Eur. J . Biochem.

47,91-97 (1974)
Proteinase K from Tritirachium album Limber
Wolfgang EBELING, Norbert HENNRICH, Michael KLOCKOW, Harald METZ, Hans Dieter ORTH, and
Hermann LANG
Biochemische Forschung, E. Merck, Darmstadt
(Received March 30/May 15, 1974)
The fungus Tritirachium album Limber was grown by submerged fermentation, investigating the
conditions for maximal secretion of proteases into the medium. Enzyme secretion starts when the
stationary phase of growth is reached, and when the culture medium is depleted of glucose and
amino acids, suggesting a catabolite repression of the enzyme.
The main proteolytic enzyme was named proteinase with respect to its keratin hydrolyzing activ-
ity. It was isolated from the culture medium and purified by crystallization and chromatography on
DEAE-Sephadex and Sephadex G-75. Homogeneity was ascertained by disc gel electrophoresis and
by isoelectric focusing. The molecular weight of proteinase K was determined by gel filtration to be
18500 f 500, the isoelectrice point was pH 8.9. The enzyme was shown to be a serine protease by
inactivation with diisopropyl phosphofluoridate and phenylmethylsulfonyl fluoride. It displayed
strong proteolytic activity on denatured but also on native proteins as demonstrated by the rapid
inactivation of bovine ribonuclease. The pH-optimum was in the range between pH 7.5-12.0. A
specificity of the enzyme for peptide bonds adjacent to the carboxylic group of aliphatic and aromatic
amino acids was observed.
Screening for microorganisms with proteolytic
activity towards native proteins wefound a strain of the
mold Tritirachium album which secrets a highly active
mixture of proteases along with an aminopeptidase
into the culture medium. The isolation of proteases
from this organism seemed of interest to us because
the organism can grow on native keratin as sole carbo-
hydrate and nitrogen source.
In this paper the cultivation of Tritirachium album
and the purification and some properties of the main
protease will be described. This enzyme was named
proteinase K with respect to its keratin hydrolyzing
activity. A characteristic property of the enzyme is
its ability to hydrolyze native proteins. This is the first
Abbreviations. Ac, acetyl-; Ac-Tyr-OEt, acetyl-L-
tyrosine ethyl ester; Bz-Arg-OEt, benzoyl-L-arginine ethyl
ester; 2 : 3-CMP, 2 : 3-cyclic cytidine monophosphate;
EDTA, ethylenediamine tetraacetate; Tos-Lys-CHzC1,
l-chloro-3-tosylamido-7-amino-2-heptanone (tosyl lysyl-
chloromethyl ketone); Tos-Phe-CHzC1, L-( I -tosylamido-
2-pheny1)ethyl chloromethyl ketone; Z-, benzyloxycar-
bonyl- ; -Nan, 4-nitroanilide.
Enzymes. Creatine kinase (EC 2.7.3.2); lactate dehydro-
genase (EC 1.1.1.27); malate dehydrogenase (EC 1.1.1.37);
ribonuclease (EC 3.1.4.22).
Definition. One Anson-U is defined as that amount of
enzyme which, under standard conditions, liberates one
millimole of Folin-positive amino acids (calculated as ty-
rosine) per min.
report on the purification of a fungal protease from
the genus Tritirachium.
MATERIALS AND METHODS
All reagents, enzymes and enzyme substrates were
products of E. Merck (Darmstadt).
Organism
Tritirachium album Limber (Merck strain No. 2429)
was isolated from soil manured with horn chips. The
strain is deposited at the Centraalbureau voor Schim-
melcultures at Baarn (CBS no. 747.69).
Cultivation
Medium for propagation cultures: 3% malt
extract, 0.3 % casein peptone. Medium for protease
production: 1 % glucose, 1 % casein peptone, 0.01 %
MgS04 . 7 H20,0.005 % CaCla . HzO, 0.28 % KHzP04
and 0.07 % Na2HP04. pH after sterilisation 5.7-5.9.
All cultures were grown at 28 C.
Conditions of aeration: Shake flasks: 1000-ml
conical flasks containing 200 ml medium, 100 strokes/
min, amplitude 5 cm. Fermenters, 8-10 1 medium:
0.3 -0.8 1 air x 1-1x min-1, stirrer speed 250-
Eur. J . Biochem. 47 (1974)
92 Proteinase K from Tvitivachiurri album Limber
500 rev./min. Propagation cultures were seeded each
by 10% of their volume and were grown in the
following manner: (a) agar slants, 10 - 14-days old;
(b) first generation of shake flasks, 48 h; (c) second
generation of shake flasks, 48 h; (d) fermenters 24 h.
Production cultures were inoculated by 5 % of their
volume. Growth rate was estimated as cell volume :
10ml of broth was centrifuged at 2000rev./min for
5 min. The sediment estimated in graduated tubes was
taken as cell volume.
The proteolytic activity of the culture fluid was
measured with hemoglobin as substrate as described
below. Aminopeptidase activity was estimated with
L-leucine-4-nitroanilide as substrate as described
elsewhere [l].
Isolation of Proteinase K
All purification procedures were carried out at
5 C. The fermentation broth (5.0 1) was separated
from mycelium by centrifugation (2800 rev./min for
20 min) and the clear culture filtrate (4.25 1) brought
to 40% saturation by addition of 285 g/1 solid ammo-
nium sulfate.
The precipitate was removed by filtration with
50 g kieselgur and discarded. Further addition of
ammonium sulfate to the filtrate (243 g/l) to a satura-
tion of 80% resulted in precipitation of the enzyme
which was collected by suction. The filter cake was
dissolved in 100 ml 0.03 M Tris-HC1 buffer, pH 7.5
and after removal of insoluble material dialyzed
against the same buffer. To the dialyzed solution
(160 ml) 15 g of DEAE-cellulose (wet form), equili-
brated with 0.03 M Tris-HC1 buffer containing 5 mM
CaC12, were added and the mixture stirred for 10 min.
Most of the dark-colored pigment was adsorbed to the
DEAE-cellulose and removed by filtration. The pale
yellow filtrate (163 ml) was saturated by addition of
561 g/1 ammonium sulfate to SO%, the precipitated
protein collected by centrifugation and redissolved in
32 ml 0.03 M Tris-HC1 buffer, pH 7.5 to a protein
concentration of 50-60 mg/ml. During dialysis of
this solution against 5 mM calcium acetate (2 1)
overnight, a heavy precipitate of crude crystallized
proteinase K appeared in the dialysis bag. The amber
crystallisate was filtered and washed twice with a
total volume of 50 ml 1 mM calcium acetate. Re-
suspended in 50ml 1 mM calcium acetate, the en-
zyme, was lyophilized to yield 820 mg protein.
Chromatography of Crystallized Proteinuse K
820 mg lyophilisate of crystallized enzyme protein
was dissolved in 60 mlO.01 M Tris-HC1 buffer, pH 7.5,
containing 1 mM calcium acetate and was passed
through a column of DEAE-Sephad2x A-50 (2.5
x 90 cm) equilibrated with the same buffer, at a flow
rate of 60 ml/h and a fraction of 15 ml. Proteinase K
was not adsorbed onto the column, but the residual
colored material and acidic protein impurities were
bound. The combined proteolytically active eluate
(210 ml) was saturated to 80% by addition of 561 g/1
ammonium sulfate and the precipitate collected by
centrifugation. The pellet was dissolved in 40 ml
0.05 M Tris-HC1 buffer, pH 8.0, placed on a column of
Sephadex (3-75 and eluted with the saine buffer at a
flow rate of 70 ml/h and a fraction volume of 15 ml.
Fractions from tubes 68 to 78 of the proteolytically
active main peak were combined (186 nil) and the en-
zyme precipitated by addition of 561 g,I solid ammo-
nium sulfate. After centrifugation, the precipitate
was taken up in 40 mlO.05 M Tris-HC1 buffer, pH 8.0
and passed again through the same column. The
eluate of the resulting single peak (190 ml) was brought
to 80% ammonium sulfate saturation, stored at 4 C
and used for all further studies.
Profein Concentration
Protein concentrations were determined by the
biuret method [2]. Protein in chromai ography frac-
tions was measured spectrophotometric illy at 280 nm
using the factor = 14.2.
Disc Electrophoresis
Polyacrylamide gel disc electrophoresis was carried
out at pH 4.3, 7.5, and 8.9 according to Maurer [3]
with 100-500~g protein for each run. Gels were
stained with a solution of 0.2% light green SF,
0.2% ponceau red, 0.1 % amido black 10 B and 5
sulfosalicylic acid in 5 % trichloroacetic acid. In addi-
tion to protein staining, proteinase K was identified
by its proteolytic activity. Disc electrophoresis was
carried out in parallel runs with 1 pg proteinase K
using polyacrylamide gel containing 0.1 % gelatine [4]
followed by incubation of the gel coluinns in 67 mM
phosphate buffer, pH 8.0 at 35 C for 45 min. Staining
was as described above.
Isoelectric Focusing
Proteinase K was subjected to isoelectric focusing,
using 1 % carrier ampholine with pH ranges of 3-10
and 7-10, stabilized by sucrose. An LK B-8101 column
was employed.
Estimation of Molecular Weight
The molecular weight of proteinase k was estimated
by Whitakers method [5] using Sephadex G-50 and
Eur. J . Biochem. 47 (1974)
W. Ebeling, N. Hennrich, M. Klockow, H. Metz, H. D. Orth, and H. Lang 93
Table 1. Ejfect of peptone and glucose concentration on production of proteinase K
Peptone Glucose Proteolytic activity after fermentation for (h)
36 48 60 72 84 96 156
% . mAnson-U/ml
Shake flasks 0.5 0.7 2.0 1.8 2.0
0.3 5.7 5.8 5.5
1 .o
1.5 0.4 4.8 5.4 5.1
0.5 1.5 1.1 1.2 2.0
15.1 5.8 1 .o 1 .o 0.0 0.0 - 1.4
1 .o 1.5 0.2 0.6 1 .o 3.4 5.9
1.5 1 .o 0.2 0.7 2.1 3.3 2.3
1.5 1.5 0.2 0.5 1.2 3.2 2.5
-
-
~
-
-
Fermenter 1 .o 1 .o 2.0 12.2 17.6 28.2 25.6 17.3
G-75 columns equilibrated with 67 mM phosphate
buffer, pH 7.6. The void volume of the column was
measured with blue dextran. Pepsin (Mr = 35000),
chymotrypsinogen ( Mr = 25000), trypsin (Mr =
23300), and lysozyme ( Mr = 14200) were used as
marker proteins.
Ultraviolet Absorption Spectrum
The spectrum was recorded with a 0.042 % solution
of proteinase K in 10 mM NaCl containing 5 mM
CaC12, pH 8.0.
Activity against Low-Molecular- Weight Substrates
Cleavage of 4-nitroanilide derivatives of amino
acids and dipeptides during incubation with proteinase
K at 25 "C was measured photometrically at 405 nm.
Incubation mixture: 3 ml 67 mM phosphate buffer,
pH 8.0, 1 mg proteinase K and substrate in the con-
centration stated in Table 3. Dipeptide-4-nitroanilides
with benzyloxycarbonyl-blocked a-amino groups were
dissolved in dimethylformamide in a ten-fold concen-
tration. After incubation of Z-L-Ala-L-Arg-Nan with
proteinase K for 5 min, 205 pg aminopeptidase K
[ I ] was added to the reaction mixture and the amount
of 4-nitroaniline released was determined at 405 nm
as described above. Esterolytic activity of proteinase K
was measured by the pH-stat method at pH 8.0 and
30 "C with the aid of an autotitrator (Metrohm AG,
Herisau) using 0,05 N NaOH as titrant. Reaction
mixture: 10 mlO.05 M Tris-HCl buffer, pH 8.0, 0.02 M
Ac-Tyr-OEt or Bz-Arg-OEt.
Activity against Protein Substrates
Proteolytic activity was assayed by the Anson
method [6] at pH 7.5 and 35.5 "C, Incubation mixture:
6.0 ml 2% urea-denatured hemoglobin and 6 pg pro-
teinase K. The activity against casein as substrate
was determined by the Kunitz method [7]. Reaction
mixture: 0.5% casein (Hammarsten) in 2 ml 0.05 M
Tris-HC1 buffer, pH 8.0 was incubated with 1 .O-3.0 pg
proteinase K at 37 "C for 20 min.
Proteolytic degradation of ribonuclease was mea-
sured by incubating proteinase K (5-20 pg/ml) in
2 ml 0.1 M NaC1, 0.1 mM EDTA, and 1.6-4.8 mg
ribonuclease/ml at pH 7.5 and 25 "C. At various
times during incubation aliquots were analyzed for
residual ribonuclease activity with 22 mM 2': 3'-CMP
as substrate as described [8].
RESULTS AND DISCUSSION
Fermentation Conditions f or Protease Production
Tritirachium album was grown by aerobic sub-
merged fermentation at 28 "C. The mold can grow
with native wool keratin as sole source of carbon and
nitrogen, but growth and protease production were
enhanced in a media containing substrates such as
peptone, milk powder, soybean meal, corn steep or
albumin. When nitrogen was supplied to the medium
as nitrates, ammonium salts or amino acids (e.g.
glutamate, alanine), only minimal proteolytic activity
could be found. I n a medium containing peptone as
carbon and nitrogen source, protease formation was
enhanced until a peptone content of 1.5 % was reached
but was decreased at higher peptone concentration
(Table 1). Addition of 1% glucose to the broth con-
taining 1 "/, peptone enhanced cell growth and protease
secretion further to about 15 mAnson U/ml medium,
but 12 h more were needed to get maximal activity.
More suitable conditions in a fermenter, e. g. better
aeration and a more homogeneous growth, caused a
further increase of the activity to 25 -28 mAnson
U/ml combined with a shorter fermentation time.
A solution of 1 "/, peptone, 1 % glucose and mineral
salts was therefore routinely used as medium for
proteinase K production. As shown in Fig. 1 the
Eur. J . Biochem. 47 (1974)
94 Proteinase K from Tritirachiiirrl album Limber
25
-
E
.
3 20
c 0
c
6
E 15
-
0 0
6.0
0
0 12 24 36 48 60 72 84
Time of cultivation (h)
Fig. 1. Fermentation diagram of Tritirachium album.
0-0 Proteolytic activity (mAnson-U/ml), o---- 0
aminopeptidase activity (mU/ml), 0-0 growth curve
(cell volume), .--a pH, a----U glucose, v-v
Folin-positive trichloroacetic-acid-soluble substances
( AAmm) . For assay procedures see Methods
Fig. 2. Disc electrophoresis of the ammonium sulfate precipi-
tate from the culture fluid. Tube 1 : polyacrylamide gel;
tube 2: polyacrylaniide gel containing 0.1 % gelatine.
Electrophoresis was conducted at pH 4.3 at 5 "C. Staining
see Methods
proteolytic activity together with an aminopeptidase
activity [l ] appeared in the medium when maximum
growth was reached and pH had passed a minimum.
At this time glucose was fully metabolized and pep-
tides and amino acids derived from peptone were
exhausted, suggesting a catabolite repression of the
protease production during the main phase of growth.
Both proteolytic and aminopeptidase activity are
stable in the cell-free liquor for days Ighen stored at
5 -8 "C. The aminopeptidase, however, is rapidly
inactivated in the presence of the intact mycelium.
Isolation of Proteinase K
Crude enzyme was separated from the culture
medium by ammonium sulfate fractionation and de-
colorized by batch-treatment with D EAE-cellulose.
Disc electrophoresis of this product in the polyacryl-
amide gel containing 0.8 % gelatine (F ig. 2) revealed
the presence of at least three distinct proteolytically
active components, the major componcnt being pro-
teinase K. The separation of the contaminating en-
zymes from proteinase K by chroma1 ographic pro-
cedures proved difficult because of very similar prop-
erties of these enzymes, e. g. molecul.ir weight and
net electric charge. A complete resolution could not
be achieved either by gel filtration or by chromatog-
raphy on DEAE-Sephadex, QAE-Sephadex or CM-
Sephadex. Yet proteinase K crystallized readily even
from crude solutions if the ionic strength was low
and the protein content adjusted to 40-50mg/ml,
forming bipyramidal prisms of very lo\v solubility in
water (Fig. 3). The purity of this cr! stallisate was
examined by disc electrophoresis at pH 4.3 and pH 8.9.
At pH 4.3 only one protein componeiit was visible,
this was proteolytically active and represented protein-
ase K. At pH 8.9 however, four additional, but proteo-
lytically inactive minor components c ould be seen
(Fig. 4, tubes 1, 2). The further purification of crystal-
line proteinase K was initially hampered by its very
low solubility. Lyophilisation of the proctuct suspended
in 1 mM calcium acetate, however, yielded a material
easily soluble in water and diluted salt jolutions. The
final purification was carried out using chromatog-
raphy on DEAE-Sephadex to remove any residual
coloring matter following by repeated gel filtration
of the enzyme on Sephadex G-75 in 0.05 M Tris-HCI,
pH 8.0. The purification is about 16-fold, the yield is
28% with respect to the total proteolltic activity of
the original culture filtrate. The enzynie is stable at
+4 "C for at least 12 months without loss of activity.
A summary of the purification procedures is given in
Table 2.
PROPERTIES OF PROTEINASE K
Homogeneity
Fig. 5 shows the profile of isoelectric focusing of
proteinase K in the pH-range 7-10. A single protein
peak in correspondence with the proteolytic activity
was found. The isoelectric point of the protein was 8.9.
Disc electrophoresis of the enzyme is shown in Fig. 4,
tubes 3, 4, 5. A single protein component was present
Eur. J . Biochem. 47 (1974)
W. Ebeling, N. Hennrich, M. Klockow, H. Metz, H. D. Orth, and H. Lang 95
Fig. 3. Photomicrograph of crystalline proteinase K. Magnification : 40
Fig. 4. Disc electrophoresis of crystallized proteinase K. Tubes 1, 2: before, tubes 3, 4, 5: after chromatography on DEAE-
Sephadex and gel filtration on Sephadex G-75. Electrophoresis was carried out at pH 4.3 (tubes 2, 3, 4) and pH 8.9
(tubes 1. 5). Tube 4: polyacrylamide gel containing 0.1 /, gelatine
Table 2. Summary of a typical purification procedure of proteinase K
Fraction Total volume Total protein Total activity Specific activity Yield
Culture filtrate
0.4-0.8 Satd ammonium sulfate
precipitate
Batch treatment with DEAE-cellulose
0.8 Satd ammonium sulfate precipitate
Crystallisate
Eluate of DEAE-Sephadex
Eluate of Sephadex G-75 chromatography
Eluate of Sephadex G-75
Chromatography
rechromatography
ml
4250
105
160
32
210
186
190
mg
16300
3 700
1630
1430
720
540
336
310
mAnson-U mAnson-U/mg
34800 2.1
29 800 8.1
25 800 15.8
24 100 16.9
16 500 22.8
14700 27.2
10 600 31.6
9 900 32.0
%
100
85
74
69
47
42
30
28
Eur. J. Biochem. 47 (1974)
Proteinase K from Tritirachiun, album Limber
10
- 9
I,
- 8
- 7
96
0.50
:
0
a, CI
m
a,
i
m
J2 L
I
3 0. 25
a
s2
0
5 10 15 20 25
Fraction no
Fig. 5. Isoelectric focusing of proteinase K. Column : LKB
8101; load: 22mg. (0-0) Protein (absorbance at
280 nm); (.-----.) proteolytic activity; (.......) pH
gradient 7-10
Table 3. Specijic activity of proteinase K against synthetic
substrates
Substrate Substrate Specific activity
concn
2-L- Ala-L- Ala-Nan
Z-L-Ala-L-Arg-Nan
L- Ala-L-Ala-Nan
L-Ala-L-Arg-Nan
Ac-L-Tyr-Nan
Ac-L-Ala-Nan
Ac-L-Leu-Nan
Ac-Tyr-OEt
Bz-Arg-OEt
~
mM
0.05
0.05
0.25
0.25
0.25
0.25
0.25
20
20
~ ~~
mU/mg
150
0
0
0
2.6
0.25
0.25
650
0
at pH 4.3 and 7.5. No protein impurities could be
observed at pH 8.9. Disc electrophoresis of the enzyme
in polyacrylamide gel containing 0.1 % gelatine accord-
ing to Stegemann [4] showed a single proteolytically
active compoiient at pH 4.3 and 7.5, respectively.
Ultraviolet-Absorption Spectrum
The absorption maximum was found to be 280 nm,
the minimum at 260 nm. The curve is a typical pattern
for protein, Azso/A260 = 2.18. A::; (280 nm) = 14.2.
A slight shoulder at around 285 nm is possible due to
absorption from tryptophan residues.
Molecular Weight
tration was estimated to be 18 500 & 500.
The molecular weight of proteinase K by gel fil-
Effect of the p H on the Enzyme Activitj
The pH activity curve of proteinase K for hydroly-
sis of urea-denatured hemoglobin showed optimal
activity in the pH range 7.5-12.0.
Effect of Inhibitors
Metal-chelating agents like EDTA and sulfhydryl
inhibitors such as monoiodoacetic acid and p-chloro-
mercuribenzoate had no inhibitor effect on the enzyme
activity. The trypsin-specific inhibitor T wLys-CHzC1
and the chymotrypsin-specific inhibitor Tos-Phe-
CHzCl showed no inhibition. Phenylmethylsulfonyl
fluoride and diisopropyl phosphorofluoridate com-
pletely inhibited the enzyme, suggesting the involvement
of serine in the mechanism of action of proteinase K.
Diisopropyl phosphorofluoridate-sensiti\ Ity is a com-
mon feature of all serine protease, e.g the alkaline
proteases of the genus Aspergillus, Bacillus or Strepto-
nzyces [9].
Enzyme Activity on Synthetic Substrates
Purified proteinase K was incubated with 4-nitro-
anilide derivatives of amino acids and di peptides with
free and blocked a-amino groups (Tahle 3). A pre-
dominant cleavage of the peptide bond a jjacent to the
carboxylic group of aliphatic and arc matic amino
acids with blocked cu-amino groups is observed. The
nitroanilide group of amino acid and dipzptide deriva-
tives with free &-amino groups is resistant to hydrolysis
by proteinase K. The specificity of thc enzyme for
alanine was further demonstrated with Z-L-Ala-L-
Arg-Nan as substrate. During incubation of 249 pmol
Z-Ala-L-Arg-Nan with proteinase K for 5 min, no
measurable liberation of 4-nitroaniline zould be ob-
served. The addition of aminopeptidase K, however,
set free 244 pmol (dA405 = 0.77) 4-nitroaniline (Fig. 6) .
This proved the nearly complete cleavage of the pep-
tide bond between alanine and arginine within the
first 5 min of incubation.
In accordance with these findings proteinase K
exhibits no esterolytic activity on Bz-l\rg-OEt, but
shows a powerful ersterolytic activity 0 1 1 the chymo-
trypsin substrate Ac-Tyr-OEt (Table 3).
Activity against Protein Substrates
Proteinase K shows strong proteolytic activity on
denatured proteins, e.g. hemoglobin and casein.
Against hemoglobin proteinase K, on a weight basis,
is about six times more active than pronase and about
three times more active than bovine trypVn. The most
interesting property of the enzyme, however, is its
capacity to degrade native proteins. Sensitivity of
native enzymes and proteins to proteolytic degrada-
Eur. J. Biochem. 47 (1974)
W. Ebeling, N. Hennrich, M. Klockow, H. Metz, H. D. Orth,
0 8 -
E
5 0.7-
0 6-
m 0.5-
2 0 4 -
\j
1
m
I=
L
2 0 3 -
4
0 2-
and H. Lang 97
1.01
0. 9 {
0 5 10 15 20
Fig. 6. Hydrolysis of Z-L-Ala-L-Arg-Nan by proteinase K.
Incubation mixture: 3 mlO.067 M phosphate buffer pH 8.0,
0.083 mM Z-L-Ala-L-Arg-Nan and 1 mg proteinase K.
After 5 min at 25 "C, 0.05 ml aminopeptidase K (4.1 mg/ml)
was added (as indicated by the arrow)
Time (min)
Table 4. Activity of proteinase K against protein substrates
Substrate Substrate Proteinase Specific
concn K concn activity
mg/ml lJ.g/ml
Hemoglobin 20 1 30 mAnson-U/mg
Casein 5 5 4.5 Kunitz U/mg
Ribonuclease 2.4 20 71 mU/mg
Lactate de-
Malate de-
hydrogenase 0.1 50 0.31 mU/mg
hydrogenase 1.3 250 0.27 mU/mg
tion by proteases of various sources has already been
observed earlier [lo-191. Depending on the structural
stability of the protein and the incubation conditions
more or less intensive proteolysis can occur.
As Table 4 shows, ribonuclease is preferentially
attacked by proteinase K. Other enzymes, e. g. lactate
dehydrogenase (rabbit muscle), malate dehydrogenase
(pig heart), creatine kinase (rabbit muscle) and phos-
phodiesterase (bovine heart) also were found to be
inactivated.
Recently Wiegers and Hilz described a method for
the rapid isolation of undegraded mRNA from poly-
somes. Proteinase K treatment of the isolated poly-
somes was superior to all other procedures, avoiding
the use of' phenol and leading to a nearly complete
yield of native mRNA [20,21]. This was due to the
rapid proteolytic inactivation of the endogenous nu-
W. Ebeling. N. Hennrich. M. Klockow. H. Metz. H. D. Orth.
cleolytic activity by proteinase K [20,22,23]. Chambon
and coworkers used proteinase K for the isolation of
high-molecular-weight DNA from mammalian cells
[24]. Cell lysis by sodium dodecylsulfate in the presence
of proteinase K yielded DNA preparations essentially
free of single-stranded nicks with a number-average
molecular weight of 200 x lo6. The high proteolytic
activity of proteinase K even in the presence of 0.5%
sodium dodecylsulfate immediately inactivated the
cellular DNA-degrading enzymes. Studies on the
specificity of proteinase K against insulin A and B
chains will be published in a separate paper.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Hennrich, N., Klockow, M., Orth, H. D., Femfert, U.,
Cichocki, P. & J any, K. (1973) Hoppe-Seyler's 2.
Physiol. Chem. 354, 1339-1346 .
Beisenherz, G., Boltze, H. J ., Buecher, Th., Czok, R.,
Garbade, K. H., Meyer-Arendt, E. & Pfleiderer, G.
(1 953) Z. Naturforsch. B 8, 555 -577.
Maurer, R. H. (1968) Disk-Elektrophorese, W. de Gruy-
ter, Berlin.
Stegemann, H. (1968) 2. Anal. Chem. 243, 573-578.
Whitaker, J . R. (1963) Anal. Chem. 35, 1950-1953.
Anson, M. L. (1939) J. Gen. Physiol. 22, 79.
Kunitz, M. (1947) J. Gen. Physiol. 30, 291.
Brummer, W. , Hennrich, N., Klockow, M., Lang, H.
& Orth, H. D. (1972) Eur. J. Biochem. 25, 129-135.
Matsubara, H. & Feder, J . (1971) The Enzymes (P. D.
Boyer, H. Lardy & K. Myrback, eds) Vol. 111,
pp. 744-765, Academic Press, New York.
Ottesen, M. (1956) Arch. Biochem. Biophys. 65, 70-77.
Pfleiderer, G., J eckel, D. & Wieland, Th. (1957) Bio-
Porter, R. R. (1959) Biochem. J. 73, 119-126.
Pfleiderer, G. & Zwilling, R. (1965) Biochem. 2. 344,
Pfleiderer, G., Zwilling, R. & Sonneborn, H. H. (1967)
Hoppe-Seyler 's Z. Physiol. Chem. 348, 13 19 - 133 1.
J eckel, G., Anders, R. & Pfleiderer, G. (1973) Hoppe-
Seyler's 2. Physiol. Chem. 354, 737 -748.
Biszku, E., Sajgo, M., Polti, M. & Szabolcsi, G. (1973)
Eur. J. Biochem. 38, 283 -292.
Pontremoli,S., Melloni,E., Balestrero, F., De Flora, A.
& Horecker, B. L. (1973) Arch. Biochem. Biophys.
Richards, F. M. & Vithayathil, P. J . (1959) J. Biol.
Klee, W. A. (1965) J. Biol. Chem. 240, 2900-2906.
Wiegers, U. & Hilz, H. (1971) Biochem. Biophys. Res.
Wiegers, U. & Hilz, H. (1972) FEBS Lett. 23, 77-82.
Wiegers, U., Kramer, G. & Hilz, H. (1973) Hoppe-
Mach, B., Faust, C. & Vassali, P. (1973) Proc. Natl.
Gross-Bellard, M., Oudet, P. & Chambon, P. (1973)
chem. Z. 329, 104-111.
127- 140.
156, 255 - 260.
Chem. 234, 1459 -- 1465.
Commun. 44, 513-519.
Seyler's Z. Physiol. Chem. 354, 1576 - 1582.
Acad. Sci. U. S. A. 70, 451 -455.
Eur. J. Biochem. 36, 32-38.
and H. Lang, Biochemische Forschung,
E. Merck b-6100 Darmstadt, Postfach.119, Federal Republic of Germany
Eur. J . Biochem. 47 (1974)