p. 3448 (1961).

~L. K. Gottwald and E. Kun, J. Org. Chem. 30~ 877 (1965).
crude fluorolactic acid. The sirup was heated under refux with anhydrous
ethanol (20 ml), benzene (60 ml), and toluenesulfonic acid (0.5 g) while
water was continuously removed by distillation. When no more water was
collected, the solvents were removed and the residue was dissolved in
ether (50 ml). The ether solution was treated with solid sodium bicar-
bonate and filtered, and the product was isolated by distillation, yielding
ethyl fluorolactate: 8.8 g, 54%, b. p. 95-98 ° at 30 ram.
Fluorolactic Acid. 45 Ethyl fluorolactate (8.8 g) was dissolved in 10%
hydrochloric acid (50 ml), and the mixture was heated on a steam bath
at 95 ° for 40 minutes. After the mixture had stood for 12 hours at room
temperature, the solvents were removed under vacuum (60 ° at 20-30
ram). Water (30 ml) was added and the distillation was repeated and
continued to dryness. The remaining sirup ~vas purified by distillation,
yielding fluorolactic acid: 4.3 g, 61%, b.p. 124 ° at 5 mm. A sample was
distilled for analysis: b.p. 122-123 ° at 3 mm.
Resolution o/3-Fluorolactic Acid 25
(--)-8-Fluorolactic acid was prepared by resolution of the raccmic
acid using morphine. (-+-)-3-Fluorolactic acid (26.5 g, 245 mmoles) and
37.9 g (125 mmoles) of morphine monohydrate were dissolved in water
and allowed to crystallize. After four fractional crystallizations, 27.3 g
(93 mmoles, 76% yield) or morphine (--)-3-fluorolactate was collected,
m.p. 222-223 ° dec, [ a ] ~ --90.8 ° (e. 5, water). The morphine was re-
generated with ammonium hydroxide, and the free acid was obtained by
ion exchange on a Dowex 50 column, giving 50 ml of a final solution
containing 8.59 g (79.5 mmoles, 65%) of (--)-3-fluorolactic acid [a]~
--2.74 ° (c. 17.18, water). The sodium salt had [a] ~ -{-8.34° (c. 10.8,
water), and the zinc salt had [~]~5 q_8.00 o (c. 5, water). Neutralization
of the acid with (+)-a-phenylethylamine afforded (~-)-a-phenylethyl-
ammonium (--)-3-fluorolactate crystallizing from ethanol as prisms of a
monohydrate; m.p. 87-88 ° [a] ~ +7.30 ° (c. 5.0, water).
(q-)-3-Fluorolactic acid was obtained from the mother liquors of the
morphine (--)-3-fluorolactate. The solution was treated with ammonium
hydroxide, and the free acid was obtained by ion exchange using a Dowex
50 column. Neutralization of the acid with (--)-a-phenylethylamine gave,
after four fractional crystallizations from ethanol, 8.20 g (35.8 mmoles,
29%) of (--)-a-phenylethylammonium (q-)-3-fluorolactate as prisms of
the monohydrate, m.p. 87-88 °, [a] ~ --7.30 ° (c. 5.0, water). Treatment
with sodium hydroxide solution, extraction of the amine with benzene,
and recovery of the free acid by ion exchange gave 3.11 g (28.8 mmoles,
24%) of (T)-3-fluorolaetic acid, [a]~ q-2.76 ° (c. 12.44, water). The
[79] ~_~voRo ANXLOGS 651
following salts were prepared: sodium salt, [a], --8.35 ° (c. 12.57, water) ;
barium salt, laiD --4.3 ° (C. 15.0, water); and calcium salt, [a]o --4.8 °
(c. 12.3, water).
Rotatory Dispersion Curves. These were measured with a Bendix
Model 460-C or Cary Model 60 spectropolarimeter using 1-mm or 1-cm
cells at 25 ° and were reproducible to within 5%. Rotations are given
below only for (1) the highest and lowest wavelengths measured, and
(2) peaks and troughs. Since measurements taken for enantiomeric pairs
agreed within 5%, the dispersion curve of only one isomer is described;
(-{-)-3-fluorolactic acid: RD (c. 2.16, water: [a]a2o ~55.5 °, [a]~24~
~1778 ° (peak), [a]2oo--2732°; (--)-sodium 3-fluorolactate from (~-)-
3-fluorolactie acid): RD (c. 1.4, water): [a] 820 --10.73 °, [61222.5 --307.5 °
(peak), [61203 --357.5 °, L-(--)-calcium lactate, [a], --4.5 ° (e. 10.0,
water); RD (e. 0.61, water): [6132o --52.3 °, [a]~4o --73.6 ° (trough),
[a] ~25 --65.4 ° (peak), [a] ~o5 --1112 °.
Fluoromalic Acid 46 (Method 9)

Methyl FIuoromaIate. Ethyl ethoxalyl fluoroacetate (10 g, 0.54 mole)
in methanol (60 ml) was added dropwise to a stirred solution of potas-
sium borohydride (6 g, 0.112 mole) in methanol (60 ml). The tempera-
ture rose to 50 ° , and dropped to 25 ° when the reduction was complete.
Stirring was continued for a further 30 minutes. Excess of methanolic
hydrogen chloride was added to the resulting pale green solution. Methyl
borate was removed by distillation of the filtered solution with repeated
addition of methanol. After neutralization with lead carbonate, filtration,
and concentration to dryness under reduced pressure, the product was
extracted with ether. Removal of the solvent gave a colorless viscous
liquid which on distillation yielded dimethyl fluoromalate (5 g), b.p. 90 °
at 0.5 mra. This gave a positive test for an a-hydroxy group with ferric
chloride-phenol. It gave an absorption maximum at 300 m~ and an
infrared band at 1050 cm-1 (C-F).
Fluoromalic Acid. ~ Dimethyl fl-fluoromalate was hydrolyzed by
dissolving 4.8 g in a mixture of 67 ml of concentrated hydrochloric acid
and 133 ml of concentrated glacial acetic acid. After standing for 3 days
at room temperature, the solution was evaporated to dryness in a
vacuum and placed in a vacuum desiccator over NaOH and P205 for 3
days. All attempts to crystallize this material were unsuccessful. The
material was then chromatographed on a silica gel column, 1.7 by 35 cm.
The column was eluted first with 15% butanol in chloroform and then
,e N. F. Taylor and P. W. Kent, J. Chem. Soc. p. 2150 (1956).

with 35% butanol in chloroform. The water layer from each fraction
(separated from the CHC13) was decolorized with Norit to remove the
phenol red indicator, concentrated to a small volume in a vacuum,
acidified with H2SO~, and extracted with ether. After drying over NaSO~,
each fraction was evaporated to dryness, and the residues were crystal-
lized from ethyl acetate-ligroin (66-75°). Only fractions A (0.097 g) and
B (1.31 g) yielded crystalline solids; the other fractions were oils and
were discarded.
The spectra of the two isomers are different, but a definite stereo-
chemical assignment cannot be made on this basis. The OH stretching
frequencies from 2.8 to 3.5 ~ are essentially identical in bot.h compounds.
The C--O frequency is 5.75 /~ for fraction A, and in fraction B this
absorption is split into two peaks at 5.64 and 5.81/~. The two compounds
differ considerably in the 8.5-10.0 /~ range which is due to the C-OH
and C-F absorptions. Compound A absorbs at 8.8 and 9.23 /~, whereas
B shows peaks at 8.56, 9.0, 9.16, and 9.89/~. When the distillation of the
dimethyl fl-fluoromalate was continued after the main fraction had
been recovered, a higher boiling fraction, b.p. 146-178 ° at 18 mm, was
obtained. When this fraction (4.61 g) was hydrolyzed and chromato-
graphed as above, fl-fiuoromalic acids A (0.047 g) and B (0.375 g) were
obtained.
Difluoromalic Acid 1T (Method 9)

Reduction of Difluorooxaloacetate to Difluoromalate. For reduction
of diethyl difluoro oxaloacetate, 11.0 g of this ester was added during
continuous mechanical stirring to a solution of sodium borohydride in
pyridine (0.47 g in 50 ml) while the temperature of the reaction mixture
was kept between 20 and 25 ° . After the addition was complete, the
mixture was stirred for 1.5 hours at room temperature. Then 40 ml of
H~O was added and the pyridine-H20 mixture was evaporated under
vacuum at approximately 70 °. A dark oil was obtained, to which 40 ml
of H20 was added. The mixture was then extracted by shaking with
chloroform. The chloroform extract was washed with one portion (40 ml)
of H~O, then dried over anhydrous MgS04. Distillation (at 0.5 ram)
yielded 3.1 g (27% yield) of diethyl difluoromalate, b.p. 70-75 ° (at 0.5
mm).
Difluoromalic Acid. Free acid was obtained by heating the ester
(1.7 g) under reflux with 10% HC1 (4.0 ml) for 3 hours. The HC1 was
removed under vacuum, leaving a thick sirup, which crystallized on
standing (0.7 g, 5 5 ~ yield). Titration yielded the following dissociation
constants: pK1 ~ 2.05, pK~ ~ 3.25. Equivalent weight: calculated 85;
found 86. The molecular weight of the anhydrous acid is 170.
[79] FLVORO XNALOGS 653
2-Fluorocitric Acid (Method i)

Diethyl 2-Fluorocitrate. 28 Diethyl fluorooxaloacetate (15.4 g 0.075
mole) was added over a period of 30 minutes to a solution of malonic
acid (8.0 g 0.075 mole) in pyridine (20 ml). The reaction mixture was
left at room temperature for 3 hours, then heated on a steam bath for
15-20 minutes until evolution of COs ceased, cooled to room temperature,
and acidified with slight excess of 10% H~SO4. The diester of monofluoro-
citric acid was extracted with three to four portions of 50 ml of diethyl
ether. This organic phase was dried with solid anhydrous Na~S04. After
removal of ether under vacuum, 10 g of brown oil (diethyl monofiuoro-
citric acid) remained. Analysis of infrared spectra revealed absorption
maxima at 2.9, 3.35, 5.75, 8.25, and 11.6 /~.
Triethyl monofluorocitrate was prepared from the diester as follows.
Diethyl monofiuorocitrate (10 g) was dissolved in 80 ml of absolute
ethanol, to which 130 ml of benzene and 0.5 g of p-toluenesulfonic acid
were added. This mixture was heated under reflux over a period of 20-30
hours. During this time almost 100 ml of distillate, containing ethanol,
benzene, and water, were removed by means of a Dean-Stark trap. The
solvent was further removed under vacuum, leaving 10.6 g of crude
triethyl fluorocitrate. This material was dissolved in 50 ml of ether,
washed with dilute (2%) aqueous solution of Na2C03, and finally dried
with anhydrous Na~SO~. The product was distilled, and yielded purified
triethyl monofluorocitrate (6.3 g, 29%), b.p. 120 ° at 0.10 ram. Refractive
index (riD28), 1.4380; infrared spectrum (neat) yielded absorption max-
ima at 2.9 ~ (strong) 3.37 ~ (strong); 5.75 ~ (strong); 6.8 ~, 6.9 ~;
7.28/~, 7.3 ~ (broad) ; 7.41 ~, 8.4 ~ (strong, broad) ; 8.8 ~, 9.1 ~ (strong) ;
and 11.6 ~ (strong). The NMR (nuclear magnetic resonance) spectrum
of the ester is shown in Fig. 4. Numerical values obtained by NMR anal-
ysis are as follows: CH3, r 1.3 multiplet; CH2, r 2.95 (singlet); CH2
(ethyl), T 4.20 (multiplet) ; OH, 7 3.95 (singlet) ; CHF, T 4.6, 5.4 (both
doublets).
Fluorocitric Acid. ~7 Triethyl fluorocitrate (3.3 g, 0.011 mole) was
dissolved in 100 ml of 0.5 N N a 0 H and left at room temperature for 24
hours. The solution was then treated with a large excess of Dowex 50-H ÷.
After removal of the resin the yellow solution was decolorized with acti-
vated charcoal and solid fluorocitrie acid obtained by freeze drying.
Cyclohexylamine Salt. ~' The acid was dissolved in ether-ethanol (4:1)
and a slight excess of purified cyclohexylamine was added, whereupon a
white precipitate was formed. After removal of excess cyclohexylamine
~7D. W. Fanshier, L. K. Gottwald, and E. Kun, J. Biol. Chem. 237, 2588 (1962).
654 P1],P,PAliA.'I"TOI~" OF COMPOUN'])8 [?9]
(by washing the precipitaLe with ether), 2.6 g of salt of fluorocitric acid
was obtained, which was recrystallized from absolute ethanol. ++
Separation of synthetic fluorocitric acid into weaker and stronger acid
components in proportion 4:1 was achieved by high voltage electro-
phoresis, as described later.
Fluorocitric Acid 28 (Enzymatic Synthesis)
Fluoroacetyl-CoA. Preparation of fluoroacetyl-CoA from fluoroacetic
anhydride was carried out by a procedure similar to that described by
Brady +9 with the modification that fluoroacetic anhydride was allowed
I000
500
250
I00
50
6.0
,ll I
5.0 4.0 3.0 2.0
,+o( ++,
1.0
,,
0 ppm { <~ }
S LANE
CRF CHF CH CH2 CHI3 I
r~J+l+,,,l,~,,l,,m+lJ ,ll~J,,It,,,l,,+,l,,,,l+,+, ,+l I,,,IIII
Fzo. 4. Numleat mal~eti+ resonance spectrum of ttiethyl fluorocitrate. Reproduced

from the J. Bial. Chem. [<239, 426 (1964)], with permission of the American Society
of Biological Chemists.
to react with CoA in place of the mixed anhydride of ethyl formate and
fluaro~e+ic acid, as done by Brady. Fluoroacetic anhydride was purified
by distillatio~a (final product: b.p. I00-105 ° at 25 ram) and stored in
sealed 5 ml vials under nitrogen. Preparation of fluoroacetyl-CoA was
done immediately before enzymatic experiments as follows. CoA was
dissolved i n 0.1 M KHCOs and the solution was chilled to 0 ° in an ice
bath. A slight excess of ltuoroacetic anhydride in ether was added and
the mixture wa+ shaken vigorously. The reaction between the acid an-
hydride and the CoA was instantaneous. The pH of the reaction mix-
= IR spectrum published in footnote 47.
'+ R. O. Brady, J. Biol. Chem. ~17, 213 (195~).
ture containing fluoroacetyl-CoA was quickly adjusted to 6.1 with dilute

HC1, excess fluoroacetic anhydride was extracted with ether, and traces
of ether were removed from the aqueous phase by a stream of nitrogen,
while the solution was kept at 10°. This method yielded fluoroacetyI-CoA
free of pyridine.
Monofluorocitrate. L-Malate, 500 micromoles, and NAD ÷, 200 micro-
moles, were dissolved in 100 ml of deionized water and the pH was ad-
justed to 7.0 with solid KHC03. Condensing enzyme (50 rag; specific
activity = 2.6 micromoles of citrate per milligram of pro%ein per minute)
and malic dehydrogenase (10 mg; specific activity = 1.45 micromoles of
NADH per milligram of protein per minute) in 10 ml of 5 mM Tris-
hydrochloric buffer (pH 7.4) were added, and the temperature was
brought to 25 ° in amber Erlenmeyer flask (250-ml volume) equipped
with magnetic stirrer, water jacket, thermometer, and pH electrodes.
An aqueous solution of freshly prepared fluoroacetyl-CoA, containing
100 mieromoles in 12.5 ml (pH 6.0), was kept separately in an ice bath,
and aliquots of 0.5-2.0 ml were added successively to the reaction mix-
ture. Samples of 1 ml were withdrawn and analyzed spectrophoto-
metrically for NADH. During the course of the reaction the pH was
8.3, and 65 micromoles of NADH were formed. After 3 hours the reaction
mixture was brought to 0 ° and added directly to a large excess of moist
Dowex 50 (H+). The mixture was stirred for 10 minutes at 10°, and the
resin then was removed by filtration, resulting in the removal of protein
and a large amount of CoA and pyridine nucleotide. The clear, acidic
filtrate was placed under vacuum for 10 minutes (to remove COs), then
lyophilized to dryness, leaving a white amorphous powder. This was
taken up in three 2-ml portions of water, and the pH adjusted to 7.0
with solid KHC03. The faintly yellow solution was decolorized by
several successive treatments with charcoal and stored at --15 ° . The
resulting solution was free of nucleotides and other ultraviolet light-
absorbing material, and analyses for fluorocitrate yielded a total amount
of 60 micromoles, in good agreement with kinetic spectrophot~metric
data. This method yields the erythro diastereoisomer.
Electrophoretic Separation. Fluorocitric acid prepared enzymatically
from monofiuorooxaloacetate and acetyl-CoA (designated as fluorocitrate
I), from fluoroacetyl-CoA and oxaloacetate, as described above (called
fluorocitrate II), and obtained by chemical synthesis were subjected to
high-voltage paper electrophoresis both on an analytical and a prepara-
tive scale. Fluorocitric acid was detected by spraying the paper with
pyridine-acetic anhydride. ResuIts of such an electrophoretic separation
are shown in Fig. 5.
Paper electrophoresis was carried out on a temperature-controlled
high-voltage apparatus at 0 ° (Pherograph; Brinkman Instruments, Inc.).

The solvent was 0.1 M ammonium formate-formic acid mixture of pH 2.8.
Current density was 50 ma with a potential gradient of 33 V/cm. Time
of electrophoresis was 1.5-2 hours. Part A shows the separation of a
mixture of chemically synthesized and enzymatically formed fluoro-
citrates. Synthetic fluorocitrate separates into components X and Y.
Enzymatically formed fluorocitrate I, from fluorooxaloacetate and acetyl-
B C
0 0 0 0 (___ Y j
¥
0 0 0 O0
000o00 X 0
0000 0 o( x 3
to
¢..)
I..4 ,.¢ +
"I"
to to to
I:). C_) ,-4 (..)
I 2 3 4 5 6 I 2 3 4 5 6 7 I 2
Fio. 5. Electrophoresis of fluorocitricacid from enzymatic and chemical syntheses.

CS: chemicallysynthesized fluorocitric acid; P: picric acid marker; fluorocitricacids
I and II (see text). For other details see text. Reproduced from J. Biol. Chem. [239,
428 (1964)] with permission of the American Society of Biological Chemists.
CoA, shows two spots, one of which is unreacted fluorooxaloacetate.

En~ymatieally formed fluorocitrate II, from fluoroacetyl-CoA and oxalo-
acetate shows a single spot. Part B shows enzymatically formed fluoro-
citrate I after treatment with 2,4-dinitrophenylhydrazine-charcoal; the
fluorooxaloacetate has been removed. The same is found after isolating
fluorocitrate I by paper chromatography in 1-butanol-pyridine-water,
3:2:15 (v/v). Part C shows a large-scale separation of components X
[79] VLUORO ANXLOGS 657
(nL-erythro) and Y (DL-threo), the diastereoisomers from synthetic

fluorocitric acid. All samples were located by spraying with pyridine-
acetic anhydride. Components X and Y were isolated by elution from
the filter paper followed by crystallization of the cyclohexylamine salt.
The latter were prepared directly from the free acids, and then analyzed
without further purifcation. Neutralization equivalents determined for
components X and Y: calculated = 70; found = 70. The characteristic
melting points of cyclohexylamine salts of fluorocitrie acid are as follows:
Unresolved mixture of components X and Y, m.p. 148-152 ° (dec.);
component X, m.p. 140-150 ° (softens at 130-140 °, dec.) ; component Y,
m.p. 190-192 ° (dec.). The melting points were found to be dependent
upon the rate of heating. The results were obtained with a temperature
increase of 1° per minute.
2,2-Difluorocitric Acid 37 (Method 2)

Diethyl 2,2-Difluorocitrate. To a mixture of 3.2 parts of malonic acid
and 7 parts of pyridine is slowly added 6.7 parts of diethyl a~-difluoro-
oxaloacetate. The mixture is then acidified with dilute sulfuric acid and
extracted with ether. Evaporation of the ether leaves 7.8 g (9270 yield) of
diethyl 2,2-difluorocitrate, HOOCCH2C (OI-I) (C00C2H5) CF~COOC2Hs.
After recrystallization from ether benzene in 84% return, it melts at 113-
114 ° .
2,2-Difluorocitric Acid Monohydrate. Diethyl difluorocitrate (31.2
parts) is dissolved in 75 parts of water containing 13.2 parts of sodium
hydroxide and allowed to stand for 65 hours at 25 ° . Hydrochloric acid
(d, 1.18, 35 parts) is then added and the solution is extracted with ether.
The ether is dried over magnesium sulfate and evaporated to give 23
parts of sirupy difluorocitric acid. This is allowed to stand until crystal-
line and is recrystallized from a mixture of ether and benzene. The
compound is the monohydrate of 2,2-difluorocitric acid, which melts at
89-90 ° .
$,4-Difluorocitric Acid 5° (Method I, Abstract)

Diethyl fluorooxaloacctate (206 g, 1 mole) was added to a saturated
aqueous solution of potassium acetate (98 g, 1 mole). After several hours
at room temperature, an oil was extracted with ether. Distillation gave
a forerun (30 g) of starting ester, and diethyl 2,4-difluorocitrate (120 g,
80%), b.p. 145-150 ° at 1 mm, m.p. 68 °. Infrared spectrum: 3495 st, 3000
st, 1745 v st, 1480 m, 1455 m, 1370, 1340, 1300 m, 1170 v st, 1015 v st,
965 m, 865, 850 st. Diethyl 2,4-difluorocitrate (50 g, 0.166 mole) was
~D. Rouge and H. Gault, Compt. Rend. Acad. Sci. 251, 94 (1960).
658 PP~PAmTION OF COMPOUNDS [79]
refluxed 6 hours in 5 N hydrochloric acid (200 ml). After vacuum evapo-

ration of the solution, hygroscopic crystals were obtained; yield was not
reported. Analysis was carried out on the barium salt, Bas(C~H30,F2)2.
7 H~O.
D. K e t o Acids
Fluoropyruvic Acid 2° (Method 8)

Diethyl fluorooxaloacetate (41.2 g, 0.2 mole) was refluxed with 3 N
hydrochloric acid (300 ml) at 105 ° for 1 hour. After standing overnight,
the solution was concentrated in vacuo to a very low volume at 40-45 ° at
5 ram. The residue was fractionated and fluoropyruvic acid condensed as
a white solid in the condenser; the solid was removed with a glass
spatula from the Claisen head, yield 17 g (80%). It was free of oxalic
acid as shown by the absence of the color reaction with diphenylamine.
The product was purified by sublimation in vacuo (at 70-80 ° at 0.5-0.7
ram) and obtained as a fine white crystalline powder. The melting point
of the product was 86 ° immediately after drying in vacuo over P20~. The
product is very hygroscopic. Semicarbazone (recrystallized from 9 5 ~
alcohol) turns brown at 198-200 ° and decomposes at 205 °. Dinitrophenyl-
hydrazone (crystallized 4 times from alcohol) begins to melt at 163 ° and
decomposes at 165 ° .
Fluorooxaloacetic Acid (Method 2)

Diethyl Fluorooxaloacetate. TM To a suspension of alcohol-free sodium
ethoxide, prepared from metallic sodium (23 g) in anhydrous ether (200
ml), freshly distilled diethyl oxalate (146 g) and, after a few minutes,
ethyl fluoroacetate (106 g) were added dropwise. The mixture was left
overnight at room temperature, and the solid yellow enolate was filtered
off, washed several times with ether until the filtrate was colorless, and
dried (yield 180 g, 7 9 ~ ) . The enolate is a cream-colored, hygroscopic
powder which becomes yellow on storage, is soluble in water and alcohol,
insoluble in ether and hydrocarbons, and gives a bluish-brown color
reaction with alcoholic ferric chloride solution.
The enolate (114 g, 0.5 mole) was washed with anhydrous ether until
the washings were colorless, suspended in ether (200 ml), and cooled to
--20 °. Then 5 N hydrochloric acid (100 ml) cooled to --20 ° was rapidly
added to the ethereal suspension and shaken well. The ether layer was
separated, and the mixture was extracted with two 50-ml portions of
ether. The combined ether extracts were washed with cold water and
dried (Na2S04), and the solvent was removed. Distillation of the residue
yielded diethyl fluorooxaloacetate as a pale yellow liquid (51 g, 50%),
b.p. 98-100 ° at 1.1 ram. Melting point of the 2,4-dinitrophenylhydrazone,

123_124 ° .20
Fluorooxaloacetic Acid (FOAA).~e Diethyl fluorooxaloacetate (25 g)
was dissolved in 240 ml of glacial acetic acid, then 120 ml of concentrated
hydrochloric acid was added. The mixture was kept at room temperature
for 3 days, then evaporated to dryness at room temperature under re-
duced pressure by means of a Rinco rotating evaporator. The residue
was 18.4 g of crude crystalline FOAA (85.5% yield). This was purified
by recrystallization from ether-petroleum ether (b.p. 30-60°). After two
recrystallizations which resulted in about 5 0 3 recovery, the m.p. was
86-87 ° (decarboxylation) as determined with a Fisher-Johns block. Anal-
ysis: calculated for C4HaOsF: 1.5 H20; C, 27.13; H, 3.41, F, 10.73.
Found: C, 27.59, H. 3.67; F, 10.7. Neutralization equivalent: calculated
88.5; found 89.0. 2,4-Dinitrophenylhydrazone: m.p. 210-214 ° (starts de-
composing between 90 ° and 150 ° , depending on the rate of heating).
An aqueous solution of FOAA gives a deep purple-red color with
aqueous FeCls. The color reaches its maximum intensity after about 10
minutes. The crystalline acid is stable for about a month at room tem-
perature, then slowly decarboxylates. FOAA is extremely soluble in
water.
Analysis and neutralization equivalent show that FOAA contains 1.5
mole of H20. Spectral evidence indicates that the keto group is in the
hydrated form. This would be expected from the strongly electron-
attracting properties of fluorine which increase the positive, character of
the neighboring carbon atoms.
The ultraviolet spectra of aqueous solutions of OAA and FOAA were
determined at pH 1.5 (OAA, E ~ = 612; FOAA, E : 96) and at pH 11
(OAA, E : 7 9 8 ; FOAA, E - - 2 4 6 ) at ) ~ a x : 2 6 5 m~ for OAk and
~,ma, : 270 ms for FOAA. It is apparent that in aqtieous solutions FOAA
is not appreciably enolized. This is further confirmed by infrared
spectra. TM
Difluorooxaloacetic Acid 1~ (Method E)

The sodium enolate of diethyl oxaloacetate (10.5 g, 0~)5 mole) was
suspended in 25 ml of absolute ethanol; the suspension, while stirred
mechanically, was brought to --15 ° by external cooling. The temperature
of the reaction mixture was kept between --10 ° and 0 ° throughout the
entire subsequent operation. Ferchloryl fluoride gas (FC1Os) was passed
into the mixture by means of a tube submerged into the suspension. After
about 25 minutes, a yellow color developed in the reaction mixture. The
pH then varied between 7 and 8. While bubbling of FC10:~ was continued,
a solution of sodium ethoxide (prepared by dissolving 1.6 g of metallic
sodium in 30 ml of absolute ethanol) was added in small portions; the pH
of the reaction mixture was brought back to 7-8 before each subsequent
addition of the base. When addition of sodium ethoxide was completed,
the reaction mixture was flushed with nitrogen and poured into 125 ml
of water in order to dissolve all inorganic salts. The aqueous solution was
extracted with 4 or 5 portions of 30-40 ml of diethyl ether, and the
ether solution in turn was washed with one portion of water and dried
over anhydrous MgS04. The diethyl ester of difluorooxaloacetic acid was
obtained by distillation of the ether extract. Yield, 6.0 g (54%) ; b.p. 64 °
at 1 mm; refractive index (no22), 1.3982. Infrared spectrum of the liquid:
~max ~ 3.35, 5.6, 5.75, 6.8, 6.9, 7.25, 7.6, 7.8, 7.9, 9.1, 9.8, and 11.5 ~.
Diethyldifluorooxaloacetic acid 2,4-dinitrophenylhydrazone has a melting
point of m.p. 102-102.5 °.
The free acid was obtained by adding 2.24 g of the ester (0.01 mole)
to 12.5 ml of concentrated HC1 and stirring at room temperature (20 °)
for 3 days. Alternatively the ester can be hydrolyzed by heating under
reflux for 1 hour with 10% HC1. The solution was then evaporated under
reduced pressure to a thick sirup. Addition of 2 ml of trifluoroacetic acid
and scratching the inside wall the glass container with a glass rod yielded
a crystalline product, which was collected and washed with small
amounts of trifluoroacetic acid. The extremely hygroscopic product, 1.0 g
(50% yield), was the nearly pure free acid, m.p. 115-120 °. Recrystalliza-
tion from trifluoroacetic acid gave the pure, less hygroscopic monohy-
drate, m.p. 119-120. Infrared spectrum (Nujol mull): Xmax= 2.9, 5.75,
7.7, 8.1, 8.35, 8.9, 9.1, and 11.0 ~. Titration of the acid yielded the follow-
ing acid dissociation constants: pK1 = 1.9, pK2 = 2.90. Equivalent
weight: calculated, 93.0; found, 93.5. The molecular weight of the mono-
hydrate is 186.
$,$-Difluoro-2-ketoglutaric Acid 37 (Method G)
Step 1. Methyl 2-Chloro-2,3,3-trifluorocyclobutanecarboxylate from
Methyl Acrylate and Chlorotrifluoroethylene. A stainless steel bomb is
charged with 300 parts of methyl acrylate, 1 part of hydroquinone, and
300 parts of chlorotrifluoroethylene. The mixture is heated at 180 ° for 10
hours. Distillation of the product gives 383 parts of methyl 2-chloro-
2,3,3-trifluorocyclobutanecarboxylate boiling at 163-165 °, n~25 1.3952.
The yield is 74~.
Step 2. Methyl 2,3,3-Trifluoro-l-cyclobutene-l-carboxylate ]rom
Methyl ~-Chloro-2,3,3-trifluorocyclobutanecarboxylate. In a glass reac-
tion vessel fitted with stirrer, reflux condenser, and addition funnel are
placed 303 parts of methyl 2-chloro-2,3,3-trifluorocyclobutanecarboxylate
and 350 parts of ether. Triethylamine (160 parts) is added and the
[79] FLUORO XNXLOGS 661
mixture is refluxed for 0.5 hour. Water (800 parts) is added and the
ether layer is washed with 107o hydrochloric acid and water. After the
ether solution is dried over magnesium sulfate, it is distilled to give 195
parts (797o yield) of methyl 2,3,3-trifluoro-l-cyclobutene-l-carboxylate,
b.p. 128-129 °, no2s 1.3850.
Step 3. Methyl 3,3-Difluoro-~-methaxy-l-cyclobutene-I-carboxylate
]rom Methyl £,8,8-trifluoro-l-cyclobutene-I-carboxylate. A glass reaction
vessel is fitted with stirrer, reflux condenser, device to add powder, and
ice bath. In the vessel are placed 178 parts of methyl 2,3,3-trifluoro-1-
cyclobutene-l-carboxylate and 350 parts of anhydrous ether. Solid
sodium methylate (58 parts) is then added in portions. After all is added,
the ice bath is removed and the mixture is stirred for 1 hour. The
sodium fuoride is filtered off and rinsed with ether. The filtrate is dis-
tilled to give 114 parts (64~ yield) of methyl 3,3-difluoro-2-methoxy-1-
cyclobutene-l-carboxylate, b.p. 179.5-181 °, n~~5 1.4302.
Step ~. Dimethyl ~,~-Difluoro-~-oxoglutarate ]rom Methyl 8,3-
Difluoro-~-methoxy-l-cyclobutene-I-carboxylate. A solution of 25 parts
of methyl 3,3-difluoro-2-methoxy-l-cyclobutene-l-carboxylate in 130
parts of methylene chloride is cooled in a dry ice-acetone mixture and
ozone is passed in until the blue color of ozone becomes evident. For
decomposing the rather stable ozonide a simple still is arranged with a
still pot in an oil bath maintained at 200 ° and an addition funnel in the
still head. The ozonized solution is added slowly. Material collected in
the receiver of the still is heated on a steam bath to evaporate methylene
chloride and then returned to the still, after which all of it remains in
the still pot. The product is distilled at 1 mm in a simple still to get rid
of considerable tar and then fractionated to give dimethyl 4,4-difluoro-2-
oxoglutarate, b.p. 89-91 ° at 3 ram, n~25 1.4064.
The ozonide may also be decomposed by dissolving it in 9 8 ~ acetic
acid and carefully adding zinc dust. This gives a product, b.p. 86--88° at
1 ram, n~~" 1.4140, in 46% yield which is a mixture of dimethyl
4,4-difluoro-2-oxoglutarate and dimethyl 4,4-difluoro-2-hydroxyglutarate.
~,~-Difluoro-P,-oxoglutaric acid is obtained from the ester by shaking
with 4 parts of concentrated hydrochloric acid for 24 hours and removal
of the hydrochloric acid under vacuum.
Ethyl $-fluorooxalsuccinate and Ethyl ~-fluoro-~,-ketoglutarate~6
(Method E)
Triethyl a-FIuorooxalosuccinate. An efficiently stirred suspension of
potassium triethyl oxalosuccinate (37.4 g) in absolute ethanol (90 ml)
was cooled with an ice-salt bath. Perchloryl fluoride was passed into the
mixture by means of a subsurface tube while the reaction temperature
TABLE IX
LISTING OF SOME MONO- AND DIFLUORO ALIPHATIC ACIDS
FOUND IN THE LITERATURE THROUGH 1966"
Category Acid Ester Method Reference°
One- and two-carbon acids

Fluoroformie bp 55.5A 1-3
Oxalyl fuoride bp 107-8
A 3
Fluoroaceticb bp 167-8 bp 114-8
A 2
B 4, 5
D 6
12 7
Difluoroacetic bp 134-5 bp 85-6 me A 2
Bromofluoroacetie bp 103 (20) bp 68 (34) G 8, 9
Chlorofluoroacetie bp 162 bp 110-1 me G 10
Iodofluoroacetie bp 60-3 9 11
Nitrofluoroacetic bp 72-5 (30) G 12
Dichlorofluoroacetic bp 116-6.5 A 2
me
Dibromofluoroacetic mp 40-1 bp 82 (33) G 13
Chloronitrofluoroacetic bp 70-1 (1) G 14
Bromodifluoroacetic bp 140 bp112 (700) G 13
Three-carbon monocarboxylic acids

2-Fluoropropionic bp 66 (13) bp 122 A 15
B 5, 16
D 6
E 6
5 6
9 2
12 7
3-Fluoropropionic bp 78.5-9.5 bp 122.5-3 9 2, 17-20
(12)
2,2-Difluoropropionic bp 140-1 G, 8 21
2-Fluoroaerylic bp 110 (728) 3, 8 22-25
9 26
3-Fluoroacrylic nd ni 12 27
3,3-Difluoroacrylie nd 9 24
Fluoropropiolic bp 128 8 28
(fluoride)
2,3-Difluoroacrylic nd 12 29
2-Fluoro-3,3-dichloroacrylic mp 68 12 30
2,3-Difluoro-3-chloroacrylic nd 12 30
2,3-Diehloro-3-fluoroacry lic nd 12 30
3,3-Difluoro-2-chloroacrylic bp 62-5 ni 12 31
3-Methoxy-2-fluoroacrylic bp 82-4 (14) 9 32
2-Amino-3-fluoropropionic mp 228-30 9 33
HCI
a Key to references starts on p. 668.

* For explanation see text following the table.
TABLE IX--Continued
3-Amino-2-fluoropropionic mp 190-5 4 34, 35
dec. HCI
3-Chloro-2-fluoropropionic bp 115-20 G 36
(22)
2-Chloro-2-fluoro-3-hydroxy- bp 82 (6) G 37
propionic
2,3-Dichloro-3,3-difluoro-2-nitro- mp 19 bp 72 (55) G 14
propionic
3-Bromo-2-fluoropropionic bp 100-10 (6) G 36
mp 74.5-5.5
2,3-Dibromo-2-fluoropropionic bp 67-7.5 A 25
(3.2)
Fluorolactic bp 124 (5) bp 95-8 (30) 9 38-43
2-Fluoro-3-hydroxypropionic mp 94.5-5.5 3, 12 35, 44-46
G 47
2,2-Difluoro-3-hydroxypropionic mp 98-9 am bp 69.5 (7) G 37
3,3-Dimethoxy-2-fluoropropionic bp 94-5 (15) 2 32
2-Fluoroformylacetic nd 2 48
Fluoropyruvic mp 86 bp 85 (14) 9, 8 39, 49, 50
me
Bromofluoropyruvic bp 90-2 (1.5) bp 90-2 (30) 8 39, 51
Three-carbon dicarboxylic acids
Fluoromalonic mp 135-6 bp 121-2 (30) 6 52, 53, 54
8
Difluoromalonic mp 205-6 am bp 94-5 (23) E 53-55
Bromofluoromalonic bp 126-8 (27) 12 56
Fluoronitromalonic bp 81-3 (15) E 6
Four-carbon monocarboxylic acids
2-Fluorobutyric bp 77-7.5 (11) B 5
D 6
9 2
12 7
4-Fluorobutyric bp 78--9 (6) 78.5 (10o) 12 17, 19
me
2-Fluoroisobutyric bp 108-9 me A 15
3-Fluoroisobutyric bp 80-2 (13) 12 57
2,2-Difluorobutyric bp 101 (8.5) bp 127-8 G 21
3,3-Difluorobutyric bp 135 C 21
2,3-Difluorobutyric mp 81 12 58
2-Fluorocrotonic mp 112 9 58
3-Fluorocrotonic mp 108-10 C, 9 21
4--Fluorocrotonic bp 66-7 (21) A 59
B 4, 17
9 39
4,4-Difluorocrotonic bp 83--4 3 60
(62-4)
(Continuad)
TABLE IX--Continued
4-Bromo-2-fluorocrotonic bp 95-7.5 (15) 9 61

3-Acetoxy-4-fluorocrotonic bp 55-7 (0.5) 9 62
4°Amino-2-fluorocrotonic mp 172-8 B, 9 61, 63
HC1
2-(Fluoromethyl)aerylic mp 81.5-2.5 bp 47.1-7.3 10 64
am (14)
2-(Difluoromethyl)acrylic bp 46.5-7 10, 9 65
(40)
2-Amino-3-fluoroisobutyric mp 219-20 10 66
HCI
2-Amino-3,3'-difluoroisobutyric mp 182-3 10 66
4-Fluoro-2-hydroxybutyrie bp 104-8 (18) 5 39
2,4-Difluoro-3-hydroxybutyric "A" 115-6 bp 51-2 9 67
am (0.06)
"B" 75-6 am
3-Fluoro-2ohydroxyisobutyric mp 101.5-2.0 bp 75 (22) 10 64, 66
me
3,3-Difluoro-2-hydroxyisobutyric mp 87.5-8.5 bp 42.5-3.5 10 65, 66
3,3'-Difluoro-2-hydroxyisobutyric mp 87-8 10 66
2-Fluoro-3-hydroxy-3,3,3-tri- bp 155-60 (2) 3 68
ehlorobutyrie
3-Fluoro-2-oxobutyric bp 70-5 (1) bp 159-61 E 69
2-Fluoroacetoacetic bp 83-5 (19) 6, 7 70
4-Fluoroacetoacetic bp 87-8 (24) 7 51, 70
2,2-Difluoroacetoacetie bp 75 (41) E 55
4,4-Difluoroacetoacetic bp 162 1 71
2,4-Difluoroacetoacetic bp 71-2 (3) 1 71, 72
2-Bromo-2,4-difluoroacetoacetic bp 100-2 (2) 9 73
4-Chloro-2,4-difluoroacetoacetic bp 101-4 (19) 7 70
Four-carbon dicarboxylic acids

Fluorosuccinie rap 144-5 bp 79-80 (1) F, 8 40, 74
5 75
2,3-Difluorosuccinic nd bp 70-1 (0.8) 12 76
2,2-Dittuorosuccinic mp 144-5 bp 53-5 (1) G 47, 77, 78
Fluoromaleic mp 128-9 G 47
Fluorofum~ric mp 236-7 G 22, 47
Difluoromaleic mp 219-20 G 47
Difluorofumaric vp 72-3 (1.1) G 47
2-Fluoro-3-ethoxyfumarie bp 75-7 11 79
(0.05)
Fluoromalic (erythro) mp 192-3 bp 125-135 9 67-80
(18)
(threo) mp 172-3 bp 146-178
(18)
2,2-Difluoromalie mp 139-40 bp 69-70 9 78, 81
(0.5)
° Key to references starts on p. 668.
[79] FLUORO ANhLOGS 665
TABLE IX--Continued
Category Acid Ester Method Reference a
Fluoro (hydroxymethyl)malonic bp 98--100 3 34

(0.2)
Fluorooxaloacetic rap 86-87 bp 121.5-2.5 2 23, 26, 49-
(9) 2 51, 82-85
Difluorooxaloacetic mp 115-6 bp 75 (41) E 55, 81, 86
B romofluorooxaloacetie 9 73
Five.carbon monocarboxylic acids

2-Fluorovaledc bp 90-90.5 5 6, 7
(12)
5-Fluorovaleric bp 90 (4) bp 72-4 (25) A 17, 19, 87
2-Fluoroisovaleric mp 35-40 5, 8 6, 7
4,4-Difluorovaleric bp 70-2 (27) F 88
Fluoroeyelobutanecarboxylic bp 75-6 (35) 5 75
2-Fluoro-4-pentenoic bp 48 (10) E, 8 89
4-Fluoro-3-methylcrotonic nd 7, 11 89
3-Fluoromethyl-3-butenoic nd 7, 11 89
4-Fluoro-3-fluoromethylcrotoni¢ bp 54-60 (10) 11 89
2-Fluoro-3-methylcrotonic bp 61 (18) 11 90
2,3-Difluoro-3-fluoromethylcrotonic bp 70-2 (30) 11 90
trans.4-Fluoro-L-proline mp 230-2 B 1
dec.
cis-4-Fluoro-L-proline mp 251 dec. B 1
5-Fluoronorvaline mp 190 5 91
5-Amino-2-fluoroo2-pentenoie mp 185 HCI 3, 8 63
2-Amino-4-fluoro-5-hydroxyvaleric mp 217-25 9 92
3-Fluoro-2-pyrrolidone-5- "A" mp mp 90-1 9 92
carboxylic 198-202
"B" nap
179-80
3-Chloro-4-fluoroisovalerie nd 9 89
5-Fluoro-4-hydroxypentanoie bp 127-9 5 39
(32) lac
2-Fluoro-,3-hydroxyisovaleric rap 68-70 bp 47-9 (0.5) 3 68
7 93
4-Fluoro-3-hydroxyisovaleric bp 70-1 (11) 7 89, 94
4,4'-Difluoro-3-hydroxyisovaleric bp 120-5 bp 90-5 (29) 3, 7, 10 94
(0.5)
3,3-Ditluoro-2-methoxy-1- bp 179.5-181 G 78
eyclobutanecarboxylate me
4-Fluoro-2-methylacetoacetic bp 90-3 (25) 7 70
2-Fluoro-3-oxovaleric bp 105-7 (32) 6 70
3-1~luoro-2-Oxovaleric bp 54-62 E, 8 69
(0.05)
4,4-Dichloro-2-fluoro-2-(hydroxy- bp 115-20 3 34
methyl)-3-oxobutanoic (0.2)
(Continued)
TABLE IX--Continued
2,4oDifluoro(hydroxymethyl)- nd 3 95
acetoacetate
3,5-Difluoro-2,4-dioxopentanoic bp 102-3 (I) 2, 8 51
pr
Five-carbon dicarboxylic acids
2-Fluoroglutaric mp 110-3 bp 110-2 (5) 4 96
E 6
E 54
Ethylfluoromalonic bp 96-99.5 5 6
(13)
2,2-Difluoroglutaric mp 103-5 bp 127-8 (28) 1 97
4 98
3,3-Difluoroglutaric bp 63-5 (2) 17 88
4-Fluoroglutamic mp 191-2 4 23, 25, 99
dec.
E 92
9 95
(2-Fluoroethyl)aminomalonic mp 74-5 ac 5 91
2-Bromo-4-fluoroglutaric mp 156-60 bp 100-4 9 95
(0.2)
2-Fluoro-4-hydroxyglutaric mp 205-10 bp 130-5 (4) 4, 8 40, 95
am
3-Fluoro-2-hydroxyglutaric nd E, 8 40
4,4-Difluoro-2-hydroxyglutaric nd G, 9 78
1-Methoxy-4,4-difluorocyclo- bp 75-7 (4) G 78
butene-2,3-dicarboxylate me
3-Fluoro-2oketoglutaric bp 107-8 (2) 4, 8 40
3-Methylfluorooxaloacetic bp 159-61 E 69
4,4-Difluoro-2-ketoglutaric bp 89-91 (3) G 78
me
FluoroacetyIfluoromalonic bp 110-1 (2) 53
Hydroxymethylfluorooxalacetic bp 120-2 (1) 100
ac
Five-carbon tricarboxylic acids
2-Fluoro-1-carboxysuccinic bp 107-9 E 74
(0.55)
1-Carboxy-2-fluoroaspartic mp 54-5 5 23
ac
Oxalofluoromalonic bp 135-6 (1) 6 51
Six-carbon monocarboxylic acids
2-Fluorohexanoic bp 106-6.5 D, 5 6
(11)
6-Fluorohexanoic bp 114 (6) bp 70-1 (9) A 17, 19, I01
me
Key to references starts on p. 668.
TABLE IX--Continued
Category Acid Ester Method Reference,
2oFIuoroisocaproic bp 132-3 ni B 5
3-Fluoropentane-3-carboxylic bp 82 (76)E 6
4-Fluoro-3,3-dimethylbutyric bp 193 A 59
2,6-Difluorohexanoic mp 47.5-50 5, 8 6
5,5-Difluorohexanoic bp 85-6 (16) bp 102 (40) C 21
2-Fluoro-3-isopropylacrylic bp 41-5 bp 51-3 (12) 3 93
3-(2-Hydroxyethyl)-2-fluoro- bp 150-60 3 56
crotonic (0.5) lac
6-Fluoronorleucine mp 244 5, 8 91
2-Fluoro-3-hydroxy-3-methyl- mp 87-90 bp 67-9 (2) 3 68
pentanoic
4-Fluoro-5-hydroxyhexanoic bp 48--9 E, 9 89
(0.05) lac
2-Fluoromevalonic mp 92-4 lac 7 11
3 94
4-Fluoromevalonic bp 124-5 7 56
(0.4) lac
6-Fluoromevalonic nd 7 102
4-Fluoro-5-oxohexanoic bp 74 (0.01) E, 8 89
2-Ethyl-4-fluoroacetoacetic bp 89-91 (22) 7 7O
Six-carbon dicarboxylic acids

Propylfluoromalonic bp 110-2 (10) 5 6
Fluoroisopropylmalonic bp 109-10.5 5 6
(15)
Allylfluoromalonie bp 110-1 (11) 5 6
3-Fluoroisobutene-l,l-dicarboxylic bp 74-5 (I) ni 3 94
2-Fluoromethylglutaconic bp 97--9(0.2) II 89
3,4-Difluoro-3-hexenedioic mp 139-44 G 103
5-Amino-2-fluoro-2-hexeneoic mp 253-5 5, 8 61
dec.
3-Fluoro-2-pyrrolidone-3,5- nd 4, 9 99
dicarboxylic
2-Fluoro-3-hydroxy-3-methyl- bp 108-10 3 94
glutaric (0.2) lac
3-(Fluoromethyl)-3-hydroxy- bp 50 (0.05) bp 59 (0.05) 7 38
glutaric me
Ethylfluorooxaloacetic bp 77 (0.25) E 69
2-Fluoro-3-hydroxy-2-oxMyl- bp 94-6 E 93
butyric (0.05)
2-Acetyl-2-fluorooxaloacetic bp 105-6 6 51
(O.5)
2-Fluoroacetyl-2-fluorooxaloacetic bp 175-7 2 51
(1.2)
(Continued)
TABLE IX--Continued
Category Acid Ester Method Reference6
~ix-carbon tricarboxyli¢ acids

4 96
2-Carboxy-2-fluoroglutaric mp 202-4 bp 130-3 E 95
am (0.5)
4-Fluoroaconitic bp 115-7 1, 8 104
(0.1)
4,4-Difluoroaconitic nd nd 1, 8, 9 78
4-Carboxy-4-fluoroglutamic nd 4 99
2-Carboxy-4-fluoroglutamic mp 103--4 ac 4 25
2-Fluorocitric (erythro ) mp 140-50 bp 120 (0.1) 1, 8 105, 106
dec.
(threo) mp 190-2 bp 120 (0.1) 7 107, 108
dec.
2,2-Difluorocitric mp 89-90 bp 125-6.5 1, 7 78
H~O (1)
2,4-Difluorocitric mp 68 1, 8 83
3-Fluoroisocitric bp 135-40 E, 9 40
(0.75)
4,4-Difluoroisocitric nd nd G 78
4-Fluorooxalosuccinic bp 123-4 E 40
(0.5)
4,4-Difluorooxalosuccinic nd G 78
Fluoroalkoxyalkanoic acids
(2-Fluoroethoxy)acetic bp 149 (13) 109
3-(2'-Fluoroethoxy)propionic bp 133-4 (12) 109
3-(3'-Fluoropropoxy)propionic bp 144-50 109
(13)
4-(T-Fluoroethoxy)butyric bp 99-100 109
(12)
4-(3'-Fluoropropoxy)butyric bp 110-11 109
(14)
° Key to References:
1. A. A. Gottlieb, Y. Fujita, S. Udenfriend, and B. Witkop, Biochemistry 4, 2507
(1965).
2. E. Gryszkiewicz-Trochimowski, A. Sporzynski, and J. Wnuk, Rec. Tray. CMm.
66, 413 (1947).
3. G. Olah, J. Kuhn, and J. Beke, Chem. Ber. 89, 862 (1956).
4. F. L. M. Pattison and J. E. Millington, J. Chem. Soc. 84, 757 (1956).
5. E. D. Bergmann and I. Shahak, Bull. Res. Council Israel &10, 91 (1961).
6. F. L. M. Pattison, R. L. Buchanan, and F. H. Dean, Can. J. Chem. 48, 1700
(1965).
7. E. D. Bergmann and I. Shahak, Israel J. Chem. 3, 73 (1965).
8. H. Machleidt, Ann. Chem. 667, 24 (1963).
9. I. L. Knunyants, E. Y. Pervova, and V. V. Tyuleneva, Izv. Akad. Nauk. $88R
Otd. Khim. Nauk, p. 843 (1956).
TABLE IX--Footno~s Continued

10. J. A. Young and P. Tarrant, J. Am. Chem. 8oc. 71, 2432 (1949).
11. G. Schmidt and H. Jahn, Ann. Chem. 644, 43 (1961).
12. A. V. Fokin, A. Skladnev, and V. A. Kor~rov, Zh. Obsheh. Khim. 38, 3271
(1~).
13. H. Cohn and E. D. Bergmann, Isra~ J. Chem. 2, 355 (1961).
14. I. V. Marlynov, Y. L. Kruglyak, and S. P. Makarov, Zh. Obsehh. Khim. $$,
3382 (1963).
15. B. C. Saunders and G. J. Stacey, J. Chem. 8oc. p. 1773 (1948).
16. E. D. Bergmann and I. Shahak, Chem. Ind. 1, 157 (1958).
17. F. L. M. Pattison, S. B. D. Hunt, and J. B. Stothers, J. Org. Chem. 21, 883
(1956).
18. E. Gryszkiewicz-Trochimowski and O. Gryszkiewicz-Trochimowski, Bull. Soc.
Chim. France, p. 928 (1949).
19. F. L. M. Pattison, J. B. Stothers, and R. G. Woolford, J. Am. Chem. Soe. 78,
2255 (1956).
20. I. L. Knunyants and G. A. Sokol'skii, Dokl. Akad. Nauk 888R 152, 602 (1960).
21. A. L. Henne and W. J. Zimmerscheid, J. Am. Chem. Soc. 69, 281 (1947).
22. E. D. Bergmann and I. Shahak, J. Chem. Soc. p. 4003 (1961).
23. M. Hudlicky, Collection Czech. Chem. Commun. 26, 1414 (1961).
24. A. L. Henne and C. J. Fox, J. Am. Chem. Soc. 76, 479 (1954).
25. A. Hudlicky, Tetrahedron Letters 14, 21 (1960).
26. H. Gault, D. Rouge, and G. Gordon, Fr. Pat. Add. 79,944 (1963); Fr. Pat.
1,255,459 (1961).
27. H. S. Davies, U.S. Patent 2,433,742 (1947).
28. W. J. Middleton, U.S. Patent 2,831,835 (1958).
29. A. Y. Yakubovich and A. P. Sergeev, USSR Patent 132,200 (1960).
30. G. S. Stoner, U.S. Patent 2,761,875 (1956).
31. W. T. Miller, U.S. Patent 2,751,414 (1956).
32. Z. Budensinsky, Collection Czech. Commun. 27, 2550 (1962).
33. C-Y. Yuan, C-H. Yao, IC-Y~ Su, and F-C. Yang, Hua Hseuh Hsueh Pao 7,
245 (1959).
34. E. D. Bergmann and S. Cohen, J. Chem. Soc. p. 4669 (1961).
35. V. Tolman and K. Veres, Collection Czech. Chem. Commun. 29, 234 (1964).
36. I. L. Knunyants, B. L. Dyatkin, L. S. German, and E. P. Machalina, Izv.
Akad. Nauk SSSR Otd. Khim. Nauk, p. 1676 (1962).
37. B. L. Dyatkin, E. P. Mochalina, and I. L. Knunyants, Dokl. Akad. Nauk SSSR
139, 106 (1961).
38. J. C. Craig, R. J. Dummel, E. Kun, and S. K. Roy, Biochemistry 4, 2547 (1965).
39. E. D. Bergmann, S. Cohen, and I. Shahak, J. Chem. Soc. p. 3448 (1961).
40. L. K. Gottwald and E. Kun, J. Org. Chem. 80, 877 (1965).
41. E. Cherbulie, A. de Picciotto, and J. Rabinowitz, Helv. Chim. Acta 43, 1143
(1960).
42. F. L. M. Pattison and J. J. Norman, J. Am. Chem. Soc. 79, 2311 (1957).
43. E. H. Eisman, H. A. Lee, Jr., and A. D. Winer, Biochemistry 4, 606 (1965).
44. P. W. Kent, G. Hebblethwaite, and N. F. Taylor, J. Chem. Soe. p. 106 (1960).
45. N. F. Taylor and P. W. Kent, J. Chem. Soc. p. 872 (1958).
46. D. H. Treble and R. J. Peters, Biochem. J. 80, 15pp. (1961).
47. M. S. Raasch, J. Am. Chem. Soc. 81~ 2678 (1959).
48. A. Y. Yakubovich and I. N. Belyaeva, USSR Patent 144,481 (1962).
670 PBEPARATION OF COMPOUNDS [79]
TABLE IX--Footnotes Conlinued

49. I. Blank, J. Mager~ and E. D. Bergmann, J. Chem. Soc. p. 2190 (1955).
50. P. V. Nair an d H . Buseh, J. Org. Chem. 28, 137 (1958).
51. I. Shahak and E. D. Bergmann, J. Chem. Soc. p. 3225 (1960).
52. E. D. Bergmann and I. Shahak, Chem. & Ind. p. 591 (1961).
53. E. D. Bergmann, S. Cohen, and I. Shahak, J. Chem. Soc. p. 4033 (1961).
54. H. Gershon, J. A. A. Renwiek, W. K. Wynn, and R. D'Ascoli, J. Org. Chem.
$1, 916 (1966).
55. C. E. Inman, R. E. Oesterling, and E. Tycykowski, J. Am. Chem. Soc. 80,
6533 (1958).
56. E. D. Bergmann and S. Cohen, J. Chem. Soe. p. 3457 (1961).
57. W. Bockmiiller, Ann. Chem. 506, 20 (1933)
58. A. Streitweiser, Jr. and D. Holtz, J. Am. Chem. Soc. 89t 692 (1967).
59. F. L. M. Pattison and B. C. Saunders, J. Chem. Soc. p. 2745 (1949).
60. A. Z. Zielinski, E. T. McBee, and H. Braenklin, Roezniki Chem. $7, 905 (1963).
61. V. Tolman and K. Veres, Tetrahedron Letters 29--$0, 1967 (1964).
62. R. Filler and S. M. Naqvi, J. Org. Chem. 26, 2571 (1961); Tetrahedron 19, 879
(1963).
63. E. D. Bergmann and S. Cohen, Tetrahedron Letters 25, 2085 (1965).
64. S T . Voong and T-C. Chiang, Hua Hsueh Hsueh Pao 24, 155 (1958).
65. A. Y. Yakubovieh, N. A. Bogoslovskii, E. P. Pravova, and S. M. Rozenshtein,
Zh. Obsheh. Khim. 28p 2288 (1958).
66. E. D. Bergmann and A. 8hani, J. Chem. Soc. p. 3462 (1963).
67. N. F. Taylor and P. W. Kent, J. Chem. Soc. p. 2150 (1956).
68. E. D. Bergmann and S. Cohen, J. Chem. Soc. p. 3537 (1961).
69. D. R. Grassetti, M. E. Brooke, and J. F. Murray, Jr., J. Med. Chem. 9, 149
(1966).
70. E. D. Bergmann, S. Cohen, and I. Shahak, J. Chem. ~qo¢.p. 3228 (1959).
71. E. T. McBee, O. P. Pierce, H. W. Kilbourne, and E. R. Wilson, J. Am. Chem.
Soc. 75, 3152 (1953).
72. J. E. G. Barnett and P. W. Kent, J. Chem. Soc. p. 2743 (1963).
73. E. D. Bergmann and I. Shahak, J. Chem. ~ c . p. 462 (1960).
74. F. H. Dean and F. L. M. Pattison, Can. J. Chem. 41, 1833 (1963).
75. E. D. Bergmann and S. Szinai, J. Chem. Soc. p. 1521 (1956).
76. M. S. Kharasch, U.S. Patent 2,426,244 (1947).
77. M. S. Raasch and J. E. Castle, Org. ~Syn. 42, 44 (1962).
78. M. S. Raasch, U.S. Patent 2,824,888 (1958).
79. H. Machleidt, Ann. Chem. 695° 134 (1966).
80. A. I. Krasna, J. Biol Chem. 286, 749 (1961); ibid. 257, 1418 (1962).
81. E. Kun, L. K. Gottwald, D. W. Fanshier, and J. E. Ayling, J. Biol. Chem. 258,
1456 (1963).
82. E. Kun, D. R. Grassetti, D. W. Fanshier, and R. M. Featherstone, Bioehem.
Pharmacol. 1, 207 (1958).
83. D. Rouge and H. Gault, Compt. Rend. Acad. ~Sci. 251, 94 (1960).
84. D. E. A. Rivett, J. Chem. Soc. p. 3710 (1953).
85. J. Blank and J. Mager, Experientia 10, 77 (1954).
86. D. W. Wiley, U.8. Patent 2 988 537 (1961).
87. F. J. Buckle, F. L. M. Pattison, and B. C. Saunders, J. Chem. Soc. p. 1471
(1949).
88. W. R. Hasek, W. C. Smith, and V. A. Englehardt, J. Am. Chem. ~So¢.$2, 543
(1960).
TABLE IX--Footnotes Continued

89. H. Maehleidt, Ann. Chem. 667, 24, 35 (1963).
90.M. W. Buxton, M. Staeey, mad J. C. Tatlow, J. Chem. Soc. p. 366 (1954).
91.M. S. Raaach, J. Org. Chem. 25, 1567 (1958).
92.V. Tolman and K. Veres, Tetrahedron Letters $2, 3909 (1966).
93.H. Maehleidt, Ann. Chem. 674, 1 (1964).
94.E. D. Bergmann, S. Cohen, and 1~. Hoffman, J. Chem. Soc. p. 3452 (1961).
95.E. D. Bergmann, S. Cohen, and A. Shani, Israel J. Chem. 1, 79 (1963).
96. L. K. Gottwald, J. E. Ayling, and E. Kun, J. Biol. Chem. 289, 435 (1964).
97. A. C. Barney and T. L. Cairns, J. Am. Chem. 8oc. 72, 3193 (1950).
98.A. G. Farbenfabriken Bayer, Ger. Patent 1,134,365 (1962).
99.R. C. Buchanan, F. H. Dean, and F. L. M. Pattison, Can. J. Chem. 40, 1571
(1962).
100. E. D. Bergmann and I. Shahak, J. Chem. Soc. p. 5261 (1960).
101. R. R. Fraser, J. E. Millington, and F. L. M. Pattison, J. Am. Chem. Soc. 79,
1959 (1957).
102. R. Tsehesche, H. Machleidt, and T. Biicher, U.S. Patent 3,075,997 (1963).
103. D. C. England, L. R. Melby, M. A. Dietrich, and R. V. Lindsey, Jr., J. Am.
Chem. 8oc. 82, 5116 (1960).
104. E. D. Bergmann and I. Shahak, Nature 186, 529 (1960).
105. D. W. Fanshier, L. K. Gottwald, and E. Kun, J. Biol. Chem. 289, 425 (1964).
106. P. J. Brown and B. C. Saunders, Chem. & Ind. p. 307 (1962).
107. D. W. Fanshier, L. K. Gottwald, and E. Kun, J. Biol. Chem. 237, 3588 (1962).
108. D. E. A. Rivett, R. A. Peters, It. W. Wakelin, and L. C. Thomas, Nature 171,
1111 (1953).
109. F. G. Buckle, R. Heap, and B. C. Saunders, J. Chem. Soc. p. 2774 (1949).
b Carboxyl derivatives of fluoroacetic acid: me b.p. 104-5 ° (77), et b.p. 117-8° (77), pr
b.p. 135-7 ° (3), t-bu b.p. 129-31 ° (19), amide m.p. 108° (11), hydrazide m.p. 116-7 °
(11), anhydride b.p. 88-9 ° (3), chloride b.p. 71.5-730 (3), bromide b.p. 95-6 ° (29),
fluoride b.p. 50.5-1 ° (3), nitrile b.p. 79-80 ° (3), hydroxamic acid m.p. 75-6 ° (86).
was m a i n t a i n e d at - - 5 ° to 0 °. After 0.5 hour, the mixture showed p H 7
on p H y d r i o n paper. Addition of perchloryl fluoride was discontinued, and
the reaction mixture was flushed with nitrogen to remove the excess per-
chloryl fluoride. T h e mixture was then poured into 300 ml of water
while stirring was continued in order to dissolve salts. T h e aqueous
mixture was t h e n extracted with three portions of e t h y l ether (100 ml)
and the ether layer was dried with a n h y d r o u s Na~SO~. Distillation of the
ether e x t r a c t afforded t r i e t h y l a-fluoro-a-oxalosucclxlate (27 g, 7 8 % ) ,
b.p. 115 ° at 0.2 ram, or 123-124 ° a t 0.5 ram, nD25 1.4300.
If the reaction t e m p e r a t u r e is allowed to rise a b o v e 0 °, diethyl fluoro-
succinate as a lower boiling fraction is obtained in 1 0 - 2 0 % yields, b.p.
79-80 ° at 1 ram, n~ 25 1.4120. T r e a t m e n t of triethyl fluorooxalosuccinate
with 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e r e a g e n t gave diethyl fluorooxalosucei-
hate 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e , which was crystallized from ethanol,
m.p. 117 °. T r e a t m e n t of t r i e t h y l a-fluoro-a-oxalosuccinate with aqueous
a m m o n i a at 50 ° gave a mixture of fumaric diamide and oxalic diamide.
D i e t h y l fluorosuccinate gave, u n d e r identical conditions, f u m a r i c diamide.
672 P~PAP~TmN OF CO~POUNDS [79]
Diethyl ,~-Oxo-fl-fluoroglutarate. Triethyl a-fluoro-a-oxalosuceinate

(20 g) and aqueous 3 7 ~ hydrogen chloride (35 ml) were mixed and left
at room temperature for 36 hours. The solution was then heated at 65-
70 ° until CO~ evolution ceased (2 hours). The solvent was removed under
vacuum (bath temperature 70°). Water was then added and the process
was repeated twice. The residue, a viscous yellow oil, was dissolved in
absolute ethanol (100 ml) containing p-toluenesulfonic acid (0.5 g), and
the mixture was heated under reflux for 24 hours. After standing an
additional 36 hours, the solution was treated with excess solid sodium
bicarbonate and then filtered. The solvent was removed by vacuum
distillation, and the residue was extracted into ether. After filtering off
insoluble salts from the ether solution and distillation, diethyl a-oxo-
fl-fluoroglutarate was obtained (10 g, 66~, b.p. 110-112 ° at 1.0-1.5 mm).
A sample was distilled for analysis: b.p. 107-108 ° at 2 mm, no 25 1.4280.
(The free acid was not reported.)
V. Compendium of Fluoro Aliphatic Acids and Esters

A list of some monofluoro and difluoro aliphatic acids and esters found
in the literature to Dec. 1966 is assembled in Table IX (p. 662). They are
first grouped according to the number of carbon atoms in the free acid.
Each group is subdivided into mono-, di-, and triearboxylic acids. Under
each subdivision, acids are listed in approximately the following order:
saturated, alicyclic, unsaturated, halogenated (nonfluoro), amino, hy-
droxy, oxo. Difluoro acids are listed after the monofluoro homolog. The
"Acid" column lists properties of free acid, unless otherwise noted. The
"Ester" column lists properties of the mono-, di- or triethyl ester, unless
otherwise noted. Abbreviations for "acetyl" and "lactone" are used in
addition to any ester groups. The "Methods" column designates one or
more synthetic reactions according to the letters and numbers used in
Tables VII and VIII. In addition, "12" is used to designate methods not
clearly belonging to one of the other nineteen categories. Alternative
syntheses are listed on separate lines.
Tile nomenclature is only partly systematic, following the conventions
of Pattison. 14 Compounds are named informally in a manner to convey
the structure without requiring illustration.
The number listed in parentheses after the boiling points is the
pressure in millimeters.
The following abbreviations are used: me, methyl ester, including
dimethyl and trimethyl; et, ethyl ester; pr, propyl ester; bu, butyl ester;
HCI, amine hydrochloride; lae, lactone; ae, acetyl or acetate; am, amide;
ni, nitrile; rid, no data for listed compound in the reference.
GLOSSARY OF ENZYME PREPAR,kTIONS 673
' ~ .~
o o
~.~o~.~
Z
0'~
i
O -~ ~
ge o
°~, ~
!
c~
i o ~.~o~
"~ ~. "2. • o
~4 GLOSSARY OF ENZYME PREPARATIONS
.2
~ E
~ - o.~
o~ 0
1:1
|
s~
o
~oi~ i~i~ ~
0
e~
III
"" ~ .~ "~ 0 0 ~ ~ 0
~ ~ ~ i~ ~ ~ ~
GLOSSARY OF ENZYME PREPARATIONS 675
e,') ~ ~I" O0
,~ ~ ~ .,~
e~
- ~
o,~. ~ ~ ~o ,
.~ .. o~.~ ~ , ,
p.,
o ~ o p~
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~ o o,..
~ ~ ~ o
.~.
..~ .~ ~ ~
~' ~ ~.~. "I. ~. ~

676 AUTHOR INDEX
Author Index
Numbers in parentheses are reference numbers and indicate that an author's work is
referred to, although his name is not cited in the text. Numbers in italics show the page
on which the complete reference is listed.
A Ayling, J. E., 629, 645, 659(17), 664(81),

Abe, S., 613 665(81), 666(96), 668(96), 670, 671
Ables, R. H., 213, 214(12), 215(12), 599
Abeliovitz, A., 133, 134(7) B
Abezgauz, F. I., 626 Bachmann, E., 11
Abrahams, M. D., 602, 603, 613 Baehmann, R. C., 398, 402(17), 403(17),
Adaehi, K., 153, 156(4), 158(4), 551 404(17), 405(17), 406(17)
Adembri, G., 598 Baehofen, R., 171, 172(4), 174(4), 177(4,
Adler, J., 69, 314, 315(2) 5), 180
Ajl, S. J., 163, 362, 363, 364, 365(14), 452 Bachstez, M., 614
Albers, R. W., 531 Badran, A. M., 557, 562
Alberts, A. W., 9 Balde, P., 430
Alberty, R. A., 92, 96, 97, 98, 577 Baltimore, B., 321
Alcock, N. W., 398, 399(11), 402(11), 403, Bambers, G., 11, 572, 584(16)
404(11), 405(11), 406(11), 408(11), Banaszak, L. J., 124
4O9 Bandurski, R. S., 277, 292
Alexander, M., 140 Banks, R. E., 624, 633(4)
Alivisatos, S. G. A., 459 Barbour, A. K., 634
Allen, S. H. G., 194, 195, 197(6), 207, 208, Bardawill, C. J., 57, 63, 155, 376
213, 216, 217, 220, 224(6), 225(6), Barker, H. A., l 1, 317, 319, 320(2), 321(1),
226(6), 227(6, 10), 228(6, 11), 263, 322(3), 325(1, 4), 326(4), 329, 330(1,
268(2), 298, 301(3) 3, 4), 331, 332, 334(1), 336, 338(1),
Allmann, D. W., 11 344, 345, 346, 347, 348, 350, 351(1, 6),
Alivisatos, S. G. A., 74 352(6), 572, 584(15)
Amarasingham, C. R., 58 Barlin, G. B., 625
Ames, B. N., 117, 210, 287 Barnaby, C., 145
Anagnostopoulos, C., 142 Barnett, L. B., 97
Andrew, I. G., 177 Barney, A. C., 666(97), 671
Andrews, P., 392 Barrett, R. J., 117
Anet, F. A. L., 576, 577(49), 580(49), Barrnett, J. E. G., 664(72), 870
584(49) Barren, E. J., 137
Anfinsen, C. B., 473 Barrow, F., 597
Ann Light, P., 11 Bartlett, G. R., 425, 426
Aogaiehi, T., 42 Baselice, 362
Arigoni, D., 90, 214, 215(18), 343 Battaile, J., 555
Arnon, D. I., 170, 171, 172(4), 174(4), Bauer, C., 430
177(2, 4, 5), 179(2), 180, 181, 315 Bechtold, M. M., 318
Asano, A., 134, 140 Beck, W. S., 209, 216
Ashworth, J. M., 170 Beenakkers, A M. Th., 393
Astwood, E. B., 117 Beevers, H., 163
Atkinson, D. E., 9, 11, 19, 25, 41 Beisenherz, G., 382
Ayenger, P., 69, 74, 459 Beke, J., 662(3), 668
AUTHOR INDEX 677
Bell, L. J., 634 Bradshaw, R. A., 96, 98(6), 99

Belyaeva, I. N., 663(48), 669 Brady, R. O., 8, 381, 531, 654
Bo~dall, D. S., 558 Brady, W. T., 56
Bender, P., 577 Braendlln, H..P., 629, 632(15)
Benedict, C. R., 250, 252(2), 256, 257(2), Braenklin, H., 663(6{)), 670
258(2), 261 Brant, D. A., 97
Benziman, M., 129, 130(1), 132(1), 133(1), Bray, G. A., 574
134(1, 4, 5, 7), 140 Brazil, H., 4, 11, 12(4), 16, 19, 22, 365,
Beppu, T., 613 546(29), 551
Berg, P., 375 Breiger, H. H., 105, 120, 124, 128(4)
Berger, R., 575, 599(33) Breusch, F. G., 574
Bergmann, E. D., 626, 627, 628, 629, 630, Breyer Bradwyic, M. G., 617
634, 638, 639(29), 641(70), 643, 647 Bridger, W. A., 70, 74(2), 460, 506
(13), 649, 658(19), 662(5, 7, 13, 16, Briggs, R. A., 398, 399(2), 402(2), 404(2),
22), 663(5, 7, 34, 49, 51, 52, 53, 56), 405(2), 414(2)
664(22, 39, 51, 63, 66, 68, 70, 73, 75), Bright, H. J., 347, 350, 351(6), 352(3, 5, 6),
665(7, 34, 39, 49, 51, 63, 68, 70, 75, 353(3, 13)
94), 666(51, 53, 95, 100), 667(5, 51, 56, Britten, J. S., 577, 584(52), 597(52)
68, 70, 94), 668(95, 104), 668, 669, Brodie, A. F., 134, 140
670, 671 Brooke, M. E., 664(69), 665(69), 666(69),
Bergmeyer, H. U., 381, 382, 386, 435, 455, 667(69), 670
479, 503, 520, 536, 540(11), 541, Brown, D. M., 38, 39(10)
545(16), 548 Brown, P. J., 668(106), 671
Berky, J. J., 170 Browning, E. T., 445
Berman, K., 309 Buchanan, B. B., 170, 171, 172(4), 173(6),
Bernath, P., 81, 82(2), 88(2, 3), 524 174(4), 177(2, 4, 5, 6), 178(7), 179(2),
Berndt, E., 479 180(6), 181
Bernstein, H. J., 576 Buchanan, R. C., 666(99), 667(99), 671
Berry, M. N., 478 Buchanan, R. L., 638, 662(6), 663(6),
Bessman, S. P., 428 665(6), 666(6), 667(6), 668
Bhattacharjee, J. K., 623 Bucher, T., 641(102), 667(102), 671
Bidwell, R. G. S., 528 Buckel, W., 3, 4(7), 389, 503, 512
Blair, .4. H., 331, 334(1), 338(1), 344, 346 Buckle, F. G, 668(109), 671
Blank, I., 629, 630, 643, 658(19), 663(49), Buckle, F. J., 627, 665(87), 670
665(49, 85), 670 Buckley, L. M., 90
Bloom, B. M., 584 Budensinsky, Z., 662(32), 663(32), 669
Bock, R. M., 52, 54, 55, 92, 98, 498, 499, Buecher, Th., 382
500(72), 537, 542 Buhler, D. A., 531
Bockmiiller, W., 663(57), 670 Bulen, W. A., 429, 528
Bogin, E., 20, 21, 25 Burdett, J. L., 631
Bogoslovskii, N. A., 664(65), 670 Burger-Rachamimov, H., 130
Boltze, H. J., 382 Burnham, B. F., 460
Bonner, W. D., 81, 82(1), 83 Burns, J. C., 398, 399(10), 402(10),
Bonting, S. L., 362 403(10), 404(10), 405(10), 414(10)
Borcic, S., 598
Burrell, R. C., 429, 528
Bortz, W. M., 547(20), 550
Botter, R., 575 Burton, K., 98
Bowen, T. J., 160, 452 Burton, R. M., 382
Bowman, R. L., 398 Busch, H., 425, 573, 574(22), 620, 623(4),
Boyer, P. D., 70, 74(2), 75(2, 11), 190, 630, 642(20), 643(20), 658(20), 659
460, 506(44), 519 (20), 663(50), 670
678 AUTHOR INDEX
Butler, L. G., 74, 75(11) Coil, N., 290

Buxton, M. W., 626, 632, 634, 665(90), 671 Collins, E. B., 160
Colowick, S. P., 41, 518, 589
C Connolly, T. N., 173, 178(11)
Cahn, R. D., 109, 114(9) Conway, E. J., 530
Cahn, R. S., 570 Cook, R. A., 46, 47, 287, 288(5)
Cairns, T. L., 666(97), 671 Coon, M. J., 75, 76(1), 77(1), 79(1), 80(1),
Cannata, J. J. B., 198, 199(2), 206(2) 81(1, 10)
C~novas, J. L., 246, 250, 252(1), 257(1), Cooper, P. M., 120, 123, 124, 125(2)
277, 286, 288(2), 289 Cooper, R. A., 309, 314(2), 317
Canvin, D. T., 398, 399(12), 402(12), 403, Cooper, T. G., 256
404(12), 405(12), 406(12) Corey, E. J., 599
Carroll, W. R.,' 55, 61(4) Cori, C., 65
Carson, S. F., 263 Cori, 0., 498
Carter, H. E., 590 Cornforth, J. W., 90, 575, 577, 596,
Castle, J. E., 634 598(45), 600(45)
Catalina, L., 252, 256(9), 257(9) Cornforth, R. H., 575, 598(45), 600(45)
Cazzulo, J. J., 250, 252(3), 258(3) Costa, C., 361
Ceci, L. N., 526, 527(5), 619 Costello, L. A., 117
Cecil, R., 96 Craig, J. C., 633, 638(25), 642(25),
Cha, C.-J. M., 65, 68(6), 69(6), 460, 506 643(25), 644(25), 647(25), 649(25),
(45), 551 650(25), 660(25), 661(25), 663(38),
Cha, S., 62, 65(1), 68(6), 69(6), 460, 667(38), 669, 671(25), 672(25)
506(45), 551 Crane, E. J., 569
Chambers, R. D., 625 Crane, P. K., 489, 491(65)
Chance, B., 12, 445 Crawford, E. J., 526
Chang, H. C., 270, 273(1, 2), 275(2), Creech, B. G., 399, 410(20), 411(20),
276(1, 2), 277(2), 279(1), 282(1) 412(20)
Changeux, J. P., 46 Czok, R., 382
Chase, J. F. A., 10, 387, 388(5), 391(5),
D
392, 505, 506, 509(81), 510, 512(81),
546(17), 549 D'Adamo, A. F., Jr., 577
Chen, Jo-Yun, 186, 187(9), 188(9), 191, Dagley, S., 160, 161(3), 452, 517
199 Daikuhara, Y., 153
Chen, R. F., 34, 35(2), 37, 38(la, 2), Dakin, H. D., 574, 584(26)
39(10), 40(2), 41(2) Dalgleish, C. E., 398, 399(16), 402(16),
Cherbulie, E., 663(41), 669 404(16), 405(16), 406(16), 407, 409
Chiang, T-C., 664(64), 670 (16), 410, 411, 412, 413, 414, 415
Chilson, O. P., 112 Daron, H. H., 160, 161(1), 169, 451, 517,
Christian, W., 43, 48, 139, 259 518(2), 540(28)
Clark, J. M., Jr., 600 D'Ascoli, R., 663(54), 666(54), 670
Clark, M. J., 188, 198 Datta, S., 452
Cleland, W. W., 33, 44, 81,230, 235 Datta, S. P., 160, 161(6), 162, 163(6)
Cloutier, A. A., 30, 447 David, M. M., 57, 63, 155, 376
Cohen, S., 634, 639, 641(70), 649, 663(39, Davies, D. D., 51, 100, 105, 121, 150
53, 56), 664(39, 63, 68, 70~, 665(39, 63, Davies, H. S., 662(27), 669
68, 70, 94), 666(53, 95), 667(53, 68, 70, Davis, B. D., 58
94), 668(95), 669, 670, 671 Davis, B. J., 33
Cohn, D. V., 134, 140 Davis, J. J., 217, 229, 248, 297, 298(la, 2),
Cohn, H., 662(13), 669 299(la), 300(la), 305(la), 306(la, 2),
Cohn, M., 309 307(la, 2), 308
AUTHOR INDEX 679
Dawes, E. A., 160, 161(3), 452, 517 Ellman, G. L., 4, 12, 19, 367, 376, 509, 536,
Dean, F. H., 634, 638, 639, 649(36), 662(6), 538(8), 544(8)
663(6), 664(74), 665(6), 666(6, 74, Elsden, S. R., 163
99), 667(6, 99), 668, 670, 671 Emery, T. F., 355, 360
del Campillo, A., 75, 76(1), 77(1), 79(1), Emmerich, R., 314
80(1), 81(1), 535 England, D. C., 634, 667(103), 671
DeLuca, H. F., 425, 547(35, 36) Englard, S., 41, 100, 104(11), 105(11),
Delwiche, E. A., 265 106(14), 120, 124, 128(4), 129(12),
Dennert, G., 59 361, 574, 576, 577, 578, 580(32),
Denstedt, O. F., 513, 514(1) 581(32, 57), 582(57), 584(52), 586,
de Picciotto, A., 663(41), 669 588(32), 589, 592, 593(86), 597(52)
Depue, R. H., 360 Englehardt, V. A., 646, 665(88), 666(88),
Der Vartanian, D. V., 83, 88(11), 90(11, 67O
20, 21), 524(4), 525 Erfle, J. D., 600
Desio, F., 598 Erickson, L. C., 20
de Torrontegui, G., 248, 252, 256(9), 257(9, Esposito, G. G., 398, 399(4), 402(4)
14) Estabrook, R. W., 436, 438(5), 481, 495
DeVay, J. E., 398, 399(3), 402(3), 403(3), Estes, F. L., 398, 402(17), 403, 404(17),
404(3), 405(3), 414 405(17), 406(17)
Dickens, F., 594 Ettinger, R. H., 523
Dickman, S. R., 30, 447, 613 Evans, H. J., 150, 559
Dietrich, M. A., 634, 667(103), 671 Evans, M. C. W., 170, 171, 173(6), 177(2,
Dilley, D. R., 557 6), 178(7), 179(2), 180(6)
Dixon, G. H., 362, 365
Dixon, M., 14 F
Doherty, D. G., 100, 105
Dolln, M. I., 263, 267, 269 Fanshier, D. W., 8, 627, 628, 629, 633,
Donninger, C., 577, 596(53), 600 653(28), 654(28, 47), 659(16, 17),
Draper, J. O., 431 664(81), 665(81, 82), 668(105, 107),
Drysdale, G., 315 670, 671
Dugger, W. M., 150 Farley, M. A., 361
Dummel, R. J., 633, 638(25), 642(25), Farr, A. G., 100
643(25), 644(25), 647(25), 649(25), Farr, A. L., 17, 23, 43, 117, 123, 130, 138,
650(25), 660(25), 661(25), 663(38), 155, 284, 289, 311, 321, 333, 345, 348,
667(38), 669, 671(25), 672(25) 351(4), 355
Dunmore, P., 23, 24(5) Featherstone, R. M., 629, 659(16),
Dyatkin, B. L., 634(43), 648, 663(36, 37), 665(82), 670
669 Fellows, R. E., 96, 98(6)
Felts, P. W., 445
E Fine, I. H., 117
Eanes, R. Z., 122 Fields, G. A., 352, 353(13)
Easterday, R. L., 277, 279(1), 282(1), 283 Filler, R., 631, 664(62), 670
Edelman, G. M., 210 Fiske, C. H., 310
Edsall, J. T., 351 Fittig, R., 600, 601, 608, 613
Eger-Neufeldt, I., 4, 11, 16(3)
Flavin, M., 191, 198, 209, 216
Eggerer, H., 3, 4(7), 9, 213, 389
Eggerer, M., 503, 512 Flodin, P., 253
Eggleston, L. V., 153, 479, 613 Floyd, N. F., 574
Eisman, E. H., 663(43), 669 Focesi, A., Jr., 198, 199(2), 206(2)
Ellfolk, N., 358, 361(11) Fokin, A. V., 662(12), 669
Elliott, W. B., 381 Folch, J., 137
680 AUTHOR INDEX
Fondy, T. P., 318, 361, 576, 577(50), Gershon, H., 663(54), 666(54), ~70
580(50), 584(50) Gibson, D. M., 69, 74, 459
Fox, C. J., 632, 643(23), 653(23), 662(24), Gibson, J., 70
669 Gill, D. M., 77
Fraenkel, G., 387, 389 Gilvarg, C., 65, 498
Francis, J. 0., 134 Giorgio, A. J., 37
Francis, M. J. 0., 135 Giovanelli, J., 369
rancisco, J., 90 Gitter, S., 643
Frank, I. R., 74, 459 Giuffrida, L., 398
Franklin, E. C., 210 Glaid, A. J., 90
Fraser, R. R., 666(101), 671 Glaid, A. J., III, 318, 576, 577(50),
Freeland, M. Q., 382 580(50), 584(50)
Freemann, T. E., 398, 399(8), 402(8), Godfrey, P., 529, 531(8)
404(8), 405(8) Goebell, I-I., 40, 41, 42(12, 15)
Frenkel, R., 481, 495(60) Goerlitz, D. F., 398, 399(6), 402(6),
Frey, A. J., 319 404(6), 405(6)
Friedemann, T. E., 310, 362 Goldbaum, L. R., 523
Frieden, C., 92, 97, 99 Goldberg, N. D., 442, 445, 447(9), 531
Friedman, L., 639 Goldfine, H., 519
Friedmann, S., 387, 389 Goldhamer, H., 132, 134(5)
Friesen, H., 117 Goldman, P., 626
Fritz, H. P., 198 Goldwhite, H., 624, 633(4)
Fritz, I. B., 9, 387, 388(3), 389, 392(3, 12), Gonen, L., 4, 11, 12(4), 16, 19, 22, 365,
393(7), 445, 505, 50ff(83) 546(29), 551
Fritz, J. S., 382 Good, N. E., 173, 178(I1)
Fuji,a, Y., 633, 662(1), 665(1), 668 Goodban, A. E., 431, 575
Fukuma, I., 318 Goodman, D. S., 575, 598(45), 600(45)
Fukunishi, K., 153, 156(4), 158(4) Gordon, G., 647, 662(26), 665(26), 669
Fukuniski, F., 551 Gornall, A. G., 57, 63, 155, 376
Fuller, R. C., 171 Gotteschalk, G., 572, 584(15)
Gottleib, A. A., 633, 662(1), 665(1), 668
G Gottschalk, E., 11
Gailiusis, J., 250, 252(2), 257(2), 258(2), GottwaId, L. K., 8, 629, 633, 645, 649, 650
261 (45), 653(28), 654(28, 47), 659(17),
Galanter, Y., 129, 130(1), 132(1), 133(1), 663(40), 664(40, 81), 665(81), 666(40,
134(1) 96), 668(40, 96, 105, 107), 669, 670,
Gamble, J. L., 425 671
Garbade, K. H., 382 Grassetti, D. R., 627, 628, 629, 659(16),
Garland, P. B., 4, 11, 12(1), 16(1), 25, 664(69), 665(69, 82), 666(69), 657
497, 505(70), 536, 539(12), 540(12), (69), 670
541(6), 542(12), 543(6), 546(6), 547 Graves, J. B., 586
(19, 31), 550, 551 Graves, J. L., 270
Gaul,, H., 647, 657, 662(~6), 665(26, 83), Green, A. A, 108, 146
668(83), 669, 670 Green, D. E., 11, 136
Gawron, O., 90, 318, 361, 576, 577(50), Green, J. P., 69
580(50), 584(5{)) Greenberg, D. M., 429
Gee, M., 398, 399(13), 402(13), 404(13), Gregory, R. P. F., 558
405 (13),~406(13) Greiner, C. M., 277, 292
Gehrke, C. W., 398, 399(6), 402(6), Griebel, C., 614, 617
404(6), 405(6) Griffin, G. E., 420
German, L. S., 663(36), 669 Grimm, F. C., 100, 105
AUTHOR INDEX 681
Grisolia, S., 10O, 105 653(23), 662(21, 24), 663(21), 667(21),

Grossman, A., 184, 186, 188(8), 189(8) 669
Grossman, L. I., 106, 116(1) Henning, H. V., 235, 236
Groth, D. P., 519 Henning, U., 59
Gruber, W., 451, 452(29), 517 Herberling, R. L., 170
Grunert, R. R., 182, 375, 376 Hersh, L. B., 77, 80(6), 81(6)
Grunewalder, C., 9 Hertel, R., 59
Gryszkiewicz-Troehimowski, E., 639, 662 Heym, G. A., 362
(1, 18), 663(2), 668, 669 Hiatt, A. J., 150
Gryszkiewicz-Trochimowski, 0., 639, 662 Hift, H., 65
(18), 669 Hill, D. L., 398, 399(10), 402(10), 403(10),
Grzybowski, A. K., 161 404(10), 405(10), 414(10)
Guiditta, A., 89(23), 90 Hill, R. L., 91, 92(3), 96(3), 97(3), 98(3, 6),
Gunsalus, C. F., 140 99
Gunsalus, I. C., 57, 70, 74, 140, 160, 161 Hirashima, M., 54, 55(3a), 542
(1), 169, 170(15), 451, 459, 517, 518 Hirschmann, H., 571, 572(12)
(2), 540(28) Hitchcock, D. I., 606
Gustavson, K. H., 555 Hixon, W. S., 526
Hjert~n, S., 143, 145(3), 201, 202(8),
H 204(8), 275, 281
Haag, V., 546(30), 551 Hobbs, M. E., 398, 402(1), 403(1), 404(1),
Hager, L. P., 57 405(1), 406(1), 414(1)
Hagre, 0. S., 11 Hoberman, H. D., 573, 574(24), 577, 578
Hamada, M., 542 Hoffman, E., 665(94), 667(94), 671
Hanahan, D. J., 137 Hofmann, A., 319
Hanson, K. R., 570, 571(11), 572, 584(11, Hohnholz, E., 100
13), 589(13), 590(13), 593, 596(90), Hohorst, H. J., 259, 260(2)
600(13), 601(90) Holloeher, T. C., 90
Harden, A., 354 Holmberg, B., 598
Harrison, B. D., 558 Holtz, D., 625, 663(58), 670
Hartree, E. F., 81, 84(5), 87, 390 Holz, G., 332
Harvey, R. J., 160 Holzer, H., 575
Hasek, W. R., 646, 665(88), 666(88), 670 Horecker, B. L., 117
Hass, G, M., 99 Horii, Z., 398, 399(14), 404(14), 405(14),
Hass, L. F., 190 406(14), 407, 409(14), 410(14), 411,
Hasselberger, F. X., 435, 436(4), 437(4), 412(14), 413
438(4), 519 Homey, D. L., 56
Hathaway, J. A., 9, 11, 19, 25, 41 Homing, E. C., 398, 399(16), 402(16),
Haugen, G. E., 310, 362 404(16), 406(16), 407(16), 409(16),
Havir, E. A., 578 410(16), 411(16), 412(16), 413(16,
Hawk, P. B.0 355 20), 414(16), 415(16)
H~yai~i, O., 369 Homing, M. G.0 398, 399(16), 402(16),
Hayakawa, T., 54, 55(3a), 542 404(16), 405(16), 406(16), 407(16),
Haynes, R. C., Jr., 528 409(16), 410(16), 411(16), 412(16),
Heap, R., 668(109), 671 413(16), 414(16), 415(16)
Hebblethwaite, G., 633, 663(44), 669 Howard, R. L~, 89(24), 90
Heev, E., 177 Howes, W. V., 163, 164, 165, 169(10),
Hegre, C. S., 213, 572, 584(16) 170(10, 18)~ 382
Heidelberger, C., 591 tisiang, M. W., 351, 352
Hele, P., 380 Hsu, C. H., 41
Henne, A. L., 627, 632, 643(23), 647, Hsu, R. Y., 231, 235
682 AUTHOR INDEX
Huang, K. Y., 431, 433, 434(6) Jones, C W., 134

Hudlicky, M., 624, 627, 662(23, 25), Jones, D. ]~., 557, 562
663(25), 665(23), 666(23, 25), 668(25), Jones, J. D., 560, 561(15, 17), 562(17)
669 Jones, M. E., 375
Hudson, P. B., 66 Joyce, B. K., 100, 105
Hfibscher, G., 537, 546(9) Juni, E., 362
Hfifner, M., 90 Jutting, G., 189
Hughes, D. E., 135, 139(2)
Hughes, L., 426 K
Hughes, W. L., 108, 146 Kahn, J., 90
Hulme, A. C., 560, 561(15, 17), 562(17) Kalb, D., 398, 399(10), 402(10), 403(10),
Hummel, J. P., 362, 526 404(10), 405(10), 414(10)
Hunig, S., 599 Kalberer, P. P., 181
Hunt, S. B. D., 627, 628, 662(17), 663(17), Kalyanpur, M. G., 623
665(17), 666(17), 669 Kanarek, L., 91, 92(3), 93, 96(3), 97(3),
Hurlbert, R. B., 425, 573, 574(22), 620, 98(3, 6)
623(4) Kaneshiro, T., 134, 140
Kaplan, N. 0., 106, 107, 109, 112(4), 113,
I 114(9), 116(1, 4, 12, 13), 145
Imai, K., 362 Karmen, A., 398
Ingold, C., 570 Karnieli, Y., 133
Ingraham, L. L., 350, 351(6), 352(5, 6), Kato, H. P., 613
353 Katsuki, H., 318
Inman, C. E., 663(55), 664(55), 665(55), Katsura, H., 610
670 Kauder, E. M., 382
Inoue, H., 153, 156(4), 158(4), 551 Kaufman, S., 65, 74, 75, 459, 498, 535
Iodice, A. A., 320 Kaziro, T., 196, 197(7), 198(7)
Irias, J. J., 244, 245(13) Kaziro, Y., 184, 186, 187(9), 188(8, 9),
Isherwood, F. A., 573 189(8), 190, 191, 193, 199
lshikawa, F., 55 Kearney, E. B., 81, 82, 88(3), 238
Itada, N., 309 Keech, D. B., 236, 243, 246, 247(2, 17),
Itzhaki, R. F., 77 248(17), 249, 257, 258, 261, 369, 519
Izawa, S., 173, 178(11) Keilin, D., 81, 82, 84(5), 87, 390
Keller, H. J., 198
J Kellerman, G. M., 198
Jackson, F. L., 134 Kellermeyer, R., 194, 197(6), 208
Jacob, F., 46 Kellermeyer, R. W., 195, 207, 208, 213,
Jacob, M., 69, 74, 459 216, 220, 227(10), 228(11), 298,
Jacobson, B., 194, 208, 217, 220, 224(6), 301(3)
225(6), 226(6), 227(6), 228(6, I1) Kent, P. W., 633, 651, 663(44, 45),
Jacobson, K., 99 664(67, 72), 669, 670
Jaenicke, L., 369, 536 Kerr, D. S., 245
Jahn, H., 662(11), 667(11), 669 Keskin, H., 574
Jakoby, W. B., 369 Kesner, L., 415, 416(1), 417(1), 420, 424
Jamieson, D., 445 Kharasch, M. S., 627, 664(76), 670
Jangaard, N. O., 25 Kier, L. B., 398, 399(8), 402(8), 404(8),
Jayasuriya, G. C. N., 294 405(8)
Jencks, W. P., 77, 80(6), 81(6) Kilbourne, H. W., 630, 635, 664(71), 670
Jenkins, W. T., 323, 325 Kimura, T., 134, 140
Johnson, B. C., 600 King, T. E., 82, 84(7), 86(7), 88, 89(24),
Johnson, M. J., 35 90, 524
AUTHOR INDEX 683
Kinnory, D. S., 429 Kumler, W. D., 629

Kirkman, S. K., 42 Kun, E., 8, 100, 105, 121, 122, 627, 628,
Kitto, G. B., 106, 107, 108(5), 112(4), 629, 633, 638(25), 642(25), 643(25),
113, 115, 116(4, 12, 13) 644(25), 645, 647(25), 649(25), 650
Klein, N., 362 (25, 45), 653(28), 654(28, 47), 659(16,
KleinzeUer, A., 489 17), 660(25), 661(25), 663(38, 40),
Klingenberg, M., 40, 41, 42(12, 15), 393 664(40, 81), 665(81, 82), 666(40, 96),
Klotzsch, H. R., 381 668(40, 96, 105, 107), 669, 670, 671,
Klyne, W., 569 672(25)
Knappe, J., 189 Kunkel, H. G., 210
Knight, E., Jr., 57 Kupieeki, F. D., 80, 81(10)
Knight, M., 190 Kupke, D. W., 61
Knox, K. L., 398, 399(16), 402(16), Kurahashi, K., 270
404(16), 405(16), 406(16), 407(16), Kurata, Y., 361
409(16), 410(16), 411(16), 414(16), t
415(16)
Knunyants, I. L., 634(43), 648, 662(9, 20), Ladd, J. N., 320, 322(3), 330(3)
663(36, 37), 668, 669 Lambeth, J., 299
Koch, B., 559 Lamprecht, W., 430
Koch, J., 369 Landau, B. R., 239, 249
Koch-Weser, D., 190 Lane, M. D., 188, 190, 213, 270, 273(1, 2),
Kohn, J., 392 275(2), 276(1, 2), 277(2), 279(1),
Kohout, P. M., 136 282(1), 283(4)
Koike, M., 54, 55(3a), 56, 61(4), 542 Lang, G., 381
Komarov, V. A., 662(12), 669 Langley, M., 55
Korkes, S., 59, 535 Lardy, H. A., 13, 69, 231,235, 314, 315(2),
Kornacker, M. S., 156 528, 530, 535(10)
Kornberg, A., 117, 344, 518, 520 Large, P. J., 292
Kornberg, H. L., 23, 135, 163, 170, 171, Lartigue, D. J., 358, 360(10), 361(~.0)
277, 286, 288(2), 289, 309, 314(2), Lascelles, J., 180
317, 362, 365 Layne, 1~., 259, 272, 276, 279, 283, 388
Kosicki, G. W., 3, 4(6), 10, 16, 365, 388 Lee, H. A., Jr., 663(43), 669
Kosow, D. P., 188 Lee, L. P. K., 10
Kosower, E., 639 Lees, M., 137
Kowala, C., 398, 399(5), 402(5), 403, Legavit, R. R., 619
404(5), 405(5), 406(5), 414 Lemieux, R. U., 576
Kramer, D. N., 362 Lengyel, P., 204
Kranz, Z. H., 398, 399(5), 402(5), 403(5), Leonard, H. A., 69
404(5), 405(5), 406(5), 414(5) Leone, E., 189
Krasna, A. I., 361, 574, 577(29), 578(29), Leonhardi, G., 526
579(29), 581(29),~628, 633(12), 651 Le Page, G. A., 519
(12), 664(80), 670 Lessard, J. R., 398, 399(2), 402(2), 404(2),
Krebs, H. A., 153, 171, 447, 479, 514, 575, 405(2), 414
613 Levin, D., 143, 145(3)
Krebs, M. A., 98 Levin, 0, 201, 202(8), 204(8), 275, 281
Kreisberg, R. A., a,45 Lewis, R. G., 115
Kruglyak, Y. L., 662(14), 663(14), 669 Lewis, Y. S., 615
Kuhn, J., 662(3), 668 Li, J. C. R., 89(24), 90
Kuksis, A., 398, 399(7), 402(7), 403, Libano, W. Y., 353
404(7), 405(7), 407(7) Lichstein, H. C., 361
Kullnig, R. K., 576 Lienhard, G. E., 41, 592, 593(87), 596(87)
684 AUTHOR INDEX
Lightbown, J. W., 134 641(89, 102), 658(22), 662(8), 664(79),

Lilienfeld, W. M., 620 665(89, 93), 667(89, 93, 102), 668, 670,
Lin, F. J., 145 671
Lindsey, R. V., Jr., 634, 667(103), 671 McIlwain, H., 594
Ling, A. M., 249 Mclntyre, R. T., 360
Linnane, A. W., 140 MeKay, R. H., 109, 114(9)
Lipmann, F., 3, 71, 153, 216, 375, 539(22), MeKenzie, A., 597
546(22), 551, 588, 619 McKeown, G. G., 398, 399(15), 402(15),
Listowsky, I., 41, 577, 584(52), 592, 403, 404(15), 405(15), 408(15)
593(86), 597(52) McLennan, D. J., 569
Littlefield, J. W., 52, 54, 55, 65, 498, 499, Maeba, P., 26, 286, 287, 288(3, 5), 292
500(72), 542 Mager, J., 629, 630, 658(19), 663(49),
I_~chmfiller, H., 217, 221, 225(12), 229 665(49, 85), 670
(12), 248, 297, 298(la), 299(la), 300 Mahler, H. R., 537
(la), 305(la), 306(la), 307(la), 308 Maitra, P. K., 436, 438(5), 481, 495(60)
Loewus, F. A., 574, 575, 586(28) Makarov, S. P., 662(14), 663(14), 669
Long, M. V., 256(1), 263, 267, 268(1) Makita, M., 398, 399(14), 404(14),
Loomis, W. D., 555, 558(1), 559(1) 405(14), 406(14), 407(14), 409(14),
Lorch, E., 189 410(14), 411(14), 412(14), 413
Losada, M., 170, 248, 250, 252(1, 9), 256 Maragoudakis, M., 619
(9), 257(1, 9, 14), 315 Marcus, A., 381
Losada, M. J., 246 Market, C. L., 46
Lovenberg, W., 180 Marler, C., 96, 98(6)
Lowenstein, J. M., 156, 171, 513, 514(3), Marlow, A. R., 426
516 Marlynov, I. V., 662(14), 663(14), 669
Lowry, O. H., 17, 23, 43, 100, 117, 123, Marquis, N. R., 388, 393(7), 509
130, 138, 155, 284, 289, 311, 321, 333, Martin, R. G., 117, 210, 287
345, 348, 351, 355, 435, 436, 437(4), Martius, C., 590, 614
438(4), 442, 445(9), 447(9), 519, 526, Maruyama, H., 270, 273(2), 275(2),
531 276(2), 277(2), 279, 282(1), 283(4)
Lugg, J. W. H., 528 Mason, H. S., 555
Lugovoy, J. K., 514, 515(4), 575 Massey, V., 43, 53, 54(3), 55(3), 91, 92, 97,
Luke, H. H., 398, 399(8), 402(8), 404(8), 98, 465, 498, 524, 536, 542(7)
405(8) Masters, R., 380
Lundin, R. E., 351, 353 Matehett, J. R., 619
Luaty, C. J., 524, 531, 535(19) Matthews, J., 56
Lynen, F., 188, 189, 190, 198, 213, 221, Maue, R., 614
225(12), 229(12), 501, 536, 547(20), Mayer, A., 602
55O Mayer, D., 436
Mayoh, H., 163
M
Mazumder, R., 65, 188, 193, 196, 197(7),
198(7), 199(2), 200, 204, 206(2), 212
McBee, E. T., 629, 630, 632(15), 635, Medes, G., 574
663(60), 664(71), 670 Megraw, R. E., 163, 362
McCaffrey, I., 398 Meister, A., 75, 485
MeCaman, M. W., 389, 392(13) Meites, L., 367
McCaman, R. E., 389, 392(13) Meites, T., 367
McFadden, B. A., 163, 164, 165, 169(10), Melby, L. R, 634, 667(103), 671
170(10, 14, 17, 18), 362 Mellanby, J., 476, 478(54), 479
Maehalina, E. P., 663(36), 669 Menon, G. K. K., 80, 81(8, 10)
Maehleidt, H., 626, 627, 630(22), 631,635, Meyer-Arendt, E., 382
AUTHOR INDEX 685
Michal, G., 386, 540(11), 541 Nassauer, M., 602

Miehejda, J., 113, 116(12) Nassauer, N., 613
Middleton, B., 10 Natelson, S., 3, 514, 515(4), 575
Middleton, W. J., 662(28), 669 Nawa, H,, 56
Mielewitz, A., 628 Neelakantan, S., 615
Mii, S., 136 Neilands, J. B., 100
Mildvan, A. S., 226, 245, 248 Nelson, E. K., 602
Miller, H. E., 600, 601, 608, 613 Nicholls, D. G., 11
Miller, R. S., 270, 273(2), 275(2), 276(2), Nief, G, 575
277(2) Nimmo, C. C., 619
Miller, S. J., 213 Nishimura, J. S., 75
Miller, W. T., 662(31), 669 Noble, R. E., 190
Millerd, A., 163 Nolan, L. S., 604
Millington, J. E., 662(4), 663(4), 666(101), Noller, C. H., 398, 399(10), 402(10),
668, 671 403(10), 404(10), 405(10), 414(10)
Mirocha, C. J., 398, 399(3), 402(3), 403, Nordal, A., 602
404(3), 405(3), 414 Norman, A. W., 425, 547(35, 36)
Moat, A. G., 360 Norman, J. J., 663(42), 669
Mobbs, R. H., 625 Northrop, D. B., 226
Mochalina, E. P., 663(37), 669 Nossal, P. M., 530
Mock, W. L., 599 Notter, G. K., 619
Moellering, H., 382, 451, 452(29), 503, Novelli, G. C., 539(22), 546(22), 551
517, 545(16), 548 Nystrom, R. F., 600
Moffitt, W., 97
Moiler, F., 46 O
Monod, J., 46
Montgomery, C. M., 519, 523 Ochoa, S., 3, 11, 12, 16, 20, 65, 141, 181,
Moore, S., 430, 575, 581(35) 184, 186, 187(9), 188(2, 8, 9), 189(2,
Morris, J. G., 177 8), 190, 191, 193, 196, 197(7/, 198(7),
Morrison, J. F., 26, 30, 444, 447, 531 199(2), 200, 204, 20fi(2), 209, 212, 216,
Mortenson, L. E., 180 230, 236, 251, 293, 365, 375, 446, 498,
Morton, R. K., 163 501, 535, 605
Moss, G., 531 O'Connell, E. L., 572, 589(19)
Moyer, R. H., 74 Oesterling, R. E., 663(55), 664(55),
Moyer, R. W., 70, 74(2), 75(2, ll) 665(55), 670
Mukherjee, B. B., 56 Ogata, S., 171, 315
Muller, H. R., 599 Ogston, A. G., 96
Muntwyler, E., 415, 416(1), 417(1), 420 Ohmura, E., 369
Murphey, W. H., 145 Okabe, K., 542
Murray, J. F., Jr., 664(69), 665(69), Olah, G., 662(3), 668
666~69), 667(69), 670 Oliver, R. M., 55
Murray, K. E., 398, 399(5), 402(5), Olmsted, M. R., 248
403(5), 404(5), 405(5), 406(5), 414(5) Olson, J. A., 169, 362, 473
Myers, W. F., 431 Olson, M. S., 430
Oser, B. L., 355
N Otauka, S., 170
Ouchterlony, O., 336
Nagai, J., 317, 318 Ouellet, L., 65
Nair, P. V., 630, 642(20), 643(20), 658(20), Overath, P., 198, 213
659(20), 663(50), 670 Overell, B. T., 528
Naqvi, S. M., 631, 664(62), 670 Owens, H. S., 431
686 AUTHOR INDEX
P Pollard, A. L., 431

Paetkau, V., 528 Popj~k, G., 575, 577, 596(53), 598(45),
Palacian, E., 248, 252, 256(9), 257(9, 14) 600(45)
Palmer, J. K., 431,573, 574(23), 609 Porath, J., 253
Pande, S. V., 17 Portsmouth, D., 599
Pappas, A., 574 Potter, V. R., 425, 573, 574(22), 591, 620,
Paran]pe, S. V., 318 623(4)
Parker, R. E., 626 Powell, R., 98
Parks, R. E., Jr., 62, 65(1), 68(6), 69(6), Pravova, E. P., 664(65), 670
460, 506(45), 551 Prelog, V., 569, 570
Parvin, R., 17, 19 Pricer, W. E., Jr., 174, 175(15), 520
Passonneau, J. V., 435, 436(4), 437(4), Pring, W., 546(30), 551
438(4), 442, 445(9), 447(9), 519, 531 Pucher, G. W., 514, 515(4), 601, 602, 603,
Pasto, D. J., 599 608, 613
Pattison, F. L. M., 624, 627, 628, 634, Pudles, J., 112
638, 639, 648, 649(36), 662(4, 6, 17, Purdy, W. C., 366
19), 663(4, 6, 17, 19, 42, 59), 664(74), Q
665(6, 17, 19, 87), 666(6, 17, 19, 74,
99, 101), 669(6, 51, 99), 668, 669, 670, Quaranta, P., 420
671
Quastel, J. H., 354
Pawollek, A., 614 Quayle, J. R., 292, 369, 370
Pearson, D. J., 4, 376, 387, 388(5), 389(2), Quin, L. D., 398, 402(1), 403, 404(1),
391(5), 503, 506, 510, 536, 540(5), 541, 405(1), 406(1), 414
545, 546(15), 548, 550(5) R
Peel, D., 292
Pennington, R. J., 270, 586 Raasch, M. S., 627, 634, 644, 657(37),
Perez, L., 132, 134(4), 140 660(37), 663(47), 664(47, 77, 78),
Perlman, D., 330 665(78, 91), 666(78, 91), 667(91),
Perrin, D. D., 625 668(78), 669, 670, 671
Perrin, J. R., 591 Rabin, R., 163, 362
Pervova, E. Y., 662(9), 668 Rabinowitz, J., 663(41), 669
Pesce, A., 109, 114 Rabinowitz, J. C., 174, 175(15), 177, 180
Peters, D. A. V., 634 Racker, E., 26, 91, 354, 446, 575
Peters, R. A., 30, 450, 627, 628, 668(108), Raeburn, S., 177
Raghavendra Rao, M. R., 318
671
Peters, R. J., 663(46), 669 Rahatekar, H. A., 318
Peterson, E. W., 555 Ramakrishna, M., 619
Pettitt, B. C., 402(21), 405(21), 406(21), Ramaley, R. F., 70, 74(2), 75(2, 11), 460,
410 5O6(44)
Pfenning, N., 180 Randall, R. J., 17, 23, 43, 100, 117, 123,
Pfleiderer, G., 100, 382 130, 138, 155, 284, 289, 311, 321, 333,
Phares, E. F., 263, 265, 267, 268(1) 345, 348, 351(4), 355
Phillips, P. H., 182, 375, 376 Rao, D. Rajagopal, 619
Phizackerley, P. J. R., 23, 134, 135 Rao, G. R., 169, 170(17)
Rao, M. R. R., 318
Pierce, O. P., 630, 635, 664(71), 670 Rather, S., 574, 578
Pierpoint, W. S., 555, 558 Read, S. I., 398, 399(15), 402(15), 403(15),
Pietropaolo, C., 361 404(15), 405(15), 408(15)
Pincus, J. B., 3, .514, 515(4), 575 Redfearn, E. R., 134
Plaut, G. W. E., 34, 35(2), 37, 38(la, 2), Reed, L. J., 55, 56, 57, 60, 61(4)
39(10), 40(2), 41(2), 42(21) Reeves, H. C., 163, 362, 363, 364, 365(14)
AUTHOR INDEX 687
Remberger, U., 9, 257, 258, 261(1) Sakaguchi, K., 613

Renold, A. E., 476(57), 479 Salvatore, F., 361
Renwick, J. A. A., 663(54), 666(54), 670 Sanadi, D. R., 52, 54, 55, 65, 69, 74, 459,
R4tey, J., 90, 188, 198, 214, 215(18) 498, 499, 500(72), 542
Rhykerd, C. L., 398, 399(10), 402(10), Sanderson, G. W., 558, 559, 560(13)
404(10), 405(10), 414(10) Sanwal, B. D., 26, 44, 46(6, 7), 47, 286,
Riegel, B., 620 287, 288(3, 5), 292
Riepertinger, C., 221,225(12), 229(12) Sasakawa, S., 196, 197(7), 198(7)
Ringelmann, E., 189 Sasakawa, T., 188, 193, 200, 212
Rinne, R. W., 250, 252(2), 257(2), 258(2), Satoshi, I., 542
261 Saunder, B. C., 626, 627, 639, 648(31),
Ristau, O., 437, 536 662(15), 663(15, 59), 665(87), 668
Rittenberg, D., 90, 578, 579 (106, 109), 669, 670, 671
Rivett, D E. A., 665(84), 668(108), 670, Savage, E., 426
671 Scaletti, J. V., 398, 399(2), 402(2), 404(2),
Roberts, G. R., 558 405(2), 414(2)
Roberts, N. R., 526 Sch~ifcr, G., 430
Robinson, G. W., 98, 99 Schmidt, G., 662(11), 667(11), 669
Roehovansky, O., 578 Schneider, M. C., 11, 75, 76(1), 77(1),
Rodwell, V. W., 65 79(1), 80(1), 81(1)
Rogers, L. J., 160, 452 Schneider, W. G., Jr., 576
Rogers, M T., 631 Schoffa, G., 437, 536
Rooze, V., 330 Scholz, R., 445
Rose, I. A., 41, 270, 572, 584(13), 589(13, Schoner, W., 546(30), 551
19), 590(13), 592, 593(87), 596(87), Schoore, G., 590
600(13) Schroepfer, G., Jr., 577, 596(53), 600
Rose, Z. B., 593, 595(88) Schultz, S. K., 387, 388(3), 389(3), 392(3,
Rosebrough, N. J., 17, 23, 43, 100, 117, 12), 505, 509(83)
123, 130, 138, 155, 284, 289, 311,321, Schulz, D. W., 435, 436(4), 437(4),
333, 345, 348, 351(4), 355 438(4), 519
Ro~en, H., 575 Schuster, C. W., 382
Rothstein, M., 163 Schwartz, P., 590
Rouge, D., 647, 657, 662(26), 665(26, 83), Schwert, G. W., 519
668(83), 669, 670 Scott, E. M., 98
Roy, S. K., 633, 638(25), 642(25), 643(25), Scott, R. M., 360
644(25), 647(25), 649(25), 650(25), Scrutton, M. C., 226, 227, 236, 244,
660(25), 661(25), 663(38), 667(38), 245(12, 13), 246, 247(2), 248(12, 15,
669, 671(25), 672(25) 18), 249, 257, 258
Rozenshtein, S. M., 664(65), 670 Searls, R. L., 55
Ruiz-Amil, M., 246, 250, 252(1), 256(9), Seibl, J., 90, 214, 353
257(1, 9) Seligson, D., 531
Rumsey, T. S., 398, 399(10), 402(10), 403, Seligson, H., 531
404(10), 405(10), 414 Senear, A. F., 612
Russell, S., 559 Sergeev, A. P., 662(29), 669
Rutter, W. J., 169, 231, 530, 535(10) Settim], G., 343
Ryback, G., 90, 577, 596(53), 600 Seubert, W., 235, 236, 257, 258, 261(1), 546
Ryhage, R., 575, 598(45), 600(45) (30), 551
Shahak, I., 626, 628, 638, 639(29), 641(70),
S 647(13), 649, 662(5, 7, 16, 22), 663(5,
Sadler, J. R., 23 7, 34, 39, 51, 52, 53), 664(22, 39, 51,
Saffran, M., 513, 514(1) 70, 73), 665(7, 34, 39, 51, 70, 73), 666
688 AUTHOR INDEX
(51, 53, 95, 100), 667(5, 51, 70), 668 Sprecher, M., 188, 198, 575, 598, 599(33),
(95, 104), 668, 669, 670, 671 60O
Shani, A., 626, 664(66), 666(95), 670, 671 Sprinson, D. B., 188, 198, 575, 579, 598,
Shanley, E. S., 591 599(33), 600
Shapiro, B., 16, 375 Srere, P. A., 3, 4(6), 9, 10, 11, 12(4), 16, 19,
Sharpies% N. E., 398 22, 26, 153, 365, 387, 388(3), 389(3),
Shavit, N., 96 392(3), 505, 509(83), 546(29), 551
Sheehan, J. C., 584 Stacey, G. J., 626, 639, 648(31), 662(15),
Shemin, D., 76, 182, 195, 208, 363 663(15), 669
Shepherd, D., 4, 11, 12(1), 16(1), 25, 536, Staeey, M., 626, 632, 665(90), 671
541(6), 543(6), 546(6) Stachow, C. S., 44, 46(6, 7), 47
Sherman, C. C., 514, 515(4) St~dler, P. A., 319
Sherman, I. W., 150 Stadtman, E. R., 22, 70, 213, 216, 289,
Shiio, I., 165, 169, 170(14) 365, 368(2), 375, 381, 382, 387, 536,
Shiio, T., 165, 169, 170(14) 538(1, 22), 546(22), 551
Shipp, W. S., 59 Stafford, H. A., 618
Shoolery, J. D., 629 Stafford, M. L., 389, 392(13)
Siebert, G., 448 Stahl, E., 431
Siegel, L., 100, 104(10, 11), 105(11), Stainer, R. Y., 140
106(14), 124, 128, 129(12) Stark, J. B., 431, 575
Siegelman, H. W., 73, 119 Stein, A. M., 42
Silverstein, E., 519 Stein, J. H., 42
Simmonds, P. G., 402, 405(21), 406(21), Stein, W. H., 430, 575, 581(35)
410 Steinberg, D., 380
Simon, E. J., 76, 182, 195, 208, 363 Steinberger, R., 630
Singer, T. P., 81, 82(2), 88(2, 3), 89(23), Stephens, N., 434
90, 238, 524, 531, 535(19) Stern, J. R., 9, 11, 16, 75, 76(1), 77(1),
Singh, R. M. M., 173, 178(11) 79(1), 80, 81(1, 8, 10), 177, 375, 468,
Sisler, E. C., 171 501, 509(77), 513, 540(27), 572,
Siu, P. M. L., 306 5s4(16), 605
Siva Raman, C., 160, 161(2) Stevens, C. M., 623
Skladnev, A., 662(12), 669 Still, J. W., 140
Slater, E. C., 83, 84 Stjernholm, R. L., 190, 194, 195, 197(6),
Sloane-Stanley, G. H., 137 207, 208, 213, 216, 217, 219, 220,
Smillie, R. M., 171 224(6), 225(6), 226(6), 227(6, 8, 10),
Smith, L. H., Jr., 523 228(6, 11), 229(8), 298, 301(3)
Smith, R. A., 74, 169, 170(15), 459 Stolzenbach, F., 109, 114(9)
Smith, W. C., 645, 665(88), 666(88), 670 Stone, R. W., 170
Smyth, R. D., 319, 320(2), 322(3), 330(3), Stoner, G. S., 662(30), 669
336, 345, 347, 348(1), 350, 351(1, 6), Stoolmiller, A. C., 599
352(6) Stoppani, A. O. M., 250, 252(3), 258(3)
Snyder, F., 434, 529, 531(8) Stothers, J. B., 627, 628, 662(17, 19),
Soderstrom, T. R., 602 663(17, 19), 665(17, 19), 666(17, 19),
Sokolov, S. V., 626 669
Sokohfkii, G. A., 662(20), 669 Strassman, M., 526, 527(5), 619, 623
Solig, H. D., 575 Straub, F. B., 100
Spahr, P. F., 351 Strecker, H. J., 455
Spencer, A. F., 513, 514(3), 516 Streitweiser, A., Jr., 625, 663(58), 670
Spizizen, J., 142 Stuart, S. C., 528, 533, 534
Sporzynski, A., 662(2), 663(2), 668 Su, IC-Y., 662(33), 669
AUTHOR INDEX 689
SubbaRow, Y., 310 Trebst, A. V., 171, 315

Subramanian, S. S., 318, 319 Tschesche, R., 641(102), 667(102), 671
Summerson, W. H., 355 Tsou, C. L., 81, 82(4), 88(4)
Sung, S.-C., 41, 42(21) Tsuboi, K. K., 66
Sutherland, E. W., 236 Tsunoda, T., 170
Suzuki, F., 153, 156(4), 158(4), 319, Tubbs, P. K., 9, 10, 387, 388(5), 391(5),
321(1), 325(1), 330(1), 551 392, 497, 505(70), 506, 510, 536,
Suzuki, I., 277 540(5), 541, 547(19, 31), 550(5), 551
Swann, M. It., 398, 399(4), 402(4), 404(4) Turner, B. C., 73, 119
Swick, R. W., 195, 213, 219, 226(9), 227(9) Turner, H. S., 570
Swim, H. E., 415, 424(2) Tuttle, L. C., 3, 71, 216, 588, 619
Switzer, R. L., 321, 325(4), 326(4), 329, Tycykowski, E., 663(55), 664(55), 665
330(4), 598 (55), 670
Szinai, S., 627, 664(75), 665(75), 670 Tyuleneva, V. V., 662(9), 668
T U
Tabor, H., 375 Udenfriend, S., 633, 662(1), 665(1), 668
Takeda, Y., 153, 156(4), 158(4), 429, 551 Udilov, G. P., 626
Tamura, Y., 398, 399(14), 404(14), Umani-Ronchi, A., 214
405(14), 406(14), 407(14), 409(14), Upper, C. D., 70, 551
410(14), 411(14), 412(14), 413 Utter, M. F., 226, 227, 236, 239, 243, 244,
Tanaka, S., 318 245(12, 13), 246, 247(2, 17), 248(12,
Tarrant, P., 662(10), 669 15, 17, 18), 249, 250, 257(4), 258,
Tarmy, E., 146 261, 270, 415, 424(2), 519, 586
Tate, S. S., 160, 161(6), 162, 163(6), 452 Uyeda, K., 177
Tatlow~ J. C., 626, 632, 665(90), 671 ¥
Taylor, G. A., 369
Taylor, N. F., 633, 651, 663(44, 45), Valentine, R. C., 244, 245(13)
664(67), 669, 670 VandenHeuvel, W. J. A., 399, 410(20),
Tehen, T. T., 277, 574, 575(28), 586(28), 411(20), 412(20)
599 VanMilligan, H., 599
Teipel, J., 96 Varner, J. E., 429, 528
Thiev, W., 599 Veeger, C., 83, 88(11), 90(11, 20, 21),
Thomas, L. C., 668(108), 671 524(4), 525
Thomas, S. L., 570 Venkitasubramanian, T. A., 17
Thomas, U., 623 Vennesland, B., 270, 277, 574, 575(28),
Thompson, V. W., 33 586(28)
Thorne, C. J. R., 100, 106(9), 116(1), 120, Veres, K., 663(35, 61), 664(61), 665(92),
123, 124, 125(2) 666(92), 667(61), 669, 670, 671
Tietz, A., 181, 188(2), 189(2) Vernon, L. P., 181
Ting, I. P., 150 Vickery, H. B., 164, 514, 515(4), 570, 600,
Tiselius, A., 143, 145(3), 201, 202(8), 601, 602, 603, 608, 613
204(8), 275, 281 Vignais, P. M., 42
Tobari, J., 134, 140 Vignais, P. V., 42
Tobin, N. F., 369 Virtanen, A. I., 358, 361(11)
Tolbert, B., 250, 257(4) Vishwakarma, P., 398, 399(7), 402(7),
Tolman, V., 663(35, 61), 664(61), 665(92), 403(7), 404(7), 405(7), 406(7)
666(92), 667(61), 669, 670, 671 Vogel, H. J., 44
Toohey, J. I., 320, 322(3), 330(3) Volcani, B. E., 320
Traynham, J. G., 351, 352(8) Volfin, P., 122
Treble, D. H., 663(46), 669 vonder Mfihll, P. A., 343
690 AUTHOR INDEX
yon Glasenapp, I., 526 Wieland, O., 4, 9, 11, 16(3), 382, 547(18),
Von Korff, R. W., 380, 429, 430(15), 519, 55O
520(9, 10), 523, 528, 539(24), 551 Wilcox, P. E., 591
Voong, ST., 664(64), 670 Wiley, D. W., 665(86), 670
Wflkson, J. S., 358, 360(12)
W Williams, G. R., 528, 533, 534
Williams, V. R., 351, 352(8), 353, 358,
Wads, A., 318 360(10, 12), 361(10)
Wakelin, R. W., 668(108), 671 Williamson, D. H., 476, 478(54), 479
Wakil, S..J., 537, 546(9) Williamson, J. R., 436, 437, 438(7), 445,
Walden,~P., 581 481, 495(60)
Walker, J. R. L., 562 Willms, C. R., 57, 60
Wallace, A., 21, 25 Wilson, A. C., 108
Wallace, J. C., 248 Wilson, D. F., 89(24), 90
Walter, P,, 528 Wilson, D. G., 602
Wang, C. C., 331, 333(2), 336(2), 337(2), Wilson, E. R., 630, 635, 664(71), 670
338(2), 340(2), 342(2) Wilson, P. W., 140
Wang, S.-F., 69, 314, 315(2) Wilson, R. M., 319, 320(2), 336, 345, 347,
Wang, T. Y., 81, 82(4), 88(4) 348(1), 351(1)
Wang, Y. L., 81, 82(4), 88(4) Winer, A. D., 663(43), 869
Warburg, O., 43, 48, 139, 259 Winger, G. D., 173, 178(11)
Warner, R. C., 186, 187(9), 188(9), 191, Winkler, M. F., 351
198, 199(2), 206(2) Winner, A. D., 519
Wassarman, P. M., 113, 116(12, 13) Winter, W., 173, 178(11)
Watt, J. M., 617 Wiskich, J. T., 561
Webb, E. C., 14 Wlslicenus, W., 602, 613
Webb, J. L., 519, 523 Witkop, B., 633, 662(1), 665(1), 668
Weber, H., 343, 592 Wnuk, J., 662(2), 663(2), 668
Webster, L. T., Jr., 375, 376, 380 Wolfe, R. G., 96, 97, 100
Wegener, W. S., 163, 362, 363, 364, 365(14) Wolfe, R. S., 165
Weinhouse, S., 574 Wolin, E. A., 165
Weiss, L., 4, 9, 11, 16(3), 547(18), 550 Wolin, M. J., 165
Welssbach, H., 319, 320(2), 322(3), Wollenberger, A., 437, 536
330(3), 336, 345, 347, 348(1), 351(1) Wong, D. T. O., 362
Weitzman, P. D. J., 4, 10, 11, 22, 23, Wood, H. G., 194, 195, 197(6), 207, 208,
24(5), 25, 366, 538(21), 551 213, 216, 217, 219, 220, 221, 224(6),
Wellman, H., 13 225(6, 12), 226(6, 9), 227(6, 8, 9, 10),
Wells, J. R. E., 163 228(6, 11), 229(8, 12), 248, 297,
Wenske, G., 40, 42(12) 298(la), 298(2), 299(la), 300(la), 301
Werkman, C. H., 277 (3), 305(la), 306(la), 306(2), 307(la,
West, P. W., 290 2), 308
Westheimer, F. W., 630 Woo[f, B., 354
Wetley, J., 299 Woo[ford, R. G., 662(19), 663(19), 665
Whatley, F. R., 181 (19), 666(19), 669
Wheat, R. W., 452 Wooltorton, L. S. C., 560, 561(15, 17),
Whlssen, N., 10, 26 562(17)
Whitaker, J. R., 336 Wosiliat, W. D., 236
White, F., 55 Wright, J. A., 26
Wichers, E., 367 Wrigley, N. G., 244, 245(13)
Wieczoreck, C. A., 119 Wu, M., 526
Wieczorek, G. A., 73 Wynn, W. K., 663(54), 666(54), 670
AUTHOR INDEX 691
Y Yphantis, D. A., 267, 392

Yakubovich, A. Y., 662(29), 663(48), Yuan, C-Y., 662(33), 669
664(65), 669, 670 Yue, S. B., 150
Yamashita, M., 619
Yang, F-C., 662(33), 669
Yao, C-H., 662(33), 669
Yarger, K., 398, 399(16), 402(16), 404(16), Zugalak, B., 213, 214(12), 215(12)
405(16), 406(16), 407(16), 409(16), Zappia, V., 361
410(16), 411(16), 414(16), 415(16) Zaugg, W. S., 181
Yates, D. W., 11, 536, 541(6), 543(6), Zeylemaker, W. P., 88, 90(21), 524(4), 524
546(6) Zielinski, A. Z., 663(60), 670
Yoshida, A., 142, 144, 145 Zimmerscheid, W. J., 627, 647, 662(21),
Young, D. A. B., 476(57), 479 663(21), 667(21), 669
Young, ft. A., 662(10), 669 Zink, M. W., 44, 46(6)
Young, M. R., 250, 257(4) Zlatkis, A., 402(21), 405(21), 406(21), 410
~92 SUBJECT INDEX
Subject Index
NOTE: In general acids are indexed under the names of their anions. Fluoro
analogs are listed separately at the end of the index (p. 724).
A dehydrogenase (FAD-linked), 130-

ADP, see Adenosine diphosphate 132
AMP, see Adenosine monophosphate Acetone powder, preparation from beef
ATP, see Adenosine triphosphate heart, 109-101
Acetate Acetyl a<lenylate, acetyl-coenzyme A
chromatography synthetase intermediate, 375, 380
ion-exchange, 426, 428 Acetylcamitine
partition column, 420, 422, 429-430 fluorometric assay for, 509-513
contamination of pyruvate by, 519, 522, levels in rat tissues, 446
523 substrate for carnitine acetyltransh,r-
product of citramalate pyruvate lyase ase, 387, 393
Acetylcarnitine transferase, fiuoromotric
reaction, 344
production from itaconate in mamma- assay with, for
acetylcarnitine, 509-513
lian liver, 315-316
L-camitine, 505-509
substrate for acetyl-coenzyme A syn-
Acetyl-coenzyme A
thetase, 375
activator of the following enzymes
substrate in assay for coenzyme A, 539
phosphoenolpyruvate carboxylase,
Acetate: coenzyme A ligase (AMP) see
Acetyl-coenzyme A synthetase 288, 292
pyruvate carboxylase, 235-237, 246,
Acetic acid(s), substituted, Taft con-
247, 251, 257, 261
stants for, 625
assays for, with
Acetic anhydride-pyridine method for
arylamine acetyltransferase, 546
determination of citrate, aconitate,
C~4-citrate formation from C'4-aspar-
and isocitrate, 514-515
tate, 546
specificity, 516
citrate synthase and dithionitroben-
Acetoacetate
zoate, 544-545, 546
enolization, 630-631
citrate synthase and malate dehydro-
fluorometric assay, 526
fluorometric enzyme assay, 444-445, genase, 501, 503, 545-548
478-481 a-ketoglutarate dehydrogenase and
inhibitor of succinate dehydrogenase, phosphotransacetylase, 497
90 phosphotransacetylase, 546, 548-549
ion-exch~mge chromatography, 427-428 assay with, for
3oketoacid-coenzyme A tra~sferase, acetylcarnitine, 512, 513
substrate for, 75, 80 carnitine, 505, 506, 508, 509
succinate dehydrogenase, 90 methylmalonyl-coenzyme A mutase,
Acetoacetyl-coenzyme A 2O7
assay, 547
methylmalonyl-coenzyme A race-
magnesium chelate, in assay of 3-keto-
acid-coenzyme A tra~sferase, 75 mase, 194-196
substrate for 3-ketoacid-coenzyme A citramalyl-coenzyme A cleavage prod-
transferase, 75 uct, 316--317
A c e l o b a c t e r x y l i n i u m , source for malate citrate cleavage product, 153
SUBJECT INDEX 693
extraction from tissues, 439 deuterium transfer by, 588

levels in rat tissues, 445 inactivation, 449~150
substrate for the following enzymes specific radioactivity determination,
acetyl-coenzyme A synthetase, 375 use in, 529-534
carnitine acetyltransfera~c, 387-389 stability, 26, 29
citrate synthase, 3, 4, 8, 9, 11, 12, 15- c/s-Aconitate
16, 19-20, 22-23, 25 assays
malate synthase, 362-363 acetic anhydride-pyridine method,
phosphotransacetylase, 381 514-515
pyruvate synthase, 172-174 pentabromoacetone method, 515
Acetyl-coenzyme A: carnitine O-acetyl- spectrophotometric method, 26--28
transferase, see Carnitine acetyl- radioactivity, by enzymatic decar-
transferase boxylation, 528-535
Acetyl-coenzyme A : orthophosphate chromatography
acetyltransferase, see Pilosl)hotrans- gas, 397, 403, 404, 407-413
acetylase partition, 422
Acetyl-coenzyme A-oxaloacetate con- thin-layer, 432-43
densing enzyme, see Citrate synthase labeled citrate synthesis from, 589
Acetyl-coenzyme A synthetase, 375-381 substrate for aconitase, 26
acetyl-adenylate intermediate, 375, 380 trans-Aconitate
assay, rationale for choice of, 375-376 assay, 514, 516
cation requirements, 380 inhibitor of fumarase, 99
mechanism, 375 partition chromatography, 423
Acetyl-3'-dephospho-coenzyme A assay, urine, occurrence in, 424
547, 548 Acyl-cocnzyme A
Acetylene dicarboxylate long-chain, assay, 547, 550-551
deuteration, 577 short-chain, assay, 546, 54~550
inhibitor of fumarase, 99 Acyl-coenzymc A syntlwtas(,, coenzyme
Acetyl phosphate A assay, use in, 537, 540
assays with, for Adenine nucleotides
citrate synthase, 3 inhibition of citrate synthase, 9-11, 16,
coenzynw A, 539 19
citrate synthesis, labeled, 584-586, 588 tissue extra(.tion for, 438
pyruvate synthase assay, use in, 172- Adenosine dil)hosphate
174 activator of NAD-specific isocitrate
substrate for phosphotransa('ctylasc, dehydrogenase, 34, 36, 3940, 42
381-382, 385, 386 fluorometric assay, 404-497
3-Acetylpyridine adenine dinucleotide levels in rat tissues, 446
cocnzyme A assay, 542 substrate for tile following enzymes
malate dehydrogenase, substrate of, propionyl-coenzyme A carboxylase,
144 181, 183, 189
3-Acetylpyridine deaminoadenine dinu- pyruvate carboxylase, 235, 246
cleotide, malate dehydrogenase, sub- succinyl-coenzyme A synthetase, 70,
75
strate of, 144
Adenosine monophosphate
Acids, aliphatie, Taft constants for, activator of N e u r o s p o r a crassa NAD-
625 specific isocitrate dchydrogenas(,,
Aeonitase, 26-30 46
activation of, 26, 27, 30, 446, 448 fluorometric assay, 494-497
citrate assay, use in, 446-449 levels in rat tissues, 446
694 SUBJECT I N D E X
phosphoenolpyruvate synthetase, prod- inhibition by ADP, 159

uct of, 309, 311, 314 organ distribution, 159
substrate for acetyl-coenzyme A syn- Adenylate kinase
thetase, 375 contaminant of isocitrate dehydrogen-
Adenosine triphosphatase ase, 42
propionyl-coenzyme A carboxylase, in- interference by, in metabolite extrac-
terference in assay of, 181-182 tion, 438
succinyl-coenzyme A synthetase, con- Adipate, inhibitor of fumarase, 99
taminant of, 74 Aeonium decorum, source of isocitrate,
Adenosine triphosphate 603
assays for, fluorometric Aerobacter aerogenes, see Klebsiella
with hexokinase and glucose 6-phos- aerogenes
phate dehydrogenase, 488--491 Alanine, thin-layer chromatography,
with phosphoglycerate kinase and 432-433
glyceraldehyde 3-phosphate dehy- Albumin, see Bovine serum albumin
drogenase, 491-494 Alcohol dehydrogenase, fluorometric
assays with, for the following metabo- assay with, for NAD, 481
lites Aldolase, interference by, in metabolite
carnitine, 505, 506, 508, 509 extraction, 438
coenzyme A, 537, 540 Aloe microstigma, source of isocitrate,
glucose, 489 6O3
succinate, 459-460, 462 Aloe saponaria, source of isocitrate, 603
inhibitor of NAD-specific isocitrate dc- Aloe spinosissima, source of isocitrate,
hydrogenase, 40 6O3
levels in rat tissues, 446 Alumina C)' gel,
protecting effect on mal~te dehydro- use in preparation of
genase, 145 acetyl-coenzyme A synthetase, 378
substrate in reactions catalyzed by citrate lyase, 156-157, 162, 518
ATP citrate lyase, 153 isocitrate dehydrogenase, 49
acetyl-coenzyme A synthetase, 375- a-ketoacid CoA transferase, 79
376, 380, 381 phosphoenolpyruvate carboxylase,
phosphocnolpyruvate synthetase, 290
309-310, 313, 314 Amino acid(s)
propionyl-coenzyme A carboxylase, citrate syntha~e composition, 10-11
181-183, 188-190 ion-exchange chromatography, 428
pyruvate carboxylase, 235-236, 244, malate dehydrogenase composition, 145
245, 247, 250, 251, 257, 261 Ammonium ions
succinyl-eoenzyme A synthetase, 70, inhibition of phosphoenolpyruvate car-
74-75 boxylase, 296
Adenosine triphosphate: citrate oxalo- requirement for phosphotransacetylasc,
acetate lyase, see Adenosine triphos- 385
phate citrate lyase substrate for aspartase, 354-356
Adenosine triphosphate citrate lyase Amytal, inhibitor of malate oxidation in
assay with, for coenzyme A, 540 A. xylinum, 134
assays Anions, effects on
hydroxamate, 153 fumarase, 97
radioactive, 154 malate dehydrogenase, 133
spectrophotometric, 154 Antisera, to malate dehydrogenases, 112,
immunology, 160 116, 147
induction in rats, 155 Apple mitochondria, 560-562
SUBJECT INDEX 695
Arachls hypooeae L-Aspartate ammonia lyase, see Aspar-

germination, 279 tase
phosphoenolpyruvate carboxylase Avidin
from, 277 effects on
Arsenate oxaloacetate transcarboxylase, 227
in assay of phosphoenolpyruvate carboxykinase,
acetyl-coenzyme A, 498, 546, 548-549 277
coenzyme A, 539 phosphoenolpyruvate carboxylase,
suecinyl-coenzyme A, 65, 547 283
in breakdown of guanosine triphos- phosphoenolpyruvate carboxytrans-
phate by succinate thiokinase, 69 phosphorylase, 307
Arsenite, action on a-ketoglutarale de- propionyl-coenzyme A carboxylase,
hydrogenase, 55 189
Arsenolysis, see Arsenate pyruvate carboxylase, 237, 243, 248,
Arylamine acetyltransferase, in assay of 251, 252, 257, 261
acetyl-coenzyme A, 546 8-Azaguanosine triphosphate, succinate
coenzyme A, 539 thiokinase, 69
Ascorbate, quinone formation, 558-560 Azotobacter agilis, malate dchydrogen-
Aspartase ase, 140
assay by ammonia determination, 355 Azotobacter vinelandii, malate dehydro-
heterotropic interactions with nucleo- genase, 134
tides, 360
homotropic interaction of substrate, 361 B
occurrence, 361 8,2 coenzymes
preparation of labeled aspartate, 578 glutamate mutase, 320, 321, 325, 329-
spectrophotometric assay, 354 330
subunit structure, 358 methylmalonyl-coenzyme A mutase,
Aspartate 194, 199, 206, 208, 209, 211-215
assay with, for acetyl-coenzyme A, 546 methylmalonyl-coenzyme A racemase,
deuterated, enzymatic synthesis, 578- 196
580 BAL, see British antilewisite
fluorometric assay, 473-476 Bacillus subtilis, source for malate de-
inhibitor of hydrogenase, 142-144, 147
phosphoenolpyruvate carboxylase, Bacterium cadaveris
288, 292 occurrence of fl-methylaspartase, 351
pyruvate carboxylase, 264 source of aspartase, 356
isolation, 428-430, 432~133, 573, 574 Bakers' yeast, source for'citrate synthase,
labeled succinate synthesis from, 596- 17-18
598 Bananas, phenol oxidase, 562
levels in rat tissues, 446 Beef heart
preparation of labeled, 598 source for the following enzymes
purification of labeled, 574 acetyl-coenzyme A synthetase, 375-
quantitative determination, 575 381
substrate for malate dehydrogenase, 100-104, 123-
aspartase, 354 128
fl-methylaspartase, 351 NAD-specific isocitrate dehydrogen-
(3R)-L-Aspartate-3-d, enzymatic synthe- ase, 34
sis, 578-580 succinate dehydrogenase, 81
(3S)-~-Aspartate-~$-d~, enzymatic syn- Beef kidney, source for malate dehydro-
thesis, 578-580 genase, 117-122
696 SUBJECT INDEX
Bicarbonate, see Carbon dioxide Butanol, use in solubilization of succi-

Bio-Gel P-10 hate dehydrogenase, 85, 87
in preparation of Butyrate, ion-exchange chromatography,
isocitrate dehydrogenase, 48-50 428
malate dehydrogenase, 148 Butyryl-coenzyme A
plant enzyme extracts, 560 assay for, with glyoxylate, 362--363
BioRad AG-50W-X8, in preparation of substrate for propionyl-coenzyme A
malate dehydrogenase, 103-104 carboxylase, 188, 189
Biotin
in oxaloacetate transcarboxylase, 225-
226, 228-229 C
in propionyl-coenzyme A carboxylase,
188 ~4C-labeled citric acid cycle intermedi-
in pyruvate carboxylase, 236, 245, 247, ates, preparation, 567-575, 591-592
261 CO~; see Carbon dioxide
Birds, galliform, malate dehydrogenases, Cadmium ions, binding to a-ketoglutar-
108 ate dehydrogenase, 55
Bovine liver, source for propionyl-coen- Calcium phosphate gel
zyme A carboxylase, 190 use in preparation of
Bovine serum albumin, in assay for carnitine acetyltransferase, 390
succinate, 524-525 citrate synthase, 6, 14
a-ketoglutarate dehydrogenase, 53 a-ketoglutarate dehydrogenase, 54
succinate dehydrogenase, 82-83 malate dehydrogenase, 143, 149
Brain, citric acid cycle compounds in, methylmalonyl-coenzyme A mutase,
445, 446 200-201, 203
British antilewisite, action on succinate methylmalonyl-coenzyme A race-
dehydrogenase, 90 mase, 192-193
y-Bromocrotonate, inhibitor of fumarasc, phosphoenolpyruvate carboxylase,
99 185
Bromosuccinate propionyl-coenzyme A carboxylase,
conversion to 238, 240
labeled malate, 584 succinate dehydrogenase, 87
labeled succinate, 598 succinyl-cocnzyme A synthetase, 73-
Brucine, oxalocitramalic acid resolution,
74
590
Brushite, in preparation of Calcium phosphate gel-cellulose column
adenosine trlphosphate citrate lyase, chromatography, general procedure,
157, 158 66
citramalate hydrolase fl-Carbethoxypropionyl chloride, homo-
component I, 335 citrate preparation, 620
component II, 338-339 Carbomethoxybiocytin isolation from
elutamate mutase, 323-325 propionyl-coenzyme A carboxylase,
Bryophyllum calycinum 189-190, 229
isocitrate source, 602, 603, 605-607, 613 Carbon dioxide (Bicarbonate entries are
propagation of, 603 also listed here)
Bryophyllum crenatum, isocitrate source, ion-exchange chromatography, 427, 428
6O3 manometric determination, in assay of
Bryophyllum tedtschenkoi, isocitrate a-ketoglutarate dehydrogenase, 52,
source, 603, 606 53, 57
SUBJECT INDEX 697
product of thioester formation, 387

itaconate metabolism in liver, 315- thiol release, 389
316 assays with, for
a-ketoglutarate dehydrogenase re- acetylcarnitine, 509-513
action, 52 carnitine, 505-509
oxalyl-coenzyme A decarboxylase re- short-chain acyl-coenzyme A, 536,
action, 369 544, 546, 549--550
substrate for the following enzymes Carnitine palmitoyltransferase for palmi-
a-ketoglutarate synthase, 177 toyl-coenzyme A assay, 544, 551
phosphoenolpyruvate carboxykinase, Catalase NADP regenerating system,
270-271, 276, 277 593-594
phosphoeuolpyruvate carboxylase, Catechol oxidase from plant tissues, 559
277, 283, 288, 292-294 Cellulose phosphate
phosphoenolpyruvate carboxytrans- use in purification of
phosphorylase, 297-298, 308 methylmalonyl-coenzyme A mutase,
pyruvate carboxylase, 235 210
pyruvate synthase, 172 methylmalonyl-coenzyme A ra~.e-
propionyl-coenzyme A carboxylase, mase, 196-197, 221,222
181, 189 oxaloacetate transcarboxylase, 218,
Carbon monoxide, use in assay of pyru- 220-222
rate synthase, 174 phosphoenolpyruvate carboxykinase,
Carbowax, use for concentrating protein, 274-275
79 phosphoenolpyruvate carboxytrans-
3-Carboxy~4-chloro-3-hydroxybutyrate, phosphorylase, 299-303
citrate synthesis from, 591-592 pyruvate carboxylase, 256
Carhoxymethyl cellulose Cellulose powder, thin-layer chromatog-
use in purification of raphy, 431
citramalate hydrolase component I, Charcoal, glutamate mutase purification,
335-336 322
glutamate mutase component S, 327- Chemical Abstracts system for naming
328 labeled compounds, 569
isoeitrate dehydrogenase, 32 Chicken heart, source for malate dehy-
malate dehydrogenase, 109-110, 121 drogenases, 107-112
Carboxymethyl Sephadex C-50, use in Chicken liver, source for pyruvate car-
purification of citramalate hydrolase boxylase, 236-244
component II, 339, 341 Chloral, use in isocitrate preparation,
Carnitine 600, 608
assay with, for acyl-coenzyme A (short Chloride ions, effect on 3-ketoacid co-
chain), 546, 549-550 enzyme A transferase, 80
derivatives, hydrolysis of, 440 4-Chloroacetoacetate, synthesis of
fluorometrie assay for 505-509 labeled citrate, 591-592
levels in rat tissues, 446 Chlorobium thiosul]atophilum
substrate for earnitine acetyltransfer- culture, 180
ase, 387 reductive carboxylic acid cycle in, 170-
Carnitine acetyltransferase, 387-393 180
assays source for ~-ketoglutarate synthase,
coupled with citrate synthase and 179
malate dehydrogenase, 388-389 source for pyruvate synthase, 174
hydroxamate, 389 p-Chloromercuribenzenesulfonate, effect
isotope exchange, 389 on isocitrate dehydrogenase, 40, 51
698 SUBJECT INDEX
p-Chloromercuribenzoate substrate for citramalate pyruvate ly-

effect on ase, 344
fumarase, 98 Citramalate hydrolase, 318, 331-344
malate dehydrogenase, 105-106, 129, component I, 332
144 component II, 338
Chloroplasts, preparation, 181 activators, 342, 344
Chlorosuccinate, labeled succinate syn- interaction of components, 331, 342-343
thesis, 596-597 Citramalate pyruvate lyase
D-Chlorosuccinate, inhibitor of succinate assay, 344-340
dehydrogenase, 90 metal ion requirement, 34{}
~,-Chlorosuccinate, substrate for succinate CitramalyNcoenzyme A metabolism
dehydrogenase, 90 in bacteria, 317--319
Chlorpromazine, FAD-linked malate de- in mammalian liver, 315-316
hydrogenase inhibitor, 133 Citramalyl-coenzyme A lyasc, 317
Chromatography Citrate
gas-liquid, 397-415 activator of NAD-specific isocitrate
application to biological materials, dehydrogenase, 46, 50
414-415 assays with, for coenzyme A, 540
methyl ester-propionyl derivatives, chemical assays
407 acetic anhydride-pyridine, 513-516
methyl esters, 399-403, 497-408 pentabromoacetone, 515-516
preparation of column and packing, chromatography
400-401, 408-412 gas-hqiud, 397, 403-404, 407-413
quantitation and identification, 412- ion-exchange, 428, 574
414 partition, 420, 422, 423
trimethylsilyl derivatives, 407, 409- thin-layer, 432-433
410 competitive inhibitor of fumarase, 97
ion-exchange, 425-430, 573-574 diurnal variation in crassulacean
paper, 424 plants, 604
partition column, 415-425 enzymatic assays with
analysis of biological fluids and aconitase and isocitrate dehydrogen-
tissues, 424-425 ase, 446, 449
elution pattern, 422 citrate lyase and malate dehydro-
thin-layer, 431-434 genase, 450
Chromic acid, effect on glassware and, citrate lyase and oxaloacetate decar-
442 boxylase, 517-518
Cinchonine, L-malate isolation, use in, esterification, 402, 407, 414, 415
574, 584 fluorometrlc assay, 527-528
Citraconase, 316, 318 isolation as guanidine salt, 574
Citraconate isolation with, of mitochondria from
metabolism in bacteria, 317-319 plant tissues, 561
metabolism in mammalian liver, 314- levels in rat tissues, 445
316 nomenclature of labeled, 571-573
partition chromatography of, 422 preparation from labeled suceinate,
Citramalate 600-601
metabolism in bacteria, 317-319 prochirality rule for, 571-572
metabolism in mammalian liver, 314-- production from itaconate, 315-316
316 protecting agent for aconitase, 26, 29
substrate for citramalate hydrolase, 331 purification, 574
SUBJECT INDEX 699
specific radioactivity determination, pig heart, 5-7

528-535 pigeon breast muscle, 7-8
substrate for rat liver, 11, 13-15
ATP citrate lyase, 153 yeast, 16-19
aconitase, 26 regulation of citric acid cycle, 11
citrate lyase, 160 of E. coli enzyme, 25-26
citrate synthaae, 3 substrate specificity, 8--9
synthesis of labeled synthesis of deuterated citrate, 584-589
chemical, 589592 tritium exchange reaction, 9
enzymatic, 584-589 Citric acid, see also Citrate
Citrate cleavage enzyme, see Adenosine Citric acid cycle compounds
triphosphate citrate lyase assay methods
Citrate condensing enzyme, see Citrate chemical, 513-516
synthase fluorometric enzyme, 434-513
Citrate (isocitrate) hydro-lyase, see specific radioactivity, 528-535
Aconitase chromatography, see Chromatography
Citrate lyase, 160-163 content of various tissues, 445-446
determination of citrate with, 450--452, fluoroanalogs, see also special index
517-518 o] Fluoro Analogs
equilibrium constant, 161 compendium, 662-668, 672
inhibition by oxaloacetate, 160 general chemistry, 624-634
Citrate oxaloacetate-lyase, see Citrate specific procedures for preparation,
lyase 638--661, 671-672
Citrate oxaloacetate-lyase (coenzyme A- methyl esters, preparation of, 399-403
acetylating), see Citrate synthase purification of, 574-575
Citrate synthase, 3-26 separation of, 573, see also Chromatog-
allosteric effectors, 11, 25--26 raphy
amino acid composition of pig heart stereospecifically labeled
enzyme, 10, 11 deuterium analysis, preparation of
assays with, for the following enzymes sample for, 575
acetyl-coenzyme A synthase, 375-376 list of compounds, 567-569
carnitine acetyltransferase, 388 nomenclature, 569--573
assays with, for the following substrates preparation of, 567-601
acetylcarnitine, 509-513 Citrogenase, see Citrate synthase
acetyl-coenzyme A, 501-504, 544-546, Citryl-coenzyme A, citrate synthasc in-
548, 550 termediate, 8-9
coenzyme A, 536, 540, 543-544, 549 Clostridium kluyveri
citryl-coenzyme A as substrate for, 8-9 cultivation, 382--383
fluorocitrate synthesis, use for, 655 source for phosphotransacctylase, 381-
inhibitors, 9-10 386
labeled citrate, preparation, use for, Clostridium pasteurlanum, ferredoxin,
584-586, 588 preparation, 177, 180
malyl-coenzyme A as substrate for, 9 Clostridium tetanomorphum
modifiers, 11, 25-26 as source for the following enzymes
occurrence, 11 citramalate hydrolase, 333-337, 338-
polarographic assay for, 365-368 341
preparation of, from citramalate pyruvate lyase, 345-346
Escherichia coli, 22-25 glutamate mutase, 321-325, 326-328
lemon fruit, 19-22 fl-methylaspartase, 348
moth flight muscle, 8 growth, 348
700 SUBJECT INDEX
Cobaitous ion Cocnzyme A transferase, in assay of

activator for phosphoenolpyruvate car- metl~yhnalonyl-coenzyme A mutase,
boxytransphosphorylase, 299, 306, 207-209
307 methylmalonyl-coenzyme A race-
requirement by citramalate pyruvate mase, 194-196
lyase, 346 Condensing enzyme, s e e Citrate synthase
Cobamide coenzymes, s e e B,, coenzymes Crassulaceae, isocitrate in, 601-603
Co-deoxyadenosylcobalamin, s e e B,2 co- Crotonate, for cultivation of C l o s l r i d i ~ m
enzyme k l u y v e r i , 382-383
Coenzyme, s e e B,, coenzymes, Coenzyme Cyanide inhibition of
A, Flavin-adenine dinucleotide, phenol oxidase, 558
Nicotinamide adenine dinucleotide, respiration in succinate dehydrogenase,
Nicotinamide adenine dinucleotide assay, 82-83
phosphate Cysteine
Coenzyme A activation of
activator of pyruvate carboxylase from aconitase, 448
yeast, 257 FAD-linked malate dehydrogenase,
assays 147
adenine absorption, 538 use for isolation of mitochondria from
dithiobis(nitrobenzoate) method, 538 plants, 561
Ellman's reagent, 538 Cytochrome c
enzymatic, 535-544 assay with, for
fluorometric, 437-441, 497-501 long-chain acyl-coenzyme A thio-
polarographic, 366-368 esters, 547
sorbate method, 537-541 succinate, 524-525
summary of procedures, 538-540 succinate oxidase, 84
assays with, for the following substrates -deficient heart muscle preparation, 84,
acetylcarnitine, 509-510 86
succinate, 460, 462 removal from heart muscle prepara-
covalently bound to 3-ketoacid-co- tion, 86
enzyme A transferase, 81 succinate assay, 524
desulfo derivative, inhibitor of citrate Cytochrome chain linkage to FAD-spe-
synthase, 10 cific malate dehydrogenase, 133-135
substrate for the following enzymes Cytochrome oxidase, interference with a-
acetyl-coenzyme A synthetase, 375 ketoglutarate dehydrogenase deter-
adenosine triphosphate citrate lyasc, mination, 53
153 Cytosine diphosphate, activator of phos-
carnitine acetyltransferase, 387-389, phoenolpyruvate carboxylase, 288
392, 393 Cytosine triphosphate, activator of phos-
citrate synthase, 4-5, 11, 16, 19, 22 phoenolpyruvate carboxylase, 288
3-ketoacid-coenzyme A transferase,
75 D
a-ketoglutarate dehydrogenase, 52, 55 DBC and DBCC, s e e B,~ coenzymes
phosphotransacetylase, 381-382, 385, D20, s e e Deuterium oxide
386 DPN, s e e Nicotinamide adenine dinu-
succinyl-coenzyme A synthetase (suc- cleotide
cinate thiokinase), 62, 68, 70, 74-75 DPNtt, s e e Nicotinamide adenine dinu-
tissue levels, 445 cleotide, reduced
Coenzyme A derivatives, hydrolysis, 439 5-Dehydroquinate, use in homocitrate
Coenzyme A thioesters, assay of, 544-551 preparation, 623
SUBJECT INDEX 701
5-Dehydroshikimate, use in labeled adenosine triphosphate citrate lyase,

citrate preparation, 589 156, 158
5-Dehydroshikimate reductase, from aspartase, 357-358
plant tissues, 559 carnitine acetyltransferase, 390
Deoxyadenosyl benzimidazolylcobamide, citrate lyase, 162-163
s e e B12 coenzymes citrate synthase, 6--8, 14, 24
Deoxyadenosylcobalamin, s e e BI: co- glutamate mutase
enzymes component E, 322
Deoxycholate, for solubilizing FAD- component S, 326-327
linked malate dehydrogenase, 131 isocitrate dehydrogenase, 36, 37, 45,
3-Deoxy-L-pentulosonate, labeled succi- 49-5(}
nate preparation from, 599 isocitrate 'yase, 167, 168
5-Deoxyquinate, homocitrate prepara- 3-ketoacid-coenzyme A transferase,
tion, 623 78-79
Deoxyribonuclease malate dehydrogenases, 109, 110, 114-
for extraction of 115, 118-119, 121, 125-126, 139-140,
FAD-linked malate dehydrogenase, 143, 146, 149, 150
142 nmlate-lactate transhydrogenase, 253
oxalyl-coenzyme A decarboxylase, malic enzyme, 233
37O methylmalonyl-coenzyme A mutase,
phosphoenolpyruvate carboxylase, 201
295 oxaloacetate transcarboxylase, 218-
3'-Dephosphocoenzyme A, assay, 547 220
Deuterium exchange, catalyzed by suc- oxalyl-coenzyme A decarboxylase,
cinate dehydrogenase, 90 371
Deuterium-labeled citric acid cycle inter- phosphoenolpyruvate carboxykinase,
mediates, 567-601 274
aspartate, 578-580 phosphoenolpyruvate carboxylase,
citrate, 584-585, 589-590 280-281, 285-286, 290-291, 295-296
malate, 580 phosphoenolpyruvate carboxytrans-
succinate, 598, 599 phosphorylase, 299--300, 303
Deuterium peromde phosphoenolpyruvate synthetase, 312
preparation of, 591 phosphotransacetylase, 383-384
use in citrate synthesis, 589-590 propionyl-coenzyme A carboxylase,
Diaphorase, use in isocitrate dehydro- 187-188
genase assay, 43 pyruvate synthase, 175, 176
Dichlorophenolindophenol suceinate thiokinase, 67, 68
assay with, for succinate, 524-525 use in removal of ferredoxin, 175
extinction coefficient, 83 Diethylaminoethyl sephadex A-50
use in assay of the following enzymes use in preparation of
citrate synthase, 4 citramalate hydrolase, 334-335
isocitrate dehydrogenase, 43 malate-lactate transhydrogenase,
a-ketoglutarate dehydrogenase, 53 265-266
malate dehydrogenase, 130, 134-137 fl-methylaspartase, 349-351
succinate, 524 pyruvate carboxylase, 260-261
Diethyl acetonedicarboxylate, for succinyl-coenzyme A synthetase, 72-
difluoroglutarate synthesis, 647 73
Diethylaminoethyl cellulose Diethyl chlorooxaloacetate, keto-enol
use in preparation of equilibrium, 629
702 SUBJECT INDEX
Diethyldithiocarbamate Dipalmityl phosphatidylethanolamine,

inhibitor of phenol oxidase, 558 FAD-linked malate dehydrogenase
use in removal of phenolic compounds, and, 136
558, 560 Diphosphopyridine nucleotide, see Nico-
Diethyl glutarate, use for homoisocitrate tinamide adenine dinucleotide
preparation, 619, 621 Diphosphothiamine, as cofactor of a-
Diethyl fl-ketoadipate, homocitrate prep- ketoglutarate dehydrogenase, 53, 55
aration and, 620-621 Dithiobis(nitrobenzoate) (Ellman's re-
Dihydrolipoyl dehydrogenase, see Lipoyl agent)
dehydrogenase assays with, for the following enzymes
Dihydrolipoyl transsuccinylase, see also acetyl-coenzyme A synthetase, 376,
a-Ketoglutarate dehydrogenase 544-546
component of a-ketoglutarate dehydro- carnitine acetyltransferaze, 376, 544-
genase, 55-57 546
4~5-Dihydroxy-2-ketovalerate, labeled citrate synthase, 4--5, 12, 16,-17, 19--20,
succinate preparation from, 599 22-23
3,4-Dihydroxymandelate, partition col- malate synthase, 365-366
umn chromatography, 422 assays with, for the following metabo-
Dihydroxytartrate, partition column lites
chromatography, 422 acetyl-coenzyme A, 544-545, 546
Diketene, assay with, for coenzyme A, camitine, 509
54O coenzyme A, 389, 536, 538
2,3-Dimercaptopropanol, reversal of ar- short-chain acyl-coenzyme A, 549-
senite inhibition of a-ketoglutarate 55O
dehydrogenase, 55 Dithiothreitol, protecting action for
2,5-Dimethoxy-2,5-dihydrofuran, labeled adenosine triphosphate citrate lyase,
malate synthesis from, 583-584 159
2,3-Dimethoxy-5-methyl-l,4-benzoqni- isocitrate dehydrogenase, 30, 31, 44
none, FAD-linked malate dehydro- tissue extractions, 439, 440
genase, relation to phospholipid ac- Dowex 1
tivation, 135 for purification of a-ketoacids, 521-523
Dimethylbenimidazole cobalamin, see B12 for separation of citric acid cycle com-
coenzymes pounds, 425-428, 573-574, 579, 581,
(5,6-Dimethylbenzimidazolyl)Co-5'-de- 588, 594--596, 609, 619-620, 623
oxyadenosine cobamide (DBC), see Dowex 50
B~ coenzymes for separation and isolation of
Dimethylbenzimidazolyl cobamide coen- aspartate, 579
zyme, see B~ eoenzymes citric acid cycle compounds, 429
Dimethylformamide, for dissociation of fluorocitrate, 653, 655
protein-phenol complexes, 558 fluorolactate, 650
Dimethylsulfoxide, for dissociation of isocitrate, 606
protein-phenol complexes, 558
2,4-Dinitrophenylhydrazine E
assays with, for E c h e v e r i a seeunda, isocitrate content, 602
a-ketoglutarate synthase, 178, 179 E c h e v e r i a weinbergii, isocitrate content,
phosphoenolpyruvate synthetase, 310 6O3
pyruvate synthase, 172, 174 ECTEOLA cellulose
identification of carbonyl compounds use in preparation of
with, 424 aspartase, 357-358
Dioldehydrase, role of Bz~ coenzyme, 214 malate dehydrogenase, 143
SUBJECT INDEX 703
phosphoenolpyruvate carboxylase, Fatty acyl-coenzyme A, assay, 437, 438,

28O 440, 498
Ellman's reagent, see Dithiobis(nitro- Ferredoxin
benzoate) preparatioa, 180
E n t e r o b a c t e r hatniac, see B a c t e r i u m ca- reduction of, 173, 174, 178
daveris reductivc carboxylic acid cycle, role in,
Enzymes, isolation from plants, 559-560 171, 177, 179-180
Epoxysuccinate, formation of, from fluo- removal of, 175, 179
romalate, 628 Ferricyanide
Escherichia coli use in assay of
growth, 23, 59, 71, 311-312 isocitrate lyase, 164
as source for a-ketoglutarate dehydrogenase, 52-
citrate synthase, 22-26 53, 56--57
glyoxylate condensing enzymes, 362- malate dehydrogenase, 129-130, 132-
365 135
a-ketoglutarate dehydrogenase, 55-61 succinate dehydrogenase, 83-84, 88-
malate dehydrogenase, 145-147 9O
phosphoenolpyruvate earboxylase, Ferrous ion activation
288--292 of aconitase, 26, 27, 29, 30, 446, 448
phosphoenolpyruvate synthetase, of citramalate hydrolase, 342, 344
309-314 Ficoll, use for concentrating protein, 79
succinyl-coenzyme A synthetase, 71 Flavin-adenine dinucleotide (FAD)
Ethanol cofactor for a-ketoglutarate dehydro-
tissue extraction, use in, 438, 439 genase, 55
use in preparation of content of succinate dehydrogenase, 88,
aconitase, 28 89
malate dehydrogenase, 102, 121 prosthetic group of malate dehydro-
malic enzyme, 232-233 genase (FAD-linked), 132, 136, 140
phosphotransacetylase, 383 Florisil, for decreasing native fluores-
propionyl-coenzyme A carboxylase, cence, 442
184-185 Fluorescence
Ethyl acetoacetate, keto-enol equilib- malate dehydrogenase, 147
rium, 631 bacterial, 147
Ethyl acrylate, use in fluoroglutarate mitochondrial, l l l
synthesis, 645-646 reduced pyridine nucleotides, 435-437
fl-Ethylaspartate, as substrate for fl- Fluorine
methylaspartase, 351 electron donation by, 625
Ethyl 2-bromoacetoacetate, keto-enol methods for introduction into carbon
equilibrium, 631 chain, 634~72
Ethyl 2-chloroacetoacetate, keto-enol Fluoro analogs, see Index of Fluoro
equilibrium, 631 Analogs
a-Ethylmalate, fluorometric assay of, Fluorometric assays, see also individual
527-528 compounds, and enzymes
fl-Ethylmalate synthesis, 362-365
using enzymatic methods, 434-513
drift, 444
F general techniques, 440-443
FAD, see Flavin-adenine dinucleotide high sensitivity, special requirements.
Fatty acylearnitines, fluorometric assay, 436
437, 438, 440, 505 labile intermediates, 441
704 SUBJECT INDEX
standard curves, 442 succinate preparation, 599

tissue extractions, 437-439 tissue levels, 445
Formate, partition column chromatog- Fumarate-d2 synthesis, 577-578
raphy, 420, 422 Fumarate-t~, synthesis, 578
Formic acid(s), substituted, Taft con-
stants, 625
Formyl-coenzyme A, product of oxalyl- G
coenzyme A decarboxylase reaction, GDP, see Guanosine diphosphate
369 GTP, see Guanosine triphosphate
Freon, tissue freezing in, 437 Garcinia cambogia, as source of hydroxy-
Fructose-l,6-diphosphate citrate, 614-617
activator of phosphoenolpyruvate car- Gas chromatography, see Chromatog-
boxylase, 288 raphy
ion-exchange chromatography, 574 Glucose 6-phosphate, ion-exchange chro-
Fumarase, 91-99 matography, 574
determination of specific radioactivity Glucose-6-phosphate dehydrogenase
of fumarate, 528-535 fluorometric assays with for
deuterium transfer by, 588 ATP, 488--491
effect of anions on activity, 91, 96, 97 NADP, 483-485
extinction coefficient, 91 Glutaconate, effect of in chemical assay
fluorometric assay of fumarate, 463-465 for citrate, 514-515
inhibitors, 99 Glutamate
preparation of deuterated malate, 579, chromatography
580-581 ion-exchange, 428-430, 573
specific radioactivity determination thin-layer, 432-433
with, 529, 531, 533-535 fluorometric assay, 471-473
stability, 91, 96 inhibitor of Neurospora crassa NAD-
subunit structure, 98 specific isocitrate dehydrogenase,
Fumarate 46
aspartase substrate, 354-355 production from itaconate in mamma-
aspartate synthesis from, 578-580 ]Jan liver, 315-316
assay for, with fumarase, 463, 465, 575 substrate for glutamate mutase, 319
chromatography tissue levels, 446
gas, 397, 402, 404, 407-415 Glutamate dehydrogenase, assays with,
ion-exchange, 428 for
partition, 422, 423 glutamate, 471-473
thin-layer, 432-433 ketoglutarate, 455-458
determination of specific radioactivity NADPH, 485-488
by enzymatic decarboxylation, 528- Glutamate mutase, 319-330
535 coenzyme specificity, 329-330
deuterated, chemical synthesis of, 577- component E, 321-325
578 component S, 325-329
esterification, 402, 463, 407, 414-415 mechanism, 214
substrate specificity, 329-330
malate formation from, 579-581
Glutamate-oxaloacetate transaminase
purification, 574 assay for aspartate, 473
substrate for assay for a-ketoglutarate, 458
aspartase, 355 synthesis of deuterated malate, 582-583
fumarase, 91 Glutamine, thin-layer chromatography,
succinate dehydrogenase, 90 432-433
SUBJECT INDEX 705
Glutamyl transferase, from plant tissues, ing), see Phosphoenolpyruvate car-

559 boxykinase
Glutarate
inhibitor of fumarase, 99 H
partition column chromatography, 422, Heart
423 citric acid cycle compounds, levels in,
Glutathione 445, 446
effect on succinate dehydrogenase spec- source for the following enzymes
trum, 90 acetyl-eoenzyme A synthetase, 375-
in propionyl-coenzyme A carboxylase 381
assay, 182-183 aconitase, 26-30
in propionyl-coenzyme A carboxylase citrate synthase, 5-11
purification, 184-188 fumarase, 91-99
Glutathione reductase, NADP regenerat- isocitrate dehydrogenase
ing system, 593 diphosphopyridine nuclcotide-spe-
Glyceraldehyde-3-phosphate dehydrogen- cific, 34--42
ase, adenosine triphosphate assay, triphosphopyridine nucleotide-spe-
use in, 491-494 cific, 30-33
a-Glycerophosphate, ion-exchange chro- 3-keto acid-coenzyme A transferase,
matography, 574 75-81
Glycerophosphate aeyltransferase, co- a-ketoglutarate dehydrogenase, 52--55
enzyme A assay, use in, 544 malate dehydrogenase
Glycolate, partition column chromatog- extramitochondrial, 101, 105, 129
raphy, 422 intramitochondrial, 99-116
Glycolyl-coenzyme A, citrate synthase, 9 propionyl-coenzyme A carboxylase,
Glyoxylate 181-190
esterification for chromatography, 403 succinate dehydrogenase, 81-90
gas chromatography, 397, 403, 406, 410 succinate thiokinase, 62-69
ion-exchange chromatographhy, 428 Heart muscle preparations
preparation with, of succinate, 599 alkali-treated, 84-85
substrate for isocitrate lyase, 163, 164, cytochrome c-deficient, 84--86, 89
169 Keilin and Hartree, 81, 84, 86
Glyoxylate condensing enzymes, radio- removal of hemoproteins from, 86
active assay, 362-365 Hematin, inhibitor of FAD-linked mal-
Graptopetalum paraguayense, isocitrate, ate dehydrogenase, 133
603 2-n-Heptyl-4-hydroxyquinoline-N-oxide,
Guanidine hydrochloride, effect on fuma- malate dehydrogenase inhibitor, 133-
rase, 98 134
Guanosine diphosphate Hexokiuase, assay for ATP, 488, 490, 491
substrate for phosphoenolpyruvate car- Hexose diphosphate, ion-exchange chro-
boxykinase, 270 matography, 573
for succinate thiokinase, 62, 69 Hibiscus sabdari~%, hydroxycitrate, 614,
Guanosine triphosphate 617-618
activator of phosphoenolpyruvate car- Hippurate, partition column chromatog-
boxylase, 288 raphy, 422
substrate for phosphoenolpyruvate car- Homoaconitate
boxykinase, 270 chemical synthesis, 619-620, 622-623
for succinate thiokinase, 62, 69 isolation from yeast mutant, 623
Guanosine triphosphate: oxaloacetate melting point of ci,~- and trans-isonwr~.
carboxy-lyase (transphosphorylat- 62a
706 SUBJECT INDEX
separation of cis- and trans-isomers, acetoacetate, 478, 480

623 D-3-hydroxybutyrate, 476, 478
Homocitrate Hydroxycitrate
chemical synthesis, 619-621 isolation from Garcinia, 615-617
isolation from yeast mutant, 623 from Hibiscus, 617-618
melting point of lactone, 621 natural occurrence, 614
Homogentisate, partition column chro- properties of ]actones, 618
matography, 422 structures of four isomers, 613-614
Homoisocitrate 7-Hydroxycoumarin, malate assay, 526
chemical synthesis, 619, 621-622 a-Hydroxyglutarate
isolation from yeast mutant, 623 ion-exchange chromatography, 428
melting point, 622 synthesis, 362-365
Homoumbelliferone, malate assay, 526 Hydroxylapatite
Hydrazine use in purification of
use in assay of isocitrate dehydrogenase, 36-38
fumarate, 463, 465 malate dehydrogenase, 119, 121
glutamate, 471, 472 methyl malonyl-coenzyme A mutase,
hydroxybutyrate, 476, 477 201-205
malate, 466, 467 phosphoenolpyruvate carboxykinase,
pyridine nucleotides, 481, 482 275
Hydrocarbons, identification of deriva- phosphoenolpyruvate carboxylase,
tives, 413-414 281
Hydrogenase, pyruvate synthase and, 174 7-Hydroxy-5-methylcoumarin, malate as-
Hydrogen peroxide, succinate synthesis say, 526
and, 593, 595, 596, 599, 600 3-Hydroxy-3-methylglutaryl-coenzyme A,
Hydroxamate assays for assay, 547
ATP citrate lyase, 153 3-Hydroxy-coenzyme A dehydrogenase
acetyl-eoenzyme A synthetase, 375 assay of coenzyme A (with diketene),
carnitine aeetyltransferase, 389 540
citrate synthase, 3 assay of coenzyme A derivatives, 547
hydroxy citrate lactones, 619 p-Hydroxyphenyllactate, partition col-
succinate thiokinase, 65, 71 umn chromatography, 422
trimethyl silyl derivatives, 407, 409-410 p-Hydroxyphenylpyruvate, partition col-
Hydroxy acids, introduction of fluorine umn chromatography, 422
in, 649-658 8-Hydroxyquinoline, FAD-linked maIate
3-Hydroxyacyl-coenzyme A dehydrogen- dehydrogenase inhibitor, 133
ass
assay of coenzyme A, 540
assay of coenzyme A derivatives, 547 I
N-Hydroxyaspartate, product of aspar- Imidazole, activator of FAD-linkcd real-
tase reaction, 360 ate dehydrogenase, 133
3-Hydroxybutyrate Inorganic phosphate, see Orthophosphate
assay, 476, 478 Inosine diphosphate, substrate for phos-
phoenolpyruvate carboxykinase, 270-
chromatography
273, 275, 277
ion-exchange, 428-430, 573 Inosine triphosphate
partition column, 420, 422, 423, 429- substrate for
430 phosphoenolpyruvat~ carboxykinase,
3-Hydroxybutyrate dohydrogenase 270
assays with, for succinate thiokinase, 69
SUBJECT INDEX 707
Iodoacetate, inhibitor of fumarase, 99 crystallization, 609

Ion-exchange chromatography, see Chro- solubility, 609
matography Isocitrate (threo- and erythro-)
Iron, non-berne, content of succinate de- chemical synthesis of lactones, 610-613
hydrogenase, 88 paper chromatography, 613
Isocitrate separation by pyridine salt precipita-
assays for tion, 612
chemical, 514, 516 Isocitrate dehydrogenase
fiuorometric, 449, 453-455 assays with, for
chromatography citrate, with aconitase, 446-449
gas liquid, 397, 403-404, 407-413 isocitrate, 453-455, 605
ion-exchange, 428, 574 radioactive citrate and isocitrate,
partition, 422 528-535
thin-layer, 432-433 deuterium transfer by, 588
diethyl lactones, preparation, 611-612 hydroxyeitrate substrate for, 618
enzymatic assay with isocitrate dehy- labeled a-ketoglutarate synthesis with,
drogenase, 453, 455 592-596
esterification, 403 specific radioactivity determination,
a-ketoglutarate synthesis from, 592-595 529-535
levels in rat tissues, 445 succinate preparation with, 595-596,
nomenclature, 569, 571 600-601
occurrence in plants, 601-603 Isocitrate dehydrogenase, NAD-specific
radioactivity by enzymatic decarboxyl- beef heart enzyme, 34--42
ation, 528-535 activation by ADP, 39-40
substrate for aconitase, 26 hydroxylapatite columns, behavior
succinate synthesis from, 592-595, 595- on, 36-38
596, 599, 600 inhibition by ATP, 40
Isocitrate (threo-v.) by mercurials, and protection by
barium salt isocitrate and Mn ÷÷, 40
decomposition with sulfuric acid, 607 by NADH, 40
precipitation in hot solution, 606 potentiation of NADH inhibition by
deuterated, chemical synthesis, 600 NADPH, 4O
monopotassium salt molecular weight, 39
crystallization from water, 605, 608 nucleotide specificity of activators
preparation from crassulacean plants, and inhibitors, 40
601-608 stability, 38
reducing malate contamination, stereochemistry of hydrogen trans-
604, 606 fer, 41
solubility, 608 substrate specificity, 41
stability, 606 Neurospora crassa enzyme, 42-47
plant species rich in, 603 activation by AMP, citrate and iso-
review of early literature, 601-602 citrate, 46-47
substrate for isocitrate dehydrogenase, growth of organism, 43
30, 34, 42, 46 inhibition by glutamate and a-keto-
synthesis, 610-613 glutarate, 46
Isocitrate (threo-~), preparation, 613 loss of regulatory site at pH 6.5, 46
Isocitrate, (threo-n.l~-) molecular weight, 46
chemical synthesis, 608-609 specificity of activators and inhibi-
contamination by erythro isomer, tors, 46
609 pea mitochondria enzyme, 47-51
708 SUBJECT INDEX
citrate as activator, 50 in bacteria, 317-319

divalent metal ion requirement, 51 in mammalian liver, 314-316
inhibition by monovalent anions, 51 Itaconyl-coenzyme A hydratase, 317
by NADH, 51
sigmoid kinetics, 50, 51 g
stability of enzyme, 50-51 Kalanchoe daigremontiana, Jedtschenkoi,
Isocitrate dehydrogenase, NADP-specific, and plnnala, isocitrate content and
30-33 propagation, 603
kinetic isotope effect in tritiated water, Keilin and Hartree heart muscle prepa-
593 ration, see Heart muscle preparation
preparation with, of deuterated or tri- Keto acids
tiated a-ketoglutarate and succi- esterification, 402
hate, 59"2-595, 595-596, 600-601 introduction of fluorine into, 658-661,
stability, 33 671-672
substrate inhibition, 33 trimethylsilyl oxime derivatives, 410
Isocitrate glyoxylate-lyase, see Isocitrate gas chromatography, 413
lyase 3-Ketoacid-coenzyme A transferase, 75-
Isocitrate lyase, 163-170 81
anaplerotic functions, 163 mechanism, 81
colorimetric assay (glyoxylate), 164 substrate specificity, 80-81
inhibitors, 170 3-Ketoacyl-coenzyme A, assay, 547
labeled succinate configuration, 601 p-Ketocaproate, coenzyme A transfera~
properties of enzyme from several substrate, 80
sources, 169 2-Ketoglutaraldehydate, succinate prep-
substrate inhibition, 170 aragon from, 599
synthesis of deuterated succinate with, ~-Ketoglutarate (2-ketoglutarate, oxo-
599 glutarate)
threo-D.-Isocitrate: NAD oxidoreductase assays
(decarboxylating), see Isocitrate de- fluorometric, 441, 442, 455--458, 543-
hydrogenase, NAD-specifie 544
threa-D.-Isocitrate: NADP oxidoreduct- spectrophotometric, 520, 521, 542-543
ase (decarboxylating), see Isocitrate assays with, for reduced pyridine nu-
dehydrogenase, NADP-specific clcotides, 485, 486, 488
Isocitric lactones, separation, 612-613 chromatography
Isoelectric precipitation of a-ketoglutar- gas-liquid, 397, 403, 406, 410, 413
ate dehydrogenase, 60-61 ion-exchange, 428, 573, 574
fl-Isopropylaspartate, substrate for fl- partition, 422, 423
methylaspartase, 351 thin-layer, 432-433
a-Isopropylmalate, fluorometric assay, esterification, 402, 403
527-528 inhibitor of citrate synthase, 9, 26
Isotope analysis, preparation of sample of isocitrate dehydrogenase, 33, 46
for, 575-576 labeled succinate synthesis from, 595-
Isozymes of malate dehydrogenase, 150 596, 6OO
Itaconate levels in rat tissues, 445
malate dehydrogenase substrate, 105,
metabolism in bacteria, 317-319 128
metabolism in mammalian liver, 314- production from itaconate, 315-316
317 purity, tests for, 521-523
succinate thiokinase substrate, 69 specific radioactivity determination,
Itaconyl-coenzyme A, metabolism 528--535
SUBJECT INDEX 709
substrate for isocitrate dehydrogenase, medium for producing high levels of

30, 33, 34, 41, 42, 46 pyruvate carboxylase in yeast, 252-
for a-ketoglutarate dehydrogenase, 253
52 production from itaconate in mamma-
trimethylsilyl derivative, 407 lian liver, 315-316
gas chromatography, 413 substrate for malate-lactate transhy-
tritiated, 592-596 drogenase, 262-263
a-Ketoglutarate decarboxylase, see a- Lactate dehydrogenase
Ketoglutarate dehydrogenase assays with, for the following
a-Ketoglutarate dehydrogenase, 53-61 citramalate pyruvate lyase, 344-345
assays with, for citrate, 518
acetyl-coenzyme A, 497-501, 543-544 malate-lactate transhydrogenase, 262-
acyl-coenzyme A, 544, 550 263
coenzyme A, 497-501, 536, 539, 541- phosphoenolpyruvate carboxy-kinase,
543, 548--549 279272
radioactive a-ketoglutarate, 497-501, propionyl-coenzyme A carboxylase,
528-535 183
succinate thiokinase, 65 pyruvate, 520, 521
characteristics of enzyme complex, 55, pyruvate synthase, 311
56, 57 succinate thiokinase, 62-63
cofactors, 55 fluorometric assays with, for the fol-
component of enzyme complex, 56 lowing
reaction sequence, 56 ADP and AMP, 494-497
a-Ketoglutarate oxidase, see a-Ketogluta- camitine, 505--509
rate dehydrogenase NADH, 485-488
a-Ketoglutarate synthase succinate, 458-462
photoreduction of ferredoxin, 181 NADP-specific activity associated with
preparation of chloroplasts, 181 malic enzyme, 235
in reductive carboxylic acid cycle, 179 tuna heart enzyme, 113, 114
requirement for thiamine pyrophos- Lactobacillus plantarum, specific radio-
phate, 179 activity determination, 530-535
sources for ferredoxin, 179 Lead acetate, hydroxycitrate isolation
fl-Ketoisocaproate, coenzyme A transfer- with, 617
ase substrate, 80 Lemon fruit, citrate synthase from, 19-22
fl-Ketovalerate, coenzyme A transferase Lipoamide oxidoreductase, isocitrate de-
substrate, 80 hydrogenase assay, 43
Kidney Lipoate, cofactor for a-ketoglutarate de-
citric acid cycle compound levels in, hydrogenase, 55
445, 446 Lipoyl dehydrogenase, see also a-Keto-
malate dehydrogenases from, 116-122 glutarate dehydrogenase
Klebsiella aerogenes, source for citrate component of a-ketoglutarate dehydro-
lyaae, 160, 517-518 genase, 55-57
sensitivity to arsenite and Cd 2., 55, 56
Lipoyl reductase4ranssuccinylase, see
L
Dihydrolipoyl transsuccinylase
Lactate Liver
chromatography citrate synthase from, 11
gas, 397, 405, 407--413 citric acid cycle compounds, levels in,
ion-exchange, 426, 428, 573 445, 446
partition, 420, 422, 423 malic enzyme from, 230-235
710 SUBJECT INDEX
mesaconate and itaconate metabolism substrate for succinate dehydrogen-

in, 314-317 ase, 90
methylmalonyl-coenzyme A mutase deuterium-labeled
from, 198--207 chemical synthesis, 581-584, 596-599
methylmalonyl-coenzyme A racemase determination of configuration by
from, 190-194 nuclear magnetic resonance, 576-
mitochondria, phosphoenolpyruvate 577
carboxykinase from, 270-277 enzymatic synthesis, 579, 580-583
pyruvate carboxylase from, 235-249 diurnal variation in erassulacean plants,
Long-chain acyl-coenzyme A, inhibitor 6O4
of citrate synthase, 9, 16, 26 esterification, 402, 403
Long-chaln acyl-coenzyme A dehydro- excretion of v-form in urine, 424
genase, assay with, for long-chain fluorometric assays for, with malate
acyl-coenzyme A, 547 dehydrogenase, 466-468
Lysozyme, malate dehydrogenase extrac- with resorcinol, 526-528
tion with, from B. subtilis, 142 inhibitor of malate dehydrogenase
from tuna, 116
M labeled, see Malate, deuterium-labeled
Magnesium ion levels in rat tissues, 445
effect on citrate lyase, 160-161 nomenclature of labeled, 569, 571-572
requirements for the following enzyme production from itaconate in mamma-
reactions lian liver, 315-316
citramalate pyruvate lyase, 346 purification, 574
isocitrate dehydrogenaee, 30, 39, 42, quantitative determination, 575
51 specific radioactivity by enzymatic de-
phosphoenolpyruvate carboxylase, carboxylation, 528-535
277, 292, 296 substrate for the following enzymes
phosphoenolpyruvate carboxytrans- fumarase, 91, 97
phosphorylase, 297 malate dehydrogenase, 99
phosphoenolpyruvate synthase, 309 malate dehydrogenase (FAD-linked),
propionyl-coenzyme A carboxylase, 129
182, 183 succinate dehydrogenase, 90, 525
pyruvate carboxylase, 235 Malate dehydrogenase
succinyl-coenzyme A synthetase, 70 activation by anions, 105
Magnesium malonie ester, homocitrate assays with, for the following
preparation, 620 acetylcarnitine, 509-513
Malate acetyl-coenzyme A, 501--504, 545-548
activator of malate dehydrogenase, 104 aeetyl-coenzyme A synthetase, 375-
assay(s) for, 466-468, 526-528 376
assay with, for aspartate, 473-476
acetyl-coenzyme A, 545-548 carnitine acetyltransferase, 388
citrate synthase, 3-4, 9 citrate, 450-452
chromatography citrate synthase, 3--4, 7, 12--13,20, 365
gas, 397, 405, 407--413
coenzyme A, 540
ion-exchange, 426, 428, 573
partition, 420, 422, 423 fumarate, 463-466
thin-layer, 432-433 a-ketoglutarate, 458--462
citrate synthesis from, 586-589 malate, 466--468
D-isomer malate-lactate transhydrogenase, 262-
inhibitor of fumarase, 99 265
SUBJECT INDEX 711
mcthylmalonyl-coenzymc A mutasc, L-Malate: NAD oxidoreductase, see Ma-

2O8 late dehydrogenase
methylmalonyl-coenzyme A race- L-Malate: NADP oxidoreductase (decar-
mase, 195 boxylating), see Malic enzyme
oxaloacetate, 468470 Malate synthase
oxaloacetate transcarboxylase, 215 polarographic assay, 365-368
phosphoenolpyruvate carboxyklnase, radioactive assay, 362-365
270-271 Maleate
phosphoenolpyruvate carboxylase, conversion to fumarate, 577
277-278, 283-284, 288-289, 292-294 inhibitor of fumarase, 99
phosphoenolpyruvate carboxytranso inhibitor of succinate dehydrogenase,
phosphorylase, 297-299 9O
pyruvate carboxylase, 235--237, 250, malate formation from, 584
251, 258-259 partition column chromatography, 422
comparison of, from different sources, Maleate hydratase, malate formation,
115-116, 147 584
fluorocitrate synthesis with, 655 Malic enzyme, 230-235
substrate specificity, 104-105, 112, 144 assays with, for
subunits, 145 fumarate
synthesis of deuterated citrate, 586-589 fluorometric, 465-466
of deuterated malate, 582-583 radioactive, 528-535
tryptophan content of, 106, 112, 116 malate, radioactive, 528-535
Malate dehydrogenase preparations coenzyme binding, 235
Bacillus, 141-145 contamination with lactate dehydro-
beef heart genase, 235
extramitochondrial, 123-128 Malonate
intramitochondrial, 99--104 chromatography
beef kidney ion-exchange, 428
extramitochondrial, 118-120 partition column, 422
intramitochondrial, 118, 120-122 coenzyme A tmnsferase substrate, 81
chicken heart, 106-112 inhibitor of fumarase, 99
Escherlchia coll, 145-147 of succinate dehydrogenase, 90
pea epicotyl, 148-150 fluorocitrate synthesis from, 653, 657
Pseudomonas, 135-140 Malonic semialdehyde, coenzyme A
tuna heart, 113-115 transferase substrate, 80-81
Malate dehydrogenase (FAD-linked), Malonyl-coenzyme A, assay, 547
129-134, 135-140 Malyl-CoA, hydrolysis by citrate syn-
anion activation, 133 thase, 9
electron transport inhibitors, 133-134 Manganous ion
Malate derivatives, a-substituted fluoro- content of pyruvate carboxylase, 245
metric assay (with resorcinol), 526- requirements for the following enzyme
528 reactions
L-Malate glyoxylate-lyase (coenzyme A- citramalate pyruvate lyase, 346
acetylating), see Malate synthase isocitrate dehydrogenase, 30, 39, 51
phosphoenolpyruvate carboxykinase,
L-Malate hydro-lyase, see Fumarase
270
Malate-lactate transhydrogenase, 262-267 pyruvate carboxylase, 245
mechanism, 267-269 Menadione, electron acceptor for FAD-
prosthetic group requirements, 267-269 linked malate dehydrogenase, 132,
substrate specificity, 267-268 134
712 SUBJECT INDEX
Mercaptoethanol, in isolation of plant methyl iodide, 402, 404-405

enzymes, 558-559 thionyl chloride, 402, 404-406
Mercury electrode, calibration for co- Methylene succinate, inhibitor of succi-
enzyme A concentration, 367-368 hate dehydrogenasc, 90
Mesaconase, see Citramalatc hydrolase Methylglutaconase, conversion of ita-
Mesaconate conyl-coenzyme A and mesaconyl-
assay with, for fl-methylaspartase, 347 coenzyme A to citramalyl-coenzyme
inhibitor of fumarase, 99 A in mammalian liver, 315-316
metabolism in bacteria, 317-319 fl-Methyl-fl-hydroxyglutarate, ion-ex-
in mammalian liver, 314-316 change chromatography, 428
spectrophotometric assay of citrama- Methylmalonate, partition column chro-
late hydrolase, 331-332 matography, 422
substrate for citramalate hydrolase, 331 Methylmalonyl-coenzyme A
for glutamate mutase, 319 ~)xaloacetate transcarboxylase sub-
Mesconyl-coenzyme A, metabolism in strate, 215-216, 227, 228
mammalian liver, 315-316 propionyl-coenzyme A carboxylase sub-
Mesoxalate, malate dehydrogenase substrate, 181, 188-190
strate, 104, 128, 144 Methylmalonyl-coenzyme A coenzyme
3-Methoxy-4-hydroxymandelate, parti- A-carbonylmutase, see Methylmal-
tion column chromatography, 422 onyl-coenzyme A mutase
Methyl acetoaeetate, keto-enol equilib- Methylmalonyl-coenzyme A isomerase,
rium, 631 see Methylmalonyl-coenzyme A mu-
a-Methylacetoacetate, eoenzyme A trans- tase
ferase substrate, 80 Methylmalonyl-coenzyme A mutase,
fl-Methylaspartase, 347-353 198-207, 207-215
assay, 347 assay, 209
assay with, for glutamate mutase, 319, assay with, for methylmalonyl-coen-
325, 327 zyme A raeemase, 190, 195
effect of divalent metal ions, 353 coenzyme content, 206, 212
inhibition by N-ethylmaleimide, 353 coenzyme protective effect on apoen-
mechanism, 353 zyme, 207
subunit structure, 352 mechanism, 213
erythro-fl-Methylaspartate, substrate for Propionlbacterium shermanii, source
fl-methylaspartase, 351 for, 207-215
lhreo-fl-Methylaspartate radioisotopie assay, 198
substrate for glutamate mutase, 319 resolution of apoenzyme and holoen-
for fl-methylaspartate, 351 zyme, 204
L-threo-3-Methylaspartate ammonia-ly- sheep liver, source for, 198-207
ase, see fl-Methylaspartase synthesis of stereospecifically deuter-
L-threo-Methylaspartate carboxy-amino- ated succinate, 600
methyl mutase, see Glutamate mu- Methylmalonyl-coenzyme A racemase
tase assay with, for methylmalonyl-coen-
Methylaspartate mutase, see Glutamate zyme A mutase, 199, 208
mutase mechanism, 193, 197
Methylation of carboxybiotin in propi- Propionibacterlum shermanii, source
onyl-coenzyme A carboxylase, 189 for, 194-198
Methylation, with sheep liver, source for, 190-104
dia~omethane, 402-406, 408, 410t 2-Methyl-l,4-naphthoquinone, FAD-
methanolic HCI, 402 linked malate dehydrogcnase, rela-
methanolic H2SO,, 402-406 tion to phospholipid activation, 135
SUBJECT INDEX 713
2-Methyl-3-phythyl-l,4-naphthoquinone, Moth muscle, citrate synthase from, 8

FAD-linked malate dehydrogenase, Mycobacterium avium, source for FAD-
relation to phospholipid activation, linked malate dehydrogenase, 134,
135 140
N-Methyl pyrrolidone, dissociation of Mycobacterium phlei, source for FAD-
protein-phenol complexes, 558 linked malate dehydrogenase, 134,
Methylsuccinate 140
D-isomer, inhibitor of succinate dehy- Myokinase, fluorometric assay with, for
drogenase, 90 AMP, 494-497
L-isomer, substrate for succinate dehy-
drogenase, 90
N
metabolism by bacteria, 317-318
by mammalian liver, 314 NAD, see Nicotinamide adenine dinu-
Methyl esters of citric acid cycle com- eleotide
pounds, 399--403 NADH, see Nicotine adenine dinucleo-
gas chromatography, 40~409 tide, reduced
preparation, 408 NADP, see Nicotinamide adenine dinu-
Mevalonate kinase, from plant tissues, cleotide phosphate
559 NADPH, see Nicotinamide adenine di-
M ~crococcus lacLilyticus nucleotide phosphate, reduced
source for the following enzymes Neurospora crassa
malate-lactate transhydrogenase, growth, 43-44
264-266 source for NAD-specific isocitrate de-
succinate dehydrogenase, 81 hydrogenase, 42
Micrococcus lysodeikticus, source of Nicotinamide adenine dinucleotide
FAD-linked malate dehydrogenase, (NAD, DPN)
134, 140 assay for, 481-483
Mitochondria assays with, for the following mctabo-
beef heart, source for lites
acetyl-coenzyme A synthetase, 376- acetoacetate, 479--481
379 acetylcarnitine, 509-513
malate dehydrogenase, 99-104 acetyl-coenzyme A, 501-504, 545, 548
succinate dehydrogenase, 81 adenosine diphosphate, 494-497
beef liver, source for propionyl-coen- adenosine triphosphate, 492-494
zyme A carboxylase, 190 aspartate, 473--476
chicken liver, source for pyruvate car- camitine, 505--509
boxylase, 238-249 citrate, 450-453, 518
extraction of, for metabolite assays, coenzyme A, 497-501, 541,543
439 fumarate, 463-465
isolation from plant tissues, 557, 560- glutamate, 471-473
562 hydroxybutyrate, 476-478
itaconate metabolism by, 314-316 a-ketoglutarate, 455--458
parapyruvate, effects on metabolism, oxaloacetate, 468-470
519 succinate, 459-462
pea, source for isocitrate dehydrogen- assays with, and substrate for the fol-
ase, 48-50 lowing enzymes
pig liver, source for phosphoenolpyru- adenosine triphosphate citrate lyase,
rate carboxykinase, 273-275 153, 154
rat liver, source for citrate synthase, carnitine acetyl transferase, 388--389
13-14 citramalate pyruvate lyase, 344-345
714 SUBJECT INDEX
citrate synthase, 3-4, 10, 11, 13, 20, assays with, and substrate for the fol-
25-26 lowing enzymes
isocitrate dehydrogenase, 34, 40-43, isocitrate dehydrogenase, 30, 33
47, 48, 51 malate dehydrogenase, 105, 128
a-ketoglutarate dehydrogenase, 52- malic enzyme, 230, 235
53, 55-57, 61 levels in rat tissues, 446
malate dehydrogenase, 99-100, 104- reduced
107, 114, 116-117, 123, 128, 134, 141, assay for, 485-488
145, 147, 148, 150 inhibition of NAD-specific isocitrate
malate-lactate transhydrogenase, 263, dehydrogenase, 40
264, 207-269 lability, 488
methylmalonyl-coenzyme A mutase, levels in rat tissues, 446
207-209 regenerating systems, 593
methylmalonyl-coenzyme A race- stability, 437
mase, 194-195 p-Nitrophenylhydrazine, for assay of
oxaloacetate transcarboxylase, 215-- glyoxylate condensing enzymes, 363-
217 364
phosphoenolpyruvate carboxykinase, Nomenclature of stereospecifically la-
270-272 beled compounds, 569-573
phosphoenolpyruvate carboxylase, Nucleoside diphosphates, ion-exchange
277-279, 263-284, 288-289, 292-294 chromatography, 573
phosphoenolpyruvate carboxytrans- Nucleoside triphosphates, ion-exchange
phosphorylase, 297-208 chromatography, 573
phosphoenolpyruvate synthetase, 311
propionyl-coenzyme A carboxylase,
181-183 O
pyruvate carboxylase, 236-237, 251, 0~, see Oxygen
257-259 Orcinol, condensation with malate, 526
suceinate thiokinase, 62-63 Orotate, partition column chromatog-
fluorocitrate synthesis and, 655 raphy, 422
levels in rat tissues, 446 Orthophosphate
reduced binding to fumarase, 96
assay, 485-488 effect on malate dehydrogenase activ-
citrate synthase inhibitor, 10, 11, 25- ity, 105, 122, 133
26 substrate for the following enzymes
isocitrate dehydrogenase inhibitor, phosphotransacetylase, 381
40, 51 pyruvate carboxylase, 235, 246
a-ketoglutarate dehydrogenase reac- succinyl-eoenzyme A synthetase, 70,
tion product, 52 75
lability, 488 succinate thiokinase, 62, 69
levels in rat tissues, 446 Orthophosphate: oxaloacetate carboxy-
stability, 437 lyase (phosphorylating), see Phos-
Nicotinamide adenine dinucleotide phosphoenolpyruvate carboxylase
phate Ostrich heart, malate dehydrogenases of,
assay for, 483-485 108
assays with, for the following substrates Oxaloacetate
citrate, 446--447, 449 assay with, for
fumarate, 465-466 acetyl-coenzyme A, 544-545, 546
isocitrate, 453-455 citrate synthase, 3-5
SUBJECT INDEX 715
chromatography specific radioactivity determination,

gas, 397, 403, 405, 410, 413 use in, 529
ion-exchange, 427 Oxaloacetate transcarboxylase, 215-230
partition column, 422, 423 assay with, for
thin-layer, 432-433 methylmalonyl-coenzyme A mutase,
esterification, 402, 403 2O8
fluorometrie assay, 441, 468-470 methylmalonyl-coenzyme A race-
inhibitor of the following reactions mase, 195
citrate synthase, 15 biotin content, 225
malate dehydrogenase, 104, 116, 144 bound metal, 226
suceinate dehydrogenase, 90, 525 effect of dianions, 217
levels in rat tissues, 445 inhibitors, 227
prevention of inhibition of citrate syn- mechanism, 228-230
thase by, 9, 16 specificity, 227
product of reduetive tricarboxylic stability of carboxylated biotin-en-
acid cycle, 171, 172 zyme, 229
product of the following enzyme reac- Oxalocitramalate lactone
tions chemical synthesis, 589-591
ATP-citrate lyase, 153, 155 properties, 591
malate dehydrogenase (FAD-linked), resolution of racemic mixture, 590
129, 135 Oxalyl-coenzyme A, substrate for oxalyl-
phosphoenolpyruvate carboxylase, coenzyme A decarboxylase, 369
277, 283, 288, 292, 294 Oxalyl-coenzyme A carboxy-lyase, s e e
specific radioactivity determination, Oxalyl-eoenzyme A decarboxylase
528-535 Oxalyl-coenzyme A decarboxylase, 369-
substrate for the following enzymes 372
citrate lyase, 160, 161 activation by divalent metal ions, 371-
citrate synthase, 3-4, 8, 1O, 12, 15, 16, 372
19-20, 22-23, 25 dependence on thiamine pyrophos-
malate dehydrogenase, 99, 104-105, phate, 371
107, 112, 116, 117, 122, 123, 128, 129, mechanism, 369
133, 135, 136, 141, 143-144, 147, 148, species distribution, 369
150 Oxamate, partition column chromatog-
malate-lactate transhydrogenase, 262- raphy, 422
265, 267-269 Oxoglutarate, s e e ~-Ketoglutarate
phosphoenolpyruvate carboxykinase, Oxoglutarate dehydrogenase, s e e a-Keto-
270--273, 276, 277 glutarate dehydrogenase
phosphoenolpyruvate carboxytrans- Oxygen
phosphorylase, 297, 306, 308 assay with, for malate dehydrogenase
pyruvate carboxylase, 235-236, 246, (FAD-linked), 130, 133-135
247, 250, 257, 258 inactivation of citramalate hydrolas~,
synthesis of labeled citrate from, 584- 342
59O
trimethylsilyl derivative, 407 P
gas chromatography of, 413
PMS, s e e Phenazine methosulfate
Oxaloacetate transacetase, s e e Citrate Palmitoylcarnitine, reversal of inhibition
synthase of citrate synthase by, 9
Oxaloacetate decarboxylasc Palnfitoyl-coenzyme A, s e e Long-ch,lin
citrate assay, 517-518 acyl-cocnzyzme A
716 SUBJECT INDEX
Paper chromatography, see Chromatog- Phosphate acetyltransferase, see Phos-

raphy photransacetylaso
Parapyruvate, formation of, 519, 523 Phosphatidylethanolamine, preparation
Partition chromatography, see Chroma- oi, 137
tography Phosphoenolpyruvate
Pea epicotyls, source for malate dehy- assays with, for the following
drogenase, 148-150 adenosine diphosphate, 494-497
Pea mitochondria, source for NAD-spe- carnitine, 505, 506, 508, 509
cific isocitrate dehydrogenase, 47 succinate, 459-460, 462
Peanut cotyledon, source for phospho- propionyl-coenzyme A carboxylase,
enolpyruvate carboxylase, 277-283 183
Penicillium purgulogenum, source for succinate thiokinase, 62-63
erythro-Ls-isocitrate, 613 inhibitor of isocitrate lyase, 170
Pentabromoacetone ion-exchange chromatography of, 573,
detection of parapyruvate, 523 574
method for citrate, 515-51{) substrate for the following enzymes
Perchlorie acid, tissue extraction by, 437- phosphoenolpyruvate carboxykinase,
439 270
Permanganate, distinction of succinate phosphoenolpyruvate carboxylase,
from methylmalonate with, 190-191, 277, 283, 288, 292
199 phosphoenolpyruvate carboxytrans-
Peroxidases, oxidation of plant phenols, phosphorylase, 297
556 phosphoenolpyruvate synthetase,
Phenazine methosulfate 3O9
assay with, for Phosphoenolpyruvate carboxykinase,
long-chain acyl-coenzyme A thio- 270-277
esters, 547 assay by
malate dehydrogenase (FAD-linked), 1*C-bicarbonate fixation, 271
130, 134, 135 1'C exchange, 273
succinate, 524-525 spectrophotometry
sueeinate dehydrogenase, 82, 88, 89, carboxylating, 271
90 decarboxylating, 272
NADP regenerating system, 593-594 specific radioactivity determination
Phenol oxidase with, 529
inhibitor, 558, 562 Phosphoenolpyruvate carboxylase
isolation from plant tissues, 562 from Escherichia coli K12, 288
oxidation of plant phenols, 556 from Escherichia coli strain W, 288-292
Phenolic compounds activation by acetyl-coen~.yme A and
conversion to quinones and reactions other acyl-coenzyme A derivatives,
with proteins, 555-556 292
enzyme inhibitors, 555 inhibition by ~,-aspartate, 292
hydrogen bonding with peptide link- requirement for divalent metal ions,
ages, 555-556 292
removal during plant enzyme isolation, from peanut cotyledons, 277-283
555-563
from Pseudomonas AM1, 292-296
Phenylacetate, partition column chroma-
tography, 422 substrate specificity, 2.q6
Phenylhydrazine, assay for glyoxylato, from Salmonella typhimurium, strain
164 LT 2, 283-288
Phosphate, see Orthophosphate activation by acetyl-coenzyme A,
SUBJECT INDEX 717
fructose-l,6-diphosphate, and coenzyme A, 539, 540, 543

nucleotides, 288 pyruvate synthase, 173
by organic solvents, 287 Pig heart
binding of polycafions, 287, 288 source for the following cnzyme~
inhibition by a.sparlate, 288 aconitase, 28-29
species distribution, 277 citrate syntha~e, 5-7
Phosphoenolpyruvate carboxytransphos- fumarase, 92-96
phorylase, 297-309 3-ketoacid-coenzyme A transferase,
effect of thiols, 298, 307 77-80
inhibition by buffers, 307 a-ketoglutarate dehydrogenase, 53-
by malate, 307 54
mechanism, 308-309 malate dehydrogenase, 100
metal ion requirements, inhibitors, and NADP-specific isocitrate dehydro-
activators, 306 genase, 30
Phosphoenolpyruvate kinase, assay with, propionyl-coenzyme A earboxylase,
for specific radioactivity of oxalo- 181-190
acetate, 528-535 succinate dehydrogenase, 85-89
Phosphoenolpyruvate synthetase, 310-314 suceinate thiokinase, 65-68
assay by Pig liver, source for phosphoenolpyru-
phosphate formation (pyruvate-de- vate, carboxykinase, 270-277
pendent), 310 Pigeon breast, source for the following
pyruvate formation (lactate dehy- enzymes
drogenase coupled), 311 camitine acetyltransferase, 387-393
pyruvate removal (ATP-dependent), citrate synthase, 7-8
309-310 Plant enzymes
mechanism, 309 isolation, 556-563
Phosphoglycerate kinase, fluorometric as- removal of phenolic compounds during
say for ATP, 491-494 isolation, 555-563
3-Phosphohistidine, succinyl-coenzyme A Plants
synthetase phosphorylation, 74-75 extraction of isoeitric acid from, 603-
D,~-Phospholactate 6O5
inhibitor of phosphoenolpyruvate car- malate dehydrogenase from
boxykinase, 277 peas, 148
of phosphoenolpyruvate carboxylase, other sources, 150
283 Polarography
Phospholipid, requirement for malate apparatus, 366-367
dehydrogenase (FAD-linked), 134- citrate synthase assay, 4, 22
136 coenzyme A assay, use in, 538
Phosphomevalonate kinase, from plant Polyamides, removal of phenolic com-
tissues, 559 pounds, use for, 557
Phosphopyruvate carboxylase, s e e Phos- Polyclar AT, removal of phenolic com-
phoenolpyruvate carboxylase pounds, use for, 557, 559-560
Phosphotransacetylase, 381-286 Polyethyleneglycols
acetyl-coenzyme A regenerating sys- for isolating plant enzymes, 557, 562
removal of phenolic compounds with,
tem, 584, 588
557, 562
ammonium ion requirement, 385 Polyvinylpyrrolidone
assay with, for inhibitor of flavoproteins, 562
aeetyl-coenzyme A, 497-501, 546, 548- isolation of mitochondria with, from
549 plant tissues, 560-562
718 SUBJECT INDEX
phenol complexing agent, in isolation Pscudomonas indigo]era, source for iso-

of plant enzymes, 557, 559-563 citrate lyasc, 165
Potassium ferricyanide Pseudomonas ovali~ (Chester), source for
assay with, for succinate dehydrogen- malate dehydrogenase (FAD-linked),
asc, 82-84, 88, 89, 90 137-140
extinction coefficient, 83 Pseudomonas oxalaticus, source for
Prochirality, 571-572 oxalyl-coenzyme A decarboxylase,
Propiona]dehyde, formation from pro- 369-372
pane-l,2-diol, 214 Pseudomonas saccharophila, dehydratase,
Propionate, partition column chromatog- succinate preparation with, 599
raphy, 420 Pyridine nucleotides, see also specific
Propionibacterium shermanli compounds
source for the following enzymes content of tissues, 446
methylmalonyl-coenzymc A mutasc, extraction from tissues, 438, 439
207-215 Pyrophosphate
methylmalonyl-coenzyme A race- inhibitor of citrate synthase, 19
mase, 196 substrate for acetyl-coenzyme A syn-
oxaloaeetate transcarboxylase, 217 thetase, 375, 380
phosphoenolpyruvate carboxytrans- phosphoenolpyruvate carboxytrans-
phosphorylase, 297--309 phosphorylase product, 297, 306
Propionyl-coenzyme A Pyrophosphate: oxaloacetate, carboxy-
citrate synthase specificity and, 9 lyase (phosphorylating), see Phos-
glyoxylate condensation with, 362 phoenolpyruvate carboxytransphos-
oxaloacetate transcarboxylase product, phorylase
215, 227-228 Pyrrolidonecarboxylate, partition column
substrate for propionyl-coenzyme A chromatography, 422
earboxylase, 181-183, 189 Pyruvate
Propionyl-coenzyme A earboxylase 181- activator of pyruvate carboxylase, 247,
190 258
assay with, for assay(s)
methylmalonyl-coenzyme A mutase, enzymatic, 520, 521
199 fluorometric, 441
methylmalonyl-coenzyme A race- assay with, for NADH, 485, 486, 488
mase, 190 chromatography
mechanism, 189-190 gas, 397, 403, 406, 410, 413
fl-Propylaspartate, substrate for fl-meth- ion-exchange, 428
ylaspartase, 351 partition, 422, 423
fl-n-Propylmalate synthesis, 362-365 thin-layer, 432-433
Protein, removal of phenolic compounds esterification, 402, 403
from 557 oxalocitramalic acid lactone synthesis
Proteus vulgaris, use in synthesis of la- from, 590
beled aspartate, 579, 598 product of oxaloacetate deearboxylase,
Pseudomonas 517-518
itaconate metabolism by, 317-319 of phosphoenolpyruvate carboxy-
source for pyruvate carboxylase, 258- transphosphorylase, 297
262 production from citramalyl-coenzyme
Pseudomonas AM1, source for phospho- A in bacteria, 317-318
enolpyruvate carboxylase, 292-296 from itaconate in mammalian liver,
Pseudomonas aeruginosa, glyoxylate con- 315-316
densing enzymes in, 363 purity and stability, 519-523
SUBJECT INDEX 719
substrate for the following enzyme re- Quenching of fluorometric assays, 441-
actions 442, 455, 469, 484~185, 502
citramalate pyruvate lyase, 344 Quinidine citrate, for isolation of citrate,
malate-lactate transhydrogenase, 263, 574
264, 266, 267, 268-269 Quinones, reactions with proteins, 556,
oxaloacetate transcarboxylase, 215- 558-559
216, 227, 228
phosphoenolpyruvate synthetase, 309 R
pyruvate carboxylase, 235, 246 Radioactive carbon, see 14C
Pyruvate: carbon dioxide ligase (ADP), Radioactive counting of column effluents,
see Pyruvate carboxylase 419
Pyruvate carboxylase, 235-262 Radioactivity of citric acid cycle com-
carboxybiotin-enzyme intermediate, pounds, determination by enzymatic
246-247 decarboxylation, 528--535
comparison of enzyme from different Rat liver, source for citrate synthase, 13-
sources, 246, 248, 250, 257-258, 261, 15
262 Reduced nicotinamide adenine dinucleo-
exchange reaction, 246 tide, see Nicotinamide adenine dinu-
inhibitors, 247-249, 257 cleotide, reduced
mechanism, 236 Reduced nicotinamide adenine dinucleo-
specificity of substrates and cofactor, tide phosphate, see Nicotinamide
245-246, 247 adenine dinucleotide phosphate, re-
Pyruvate dehydrogenase duced
induction by pyruvate, 59 Reductive tricarboxylic acid cycle, 170-
separation from a-ketoglutarate dehy- 181
drogenase, 61 Resorcinol, fluorometric assay with, for
Pyruvate kinase malate, 526-528
assays with, for the following metabo- Rhodospirillum rubrum
lites source for a-ketoglutarate synthase,
ADP, 494-497 179
AMP, 494-497 for pyruvate synthase, 175
carnitine, 505-509 Rose petal mitochondria, 561
assays with, for the following enzymes
phosphoenolpyruvate carboxykiuase, $
270-272 Saccharomyces cerevisiae, source for
propionyl-coenzyme A carboxylas(,, pyruvate carboxyla~e, 250--258
181, 183 Sagarose 8, pyruvate carboxylase purifica-
succinate, 458-462 tion, use in, 256
succinate thiokinase, 62-63 Salmonella tgphimurium, strain LT 2,
Pyruvate synthase, 173-177 source for phosphoenolpyruvate car-
ia reductive carboxylic acid cycle, 172 boxylase, 283-288
occurrence, 177 Samia cynthia, source for citrate syn-
requirement for ferredoxin, 175 thase, 8
for thiamine pyrophosphate, 176 Sedum, isocitrate content, 602, 603
Sephadex G-25
O use in preparation of
Quinate, starting material for homoci- citrate synth~e, 6
trate preparation, 623 isocitrate dehydrogenase, 32, 39
(6S)-Quinate-6-t, citrate preparation a-ketoglutarate synthase, 179
from, 589 malate dehydrogenase, 131-132
720 SUBJECT INDEX
malate-lactate transhydrogenase, 253 Sorbate, assay with, for coenzyme A,

pyruvate carboxylase, 255-256, 261 537-541
Sephadex G-50 Spectrophotometric standardization of
use in preparation of fluorometric assays, 443--444
oxaloacetate transcarboxylase, 221, Squalene, succinate preparation from, 600
226 Starch block electrophoresis of malate
oxalyl-coenzyme A decarboxylase, dehydrogenase, 102, 126
371 Starch gel electrophoresis of malate de-
phosphoenol carboxytransphospho- hydrogenases, 108-110, 114, 116, 117
rylase, 298, 302, 303 Stereochemical configuration, determina-
plant enzyme extracts, 560 tion of, 576--577
succinyl-coenzyme A synthetase, 72, Subunits of fumarase, 98
73 Succinate
Sephadex G-100 assays
use in preparation of enzymatic, 524-525, 575
acetyl-coenzyme A synthetase, 378, fluorimetric, 444-445, 458-462
379 assay with, for carnitine, 505, 506, 508,
aconitase, 28-29 509
carnitine acetyltransferase, 390-392 brominated and deuterated, 584
glutamate mutase components S, 327 chlorinated and deuterated, 597
succinate thiokinase, 68 chromatography
malate dehydrogenases, 110-111, 115, gas, 397, 404, 407-413
146 ion-exchange, 428, 573
fl-methylaspartase, 349 partition, 422, 423
Sephadex G-150, glutamate mutase prep- thin-layer, 432--433
aration, 323 contamination of a-ketoglutarate by,
Sephadex G-200 520, 523
use in preparation of deuterated
adenosine triphosphate citrate lyase, chemical synthesis, 596-598, 596-601
158 determination of configuration, 577
citramalate hydrolase component II, enzymatic synthesis, 599-600
339-341 esterification, 402, 403, 415
citrate synthase, 18, 24-25 inhibitor of fumarase, 99
phosphoenolpyruvate carboxylase, isocitrate synthesis from 608
281-282 labeled, see Succinate, deuterated or
phosphoenolpyruvate synthetase, 313 Succinate, tritiated
pyruvate carboxylase, 242 levels in rat tissues, 445
Sepharose 2B, oxaloacetate transcarbox- metabolism of radioactive, 533-535
ylase preparation, use in 223-224 nomenclature of labeled, 571--572
~erratia marcescens, malate dehydrogen- production from citramalate and suc-
ase from 140 cinyl-coenzyme A in bacteria, 317
Silica gel, partition column chromatog- purification, 574-575
raphy, 416, 419-420, 429-430, 573 quantitative determination, 575
Sodium borohydride reduction of keto- substrate for the following enzymes
acids, in ion-exchange chromatog- coenzyme A transferase, 81
raphy, 428 3-ketoaeid-coenzyme A transferase,
Sodium chloride, aconitase activation by, 75
30 a-ketoglutarate synthase, 178
Sodium cyanide, labeled citrate synthesis succinate dehydrogenase, 82-84
from 591-592 succinate oxidase, 84
SUBJECT INDEX 721
succinate thiokinase, 62, 69 methyhnalonyl-coenzyme A mutase,

succinyl-coenzyme A synthetase, 70, 198, 207-208
75 succinate thiokinase, 62-63, 65, 69
tritiated, preparation of, 592-595, 595- succinyl-coenzyme A synthetase, 70-
596, 598 71, 75
Succinate: coenzyme A ligase (GDP), Succinate: eoenzyme A ligase (ADP),
s e e Succinate thiokinase see Suceinyl-eoenzyme A synthetase
Succinate dehydrogenase, 81-90 Succinyl-eoenzyme A: 3-oxoacid coen-
assay with, for succinate, 524-525 zyme A-transferase, s e e 3-Ketoa<,id
enzyme-inhibitor complexes, 90 coenzyme A transferase
inhibitors, 88, 90, 525 Succinyl-coenzyme A synthetase, 70-75
reconstitution activity of purified en- ATPase activity, 74
zyme, 82, 88 assay with, for succinyl-coenzymc A,
substrates, 90, 525 547
Succinate oxidase nucleotide specificity, 74
assay, 84-85 phosphoenzyme, 74-75
assay with, for succinate, 575 succinyl phosphate intermediate, 75
Keilin and Hartree heart muscle prep- substrate for a=ketoglutarate syntha~e,
aration, 85 178
reconstitutive activity of alkali-treated Succinyl phosphate, intermediate in sue-
heart muscle preparation, 82, 84- cinyl-coenzyme A synthetase reac-
85, 88 tion, 75
Succinate : (acceptor) oxidoreductase, s e e Suceinyltransferase, s e e Lipoyl dehydro-
Succinate dehydrogenase genase
Succinate thiokinase, 62-69 Sucrose gradient centrifugation, methyl-
arsenolysis reactions, 69 malonyl-coenzyme A mutase prep-
fluorometric assay with, for aration, 210-211
carnitine, 505-509 Sulfhydryl, s e e Thiol
succinate, 458-462 Sulfide, labile, in succinate dehydrogen-
mechanism, 69 ase, 88
metabolism of itaconate and mesacon-
ate with, in mammalian liver, 315- T
316 TPN, s e e Nicotinamide adenine dinu-
Succinyl-coenzyme A cleotide phosphate
arsenolysis, 69, 547 TPNH, s e e Nicotinamide adenine dinu-
assay with, for coenzyme A, 500 cleotide phosphate, reduced
assay for, with permanganate, in pres- TPP, s e e Thiamine pyrophosphate
ence of methylmalonyl-CoA, 190- Taft constants for aliphatic acids, 625
191 Tartrate
formation with, of oxalyNcoenzyme A, citrate assay, interference with, 514-515
369 inhibitor of fumarase, 99
metabolism in bacteria, with methyl malate dehydrogenase substrate, 105,
succinate, mesaconate and citra- 128, 134, 144
malate, 317 partition column chromatography, 422
product of a-ketoglutarate dehydro- Tartronatc
genase reaction, 52 malate dehydrogenase substrate, 128,
substrate for the following enzymes 144
CoA transferase, 194, 195 partition column chromatography, 422
3-ketoacid-conenzyme A transferase, Tea leaves, enzyme isolation from, 559-
75, 80 56O
722 SUBJECT INDEX
Tetraethyl ethane-l,l,2,2-tetracarboxyl- Triethyl ethane-l,l,2-tricarboxylate, suc-

ate, succinate preparation from, 598 cinate preparation from, 598
Tetrazolium salt, malate dehydrogenase Triethyl 2-oxaloglutarate, intermediate of
activity, 117 homoisocitrate preparation, 619, 621-
Thiamine pyrophosphate 622
cofactor for Triethyl oxalosuccinate
a-ketoglutarate dehydrogenase, 55 isocitrate synthesis from 611-613
a-ketoglutarate synthase, 178, 179 potassium enolate, preparation, 639,
oxalyl-coenzyme A decarboxylase, 642
371 synthesis, 610-611
pyruvate synthase, 173, 174, 176--177 Trimethylsilyl derivatives, preparation
Thin-layer chromatography, see Chroma- and separation, 401, 404--407, 409-413
tography Triphosphopyridine nueleotide, see Nico-
Thiol activators of tinamide adenine dinucleotide phos-
aconitase, 26, 27, 29-30 phate
citramalate hydrolase, 342 Tritium labeled citric acid cycle inter-
Thiol groups mediates, preparation, 567-577, 589,
determination of, with p-chloromer- 592-596
curibenzoate, 98 Triton X-114, phcnol oxidase extraction,
of fumarase, 98-99 562
Thiol reagents, inactivation of Trypsin
fumerase, 98 fumarase hydrolysis with, 98
isocitrate dehydrogenase, 51 lipoyl dehydrogenase isolation with, 55
Thiocyanate, inhibitor of fumarase, 99 Tryptophan, malate dehydrogenase lack
Thiomalate of, 106, 111, 116, 147
activator of aconitase, 27, 29 Tuna heart
partition column chromatography, 422 source for
Tissues lactate dehydrogenase, 113-114
acid extraction, 437-438 malate dehydrogenase, 113-115
alkaline extraction, 439 Tungstic acid, partition column chroma-
dry weight, determination, 444 tography, 422
extracts, fluorescence of, 441-442 Tyrosine, quinone formation from, 562-
Transacetylase, assay with, for citrate 563
synthase, 3
Tricarballylate, inhibitor and U
protecting agent for aconitase, 26, 28, Ubiquinone, malate dehydrogenase co-
29, 447 factor, 132-135, 137
Trichloroacetate, partition column chro- Ultracentrifugafion, in a-ketoglutarate
matography, 422 dehydrogenase preparation, 60, 61
Trichloromethylparaconic acid, interme- Ultrafiltration, in glutamate mutase prep-
diate in isocitrate synthesis, 608-609 aration, 323, 324
Triethylaminoethyl cellulose Utraviolet recording, partition column
chromatography, 418
use in preparation of
Umbelliferone, malate assay using, 526
acetyl-coenzyme A synthetase, 379 Urate, partition column chromatography,
methylmalonyl-coenzyme A mutase, 422
203-204, 210 Urea
oxaloacetate transcarboxylase, 218, denaturation of fumarase, 98
221-223 in thiol group titration, 98
SUBJECT INDEX 723
oxaloacetate transcarboxylase subunits, Y

226 Yeast
Urine, citric acid cycle compounds in, citrate synthase
415, 424 assay, 16-17
Y properties, 19
purification, 17-18
Vaieryl-coenzyme A, glyoxylate conden- homocitrate accumulation, 623
sation with, 362-363
Veillonella gazogenes, see Micrococcus Z
lactilyticus Zeocarb-215, in hydroxycitrate isolation,
Vitamin K, FAD-specific malate dehy- 616, 618
drogenase and, 134, 135, 137, 140 Zinc ion
activator and inhibitor of isocitrate
W dehydrogenase, 51
Water, tritiated, sublimation of, 575-576 protector of malate dehydrogenase, 145
724 INDEX OF FLUORO ANALOGS
INDEX OF FLUORO ANALOGS
3-Acetoxy4-fluorocrotonate, 664 Dibromofluoroacetate, 662

2-Acetyl-2-fluorooxaloacetate, 667 2,3-Dibromo-2-fluoropropionate, 663
Acrylic acid 2,3-Dichloro-3,3-difluoro-2-nitropropion-
acid strength of, 632 ate, 663
Allylfluoromalonate, 667 Dichlorofluoroacetate, 662
2-Amino-3,3'-difluoroisobutyrate, 664 2,3-Dichloro-3-fluoroacrylate, 662
2-Amino-2-fluorocrotonate, 664 4,4-Dichloro-2-fluoro-2- (hydroxy-
5-Amino-2-fluoro-2-hexenoate, 667 methyl)-2-ketobutanoate, 665
2-Amino-4-fluoro-5-hydroxyvalerate, 665 Diethyl monofluorosuccinate, physical
2-Amino-3-fluoroisobutyrate, 664 properties of, 643
5-Amino-2-fluoro-2-pentenoate, 665 Difluoroacetic ester, 662
2-Amino-3-fluoropropionate, 662 2,2-Difluoroacetoacetate, 664
3-Amino-2-fluoropropionate, 663 2,4-Difluoroacetoacetate, 664
Bromodifluoroacetate, 662 4,4-I)ifluoroacetoacetate, 664
2-Bromo-2,4-difluoroacetoaCetate, 664 4,4-Difluoroaconitate, 668
Bromofluoroacetate, 662 2,3-Difluoroacrylate, 662
4-Bromo-2-fluorocrotonate, 663 3,3-Difluoroacrylate, 662
2-Bromo-4-fluoroglutarate, 686 acidity of, 632
Bromofluoromalonate, 663 2,2-Difluorobutyrate, 663
Bromofluorooxaloacetate, 665 2,3-Difluorobutyrate, 663
3-Bromo-2-fluoropropionate, 663 3,3-Difluorobutyrate, 663
Bromofluoropyruvate, 663 2,3-Difluoro-3-chloroacrylate, 662
1-Carboxy-2-fluoroaspartat e, 666 3,3-Difluoro-2-chloroacrylate, 662
2-Carboxy-4-fluoroglutamate, 668 2,2-Difluoroeitrate, 668
4-Carboxy-4-fluoroglutamate, 668 optical isomers of, 633
2-(Jarboxy-2-fluoroglutarate, 668 2,4-Difluorocitrate, 668
4-Chloro-2,4-difluoroacetoacetate, 664 optical isomers of, 633
Chlorofluoroacetate, 662 4,4-Difluorocrotonate, 663
2-Chloro-3-fluoro-3-hydroxypropionate, 3,5-Difluoro-2,4-diketopentanoate, 666
663 2,3-Difluoro-3-fluoromethylcrotonate, 665
3-Chloro-4-fluoroisovalerate, 665 Difluorofumarate, 664
3-Chloro-2-fluoropropionate, 663 2,2-Difluoroglutarate, 666
Chloronitrofluoroacetate, 662 3,3-Difluoroglutarate, 666
Decarboxylation, 630-631, 635 2,6-Difluorohexanoate, 667
fluoroacetate, 630 5,5-Difluorohexanoate, 667
Decomposition of 3,4-Difluoro-3-hexenedioate, 667
2-amino-3-fluoroisobutyrate, 626 2,4-Difluoro-3-hydroxybutyrate, 664
2,4-difluoro-2-alkyloxaloacetate, 632 4,4-Difluoro-2-hydroxyglutarate, 666
fluoroacetate, 626 3,3-Difluoro-2-hydroxyisobutyrate, 664
fluoroacetoacetate, 626 3,3'-Difluoro-2-hydroxyisobutyrate, 664
2-fluoro-2-alkylacetoacetate, 631 4,4'-Difluoro-3-hydroxyisovalerate, 665
2-fluorocrotonate, 625
2,4-Difluoro (hydroxymethyl) acetoace-
2-fluoro-2-hexenoate, 626
ethyl fluoroacetate, 626 tate, 666
ethyl fluoromalonate, 626 2,2-Difluoro-3-hydroxypropionate, 663
trifluoromethylacrylate, 626 4,4-Difluoroisocitrate, 668
2-trifluoromethylpropionate, 626 4,4-Difluoro-2-ketoglutarate, 668
INDEX OF FLUORO ANALOGS 725
Difluoromalate, 664 2-chloro-l,l,2-trifluoro ethylamine, 634,

difluoromalate synthesis using difluoro- 637
oxaloacetate, 652 enolates, 634, 636, 639-644, 659--661,671
Difluoromaleate, 664 epoxides, 634
Difluoromalonate, 663 fluorohalo olcfins, 634
3,3-Difluoro-2-methoxy-l-cyclobutane- fluoro olefins, 634, 637, 644
carboxylate, 665 perchloryl fluoride, 634, 636, 642, 645,
2 (Difluoromethyl) acrylate, 664 659, 661, 671
Difluorooxaloacetate, 665 unsaturated acids, 647--649
difluorooxaloacetate synthesis using sulfur tetrafluoride, 634, 637, 647
diethyl a,a-difluorooxaloacetate, 657 trifluorochloroethylene, 635, 644, 660
diethyl oxaloacetate sodium enolate, trifluoroethylene, 634
659-660 Fluoroacetate, 662
4,4-Difluorooxalosuccinate, 668 partition column chromatography, 422
22-Difluoropropionate, 662 Fluoroacetoacetate, 664
2,2-Difluorosuccinate, 664 Fluoroacetic anhydride, reaction with co-
2,3-Difluorosuccinate, 664 enzyme A, 654-655
DifluorovMerate, 685 Fluoroacetyl-coenzyme A, citrate syn-
3,3-Dimethoxy-2-fluoropropionate, 663 thase substrate, 8, 655
Effect of fluoro substitutions on enoliza- Fluoroacetylfluoromalonatc, 666
tion, 629-630 2-Fluoroacetyl-2-fluorooxaloacetate, 667
Ethyl ethoxalyl fluoroacetate, use in fluo- Fluoroaconitate, 668
romalate synthesis, 651 2-Fluoroacrylate, 662
Ethyl 4-fluoroacetoacetate, 667 acidity of, 632
Ethyl fluoromalonate, 686 Fluoroacrylate synthesis using
Ethyl fluorooxaloacetate, 667 butyl fluorooxaloacetate, 647
Ethyl lactate toluene sulfonate, fluoro- 2,3-dibromo-2-fluoropropionate, 647
propionate synthesis, 638 Fluoro aliphatic ester syntheses, from
Ethyl oxalate, fluorocrotonate synthesis, acid ha]ides, 635, 641
647 aldehydes, 634, 635, 640
Fluorine elimination from alkyl ha]ides, 635, 640
2-amino-2-carboxy-4-fluorobutyrate, 627 difluoroacetic ester, 638
2-carboxy-3ofluoroaspartate, 627 fluoroacetate, 635-638
3,3-difluorobutyrate, 627 Fluoro aliphatic ester syntheses using
2,3-difluorosuccinate, 627 Claisen condensations, 635, 640, 653,
4-fluoro-2-alkylacetoacetate, 627, 632 657-659
3-fluoroaspartate, 627, 628 decarbonylation reactions, 635, 658
4-fluorobutyrate, 627 Grignard reactions, 635, 641
7-fluoroheptanoate, 627 Michael condensations, 635, 640, 645-
4-fluoro-5-ketohexanoate, 627 646
fluoromalate, 628 Reformatsky reactions, 635, 641
fluorosuccinate, 627 Wittig reactions, 635, 641
5-fluorovalerate, 627 4-Fluorobutyrate, 663
Fluorine introduction using Fluorocarboxylic acids
acrylic esters, 634 general chemistry of, 624-634
bromonium fluoride, 634, 636 stability of C-F bond in, 626-628
N-bromosuccinamide, 634 2-Fluoro-l-carboxysuccinate, 666
carbonyl groups, 628-631, 634,637, 647 Fluorocitrate, 668
carboxyl groups, 634 electrophoretic separation, 655-657
fluorometric assay, 450 2-Fluoroisobutyrate, 663

syrt]lesis using diethyl fluorooxaloacc- 3-Fluoroisobutyrate, 663
tate, 653 2-Fluoroisocaproate, 667
2-Fluorocitrate, optical isomers of, 633 3-Fluoroisocitrate, 668
2-Fluorocrotonate, 663 2-Fluoro-3-isopropylacrylate, 667
3-Fluorocrotonate, 663 Fluoroisopropylmalonate, 667
4-Fluorocrotonate, 663 2-Fluoroisovalerate, 665
Fluorocyclobutanecarboxylate, 665 3-Fluoro-2-ketobutyrate, 664
Fluorodicarboxylic acid preparation using Fluoroketoglutarate, 666
cyclobutanes, 634, 637 4-Fluoro-5-ketohexanoate, 667
2-Fluoro-3,3-dichloroacrylate, 662 Fluoro ketones, condensation or addition
4-Fluoro-3,3-dimethylbutyrate, 657 reactions of, 635, 641
(2-Fluoroethoxy) acetate, 668 2-Fluoro-3-ketovalerate, 665
4-(2'-Fluoroethoxy)butyrate, 668 3-Fluoro-2-ketovalerate, 665
3- (2'-Fluoroethoxy) propionate, 668 Fluorolactate, 663
(2-Fluoroethyl) aminomalonate, 666 configuration of, 633
4-Fluoro-3-fluoromethylcrotonate, 665 optical rotatory dispersion curves, 651
Fluoroformate, 662 resolution, 650-651
2-Fluoroformylacetate, 663 synthesis using
Fluorofumarate, 664 4-fluoromethyl-2,2-dimethyl-l,3-di-
synthesis using 2,2-difluorosuccinate, oxolane toluenesulfonate, 649
648 1-fluoropropane-2,3-diol, 649
Fluoroglutamate, 666 Fluoromalate, 664
Fluoroglutarate, 666 isomers, 633
physical properties of, 646 Fluoromaleate, 664
synthesis using Fluoromalonate, 663
diethyl fiuoromalonate, 645-646 stability of C-F bond in, 626
dimethyl a-fluoro-a-carbomethoxy- synthesis using diethyl oxalofluoroace-
glutarate, 644--645 tare, 639
2-Fluorohexanoate, 666 4-Fluoro-2-methylacetoacetate, 665
6-Fluorohexanoate, 666 2 (Fluoromethyl) acrylate, 664
Fluorohydroxybutyrate, 664 3-Fluoromethyl-3-butenoate, 665
2-Fluoro-4-hydroxyglutarate, 666 2-Fluoro-3-methylcrotonate, 665
3-Fluoro-2-hydroxyglutarate, 666 4-Fluoro-3-methylcrotonate, 665
4-Fluoro-5-hydroxyhexanoate, 667 2-Fluoromethylglutaconate, 667
3-Fluoro-2-hydroxyisobutyrate, 664 3- (Fluoromethyl)-3-hydroxyglutarate, 667
2-Fluoro-3-hydroxyisovalerate, 665 Fluoromevalonate, 667
4-Fluoro-3-hydroxyisovalerate, 665 Fluoronitromalonate, 663
2-Fluoro-3-hydroxy-3-methylglutarate, 6-Fluoronorleucine, 667
667 5-Fluoronorvaline, 665
Fluoro (hydroxymcthyl) malonate, 665 Fluoroxaloacetate, 665
2-Fluoro-3-hydroxy-3-methylpentanoate, citrate synthase substrate, 8
667 ketone cleavage, 630
2-Fluoro-3-hydroxy-2-oxalylbutyrate, 667
synthesis using diethyloxalate, 658
5-Fluoro-4-hydroxypentanoate, 665
2-Fluoro-3-hydroxypropionate, 663 4-Fluorooxalosuccinate, 668
resolution of, 633 3-Fluoropentane-3-carboxylate, 667
2-Fluoro-3-hydroxy-3,3,3-trichlorobuty- 2-Fluoro-4-pentenoate, 665
rate, 664 4-Fluoro-L-proline, 665
3-Fluoroisobutene-4,1-dicarboxylate, 667 Fluoropropiolate, 662
INDEX OF FLUORO ANALOGS 727
a-Fluoropropionate, 662 Morphine, fluorolactate resolution and,

stability, 627 650
fl-Fluoropropionate, 662 Nitrofluoroacetate, 662
stability, 627 Olefins, terminal, introduction of fluorine
4-(3'oFluoropropoxy)butyrate, 668 and, 634, 636
3-(3'-Fluoropropoxy) propionate, 668 Oxaloacetate, decarboxylation, fluoro sub-
3-Fluoro-2-pyrrolidone-5-carboxylate, 665 stitutions and, 630
3-Fluoro-2-pyrrolidone-3,5-dicarboxylate, Oxalofluoromalonate, 666
667 Oxalyl fuoride, 662
Fluoropyruvate, 663 Ozone, fluoroketoglutarate synthesis and,
synthesis using diethyl fluorooxaloace- 661
rate, 658 a-Phenylethylamine, fluorolactate resolu-
Fluoro substitution, effects on neighbor- tion and, 650
ing functional groups, 628-633 Propylfluoromalonate, 667
Fluorosuccinate, 664 Silica gel, fluoromalate preparation, 651-
synthesis using 652
diethyl oxaloacetate, 642 Stereochemistry, 633
diethyl succinate, 642 Sulfonate ester, introduction of fluorine
2-Fluorovalerate, 665 and, 634, 636, 638-639, 649
5-Fluorovalerate, 665 Synthesis and preparation of
Formaldehyde reaction with fluoro ole- diethyl 3,3-difluoroglutamate, 646-647
fins, 634 diethyl fluorooxaloacetate, 635
3- (2-Hydroxyethyl)-2-fluorocrotonate, 667 2,2-difluorocitrate, 657
Hydroxymethylfluorooxaloacetate, 666 2,4-difluorocitrate, 657-658
Iodofluoroacetate, 662 difluorofumarate, 648-649
Keto-enol equilibrium of 4,4-difluoro-2-ketoglutarat e, 660-661
diethyl fluorooxaloacetate, 629-630 difluoromalate, 652
ethyl 4,4-difluoroacetoacetate, 631 difluoromaleate, 649
ethyl 2-fluoroacetoacetate, 631 difluorooxaloacetate, 659-660
ethyl 4-fluoroacetoacetate, 631 22-difluorosuccinate, 644
ethyl 4,4,4-trifluoroacetoacetate, 631 ethyl 4-fluorooxalosuccinate, 661, 671
Methanol borontrifluoride, methyl esters ethyl 2-fluoropropionate, 638
and, 402--406, 415 fluoroacetoacetate, 638
1-Methoxy4,4-difluorocyclobutene-2,3- fluoroacetone, 638
dicarboxylate, 666 fluoroacetyl-coenzyme A, 654-655
3-Methoxy-2-fluoroacrylate, 662 fluoroaconitate, 638
Methyl acrylate, fluoroketoglutarate syn- fluoroacrylate, 638, 647
thesis and, 660 fluoroaliphatic esters, 635-638
Methyl "},-bromocrotonate, fluoroeroton- fluoroaminoisobutyrate, 638
ate synthesis and, 648 fluorocitrate, 638, 654-657
Methyl-2-chloro-2,3$-trifluorocyclobu- 2-fluorocrotonate, 647-648
tanecarboxylate, fluoroketoglutarate fluorodicarboxylic acids, 634
synthesis and, 660-661 fluorofumarate, 648
Methyl-3,3-difluoro-2-methoxy-l-cyclo- fluoroglutamate, 638
butene-l-carboxylate, fluoroketoglu- fluoroglutarate, 638, 644-646
tarate synthesis and, 661 a-fluoroglutaric diamide, 646
Methyl 4-fluorocrotonate, synthesis of, fluorohydroxybutyrate, 638
648 fluoroketoglutarate, 638, 672
3-Methylfluorooxaloacetate, 666 fluoromalate, 638, 651-652
fluoromalonate, 638, 639 Triethyl oxalosuccinate, introduction of

fluoromevalonate, 638 fluorine in, 661
fluorooxaloacetate, 638, 658-659 2,3,3-Trifluoroacrylate, acidity, 632
4-fluoro-L-proline isomers, 633 2,2,4-Trifluorocitrate, optical isomers of,
a-fluoropropionate, 638--639 633
fluoropyruvate, 638, 658 4,4,4-Trifluorocrotonate, acidity of, 632
fluorosuccinate, 638, 639, 642-644 2-Trifluoromethylacrylate, stability, 632-
Tetrafluorocitrate, isomers of, 633 633
Tetrafluoroethylene, introduction of fluo- 2-Trifluoromethylpropionate, stability,
rine and, 634 633
1,1,2-Trichloro-2,3,3-trifluorocyclobutane, Trifluorosuccinate, difluorofumarate syn-
difluorosuccinate synthesis and, 644 thesis from, 648-649
Triethyl a-fluoro-a-oxalosuccinate, fluoro- Triple bonds, introduction of fluorine and,
ketoglutarate synthesis and, 672 634, 636
Triethyl monofluorocitrate, preparation Vinylidene chloride, difluorosuccinate
of, 653 synthesis and, 644
ISBN 0 - 1 2 - 1 8 1 8 7 0 - 5

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Contributors to Volume X I I I

Article numbers are shown in parentheses followlng the names of contributors.

S. J. AJL (55), Department o] Bio- vania School o] Medicine, Philadelphia,

JUDXTH J. DAvls (47), Department o] chemistry, Dental School, Osaka Uni-

chemistry and Biophysics, Oregon State versity School o] Medicine, Cleveland,

Vlll CONTRIBUTORS TO VOLUME XIII

o] Microbiology, Albert Einstein Medi- serve University School o/ Medicine,

I. Preparation and Assay of Enzymes

VOLUME IX. Carbohydrate Metabolism

VOLUME X. Oxidation and Phosphorylation

VOLUME XI. Enzyme Structure

VOLUME XII. Nucleic Acids (Parts A and B)

VOLUMEXIII. Citric Acid Cycle

VOLUME XIV. Lipids

VOLUME XV. Steroids and Terpenoids

VOLUMEXVI. Fast Reactions

VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B)

VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C)

VOLUMEXIX. Proteolytic Enzymes

VOLUME XX. Nucleic Acids and Protein Synthesis (Part C)

VOLUME XXI. Nucleic Acids (Part D)

VOLUME XXlI. Enzyme Purification and Related Techniques

VOLUME XXlII. Photosynthesis (Part A)

VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B)

VOLUME XXV. Enzyme Structure (Part B)

VOLUME XXVI. Enzyme Structure (Part C)

VOLUME XXVII. Enzyme Structure (Part D)

VOLUME XXVIII. Complex Carbohydrates (Part B)

VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E)

VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F)

VOLUME XXXI. Biomembranes (Part A)

VOLUME XXXII. Biomembranes (Part B)

VOLUME XXXIII. Cumulative Subject Index Volumes I-XXX

VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B)

VOLUME XXXV. Lipids (Part B)

VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)

VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones)

VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides)

VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and

VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function)

VOLUME XLI. Carbohydrate Metabolism (Part B)

VOLUME XLII. Carbohydrate Metabolism (Part C)

VOLUME XLIII. Antibiotics

VOLUME XLIV. Immobilized Enzymes

VOLUME XLV. Proteolytic Enzymes (Part B)

VOLUME XLVI. Affinity Labeling

VOLUME XLVII. Enzyme Structure (Part E)

VOLUME XLVIII. Enzyme Structure (Part F)

VOLUME XLIX. Enzyme Structure (Part G)

VOLUME L. Complex Carbohydrates (Part C)

VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism

VOLUME LII. Biomembranes (Part C: Biological Oxidations)

VOLUME LIII. Biomembranes (Part D: Biological Oxidations)

VOLUME LIV. Biomembranes (Part E: Biological Oxidations)

VOLUME LV. Biomembranes (Part F: Bioenergetics) (in preparation)

VOLUME LVI. Biomembranes (Part G: Bioenergetics) (in preparation)

VOLUME LVII. Bioluminescence and Chemiluminescence

VOLUME LVIII. Cell Culture

VOLUME LIX. Nucleic Acids and Protein Synthesis (Part G)

VOLUME 61. Enzyme Structure (Part H) (in preparation)

VOLUME 62. Vitamins and Coenzymes (Part D) (in preparation)

VOLUME 63. Enzyme Kinetics and Mechanism (Part A) (in preparation)

Vol. I [199]. Deacylases (Thiol E&erase). John Gergely.

Utilization o] Acetyl Phosphate