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BIOCHEMISTRY
202,348-355
(1992)
Riboflavin 5Pyrophosphate:
A Contaminant of
Commercial FAD, a Coenzyme for FAD-Dependent
Oxidases, and an Inhibitor of FAD Synthetase
Holly A. Hartman,
Department
Received
Dale E. Edmondson,
of Biochemistry,
December
Rollins
University,
Atlanta,
Georgia 30322
3,1991
synthetase (ATP:FMN
Commercially available preparations of flavin adenine dinucleotide (FAD) have been found to be 94%
pure, the remaining 6% being composed of four or five
minor contaminants which can be separated from FAD
byreverse-phasehigh-performanceliquidchromatography. FAD purified in this manner has been shown to be
100% pure. One of the contaminants has been identified
as riboflavin 5-pyrophosphate (RPP) by spectroscopic
and chemical methods of analysis. This compound has
been shown to exhibit biological activity as a weak cofactor for two FAD-requiring
enzymes. With the apoprotein of porcine D-amino-acid oxidase, values determined for RPP were 8.4 pM for X, and 0.10 for V,,,,
compared to 0.47 NM and 0.28 (36 Ulmg), respectively,
for FAD. With fungal glucose apooxidase, values determined for RPP were 474 nM for K, and 0.02 for V,,
and 45 IIM and 0.09 (106 U/mg), respectively, for FAD.
RPP can also inhibit FAD biosynthesis. For bovine
liver FAD synthetase, a Ki value for RPP against FMN
was determined to be 9 PM where K,,, for FMN was 5.5
PM. These studies illustrate the value of riboflavin 5pyrophosphate as a flavin analog for use in the study of
structure/function
relationships within certain flavindependent enzymes. o 1992 Academic PXW, IDC.
adenylyltransferase
EC 2.7.7.2),
(1,2). However, the enzyme is present in low abundance
and is therefore difficult to obtain in high yield. Further,
the stability of the purified enzyme becomes a limiting
factor in the production of large quantities of FAD.
Alternatively,
the coenzyme can be purified from
commercially
available preparations of chemically synthesized FAD. Massey and Mendelsohn
(3) have developed a successful purification
method using a column of
immobilized
FAD-specific
apoenzyme. However, this
procedure is limited in its utility by low yields of pure
coenzyme and by the extensive preparations
involved.
More efficient methods for purification
of FAD from
commercial preparations by ion-exchange and dynamic
complex-exchange
(or ion-paired) HPLC have been devised by Entsch and Sim (4). These authors concluded
that commercial FAD obtained from Sigma was about
87% pure and contained four or five minor contaminants which were not identified. We have foundit convenient to use a modification
of the reverse-phase HPLC
procedure published by Light et al. (5) for purification of
FAD from commercial preparations, since a pure coenzyme fraction having low salt content can be obtained
by this method.
In this paper, we confirm the findings of Entsch and
Sim (4) and report our identification
of a contaminant
of commercial
FAD as riboflavin
5-pyrophosphate
(RPP) by spectroscopic and chemical methods. Further,
we describe the biological activity of this compound as a
coenzyme for D-amino-acid
apooxidase and glucose
apooxidase and as an inhibitor of FAD synthetase.
MATERIALS
AND METHODS
Materials
1 To whom correspondence
should be addressed.
2 Abbreviations
used: FMN,
flavin mononucleotide;
vin 5-pyrophosphate;
Pipes,
1,4-piperazinediethanesulfonic
SDS, sodium dodecyl
sulfate.
RPP,
riboflaacid;
348
Copyright
All
rights
0 1992
of reproduction
$3.00
by Academic
Press, Inc.
in any form reserved.
SPECTROSCOPIC
STRUCTURE/FUNCTION
STUDIES
crystalline D-amino-acid
oxidase from porcine kidney
were also obtained from Sigma. D-Glucose was from
Mallinkrodt;
EDTA and cellulose TLC plates were from
Kodak. D-Phenylglycine
was purchased from Aldrich.
Glucose apooxidase from Aspergillus niger was provided
by Dr. D. L. Morris of Miles Laboratories.
The 2,3,4,5-tetra-O-acetylriboflavin
and lo-formylmethylflavin were prepared from riboflavin by peracetylation
(6)
and periodate oxidation (7), respectively. Bovine liver
was obtained immediately
following sacrifice of the animal and kept on ice until frozen at -20C in packages
containing about 2 kg of tissue. All other chemicals and
reagents were of the highest purity available from
Sigma, Fisher, or Aldrich.
OF RIBOFLAVIN
5-PYROPHOSPHATE
349
Spectral Analysis
The spectra of compounds dissolved in 0.1 M potassium phosphate buffer, pH 7.0, were obtained using a
Milton Roy Spectronic 3000 array spectrophotometer.
For analyses, samples were placed in quartz cuvettes
having pathlengths
of 1 cm. The absorbance of each
compound between 200 and 600 nm was measured.
Photolysis
Purification
of Commercial FMN
Peracetylation
reactions were performed as described
by McCormick
for synthesis of 2,3,4,5-tetra-o-acetylriboflavin
(6) both with and without the addition of
perchloric acid catalyst. Solutions containing 25 nmol of
Aavin in distilled water were pipetted into polypropylene microtubes (30 X 8 mm) and evaporated to dryness
using a Savant speed vat concentrator equipped with a
Savant refrigerated condensation
trap and a Savant
VP100 two-stage vacuum pump. The compounds were
resuspended in 50 ~1 of glacial acetic acid: acetic anhydride (l:l, v/v). Zero-time aliquots were applied to cellulose TLC plates before the addition of 0.5 ~1 of 70%
perchloric acid to the reactions. Aliquots were then
taken at lo-min intervals during incubation of the samples at 40C and immediately
applied to the TLC plates.
Acid Hydrolysis
350
HARTMAN,
EDMONDSON,
ing water bath for 4 h. Aliquots taken at 60-min intervals were immediately
applied to cellulose TLC plates.
AND
MCCORMICK
Purification
of FAD synthetase was based upon previously published procedures for isolation of this enzyme
from rat liver (2,13) with modifications
being made as
necessary due to the larger scale of the preparation
and
3 The catalytic
activity
of the bovine
as compared
to 7.1 for the rat enzyme
differences
in the amino acid composition
enzyme
is maximal
at pH 6.1
(2), and there are apparent
of the two synthetases
(25).
SPECTROSCOPIC
STRUCTURE/FUNCTION
STUDIES
OF
RIBOFLAVIN
351
5-PYROPHOSPHATE
20
40
60
60
MINUTES
FIG.
1. Reverse-phase
mercial
FAD.
HLPC
separation
of compounds
in com-
of FMN-Sepharose
352
HARTMAN,
TABLE
Composition
EDMONDSON,
AND MCCORMICK
of Commercial
TABLE
FAD
RI Values of Flavins
Fractions as % composition
Supplier
Fluka
Boehringer-Mannheim
Sigma*
1
1
0.4
0.4
1
1.9
1.4
1.7
1.2
94.2
93.7
94.7
3
2.6
0.7
a Based on integrated areas from HPLC chromatograms where retention times in minutes were 24 (A), 37 (B), 43 (C), 46 (D), and 51
03).
Contains two additional contaminants, one eluting between A and
B and comprising 1.1% of the preparation, and the other as a trace
contaminant eluting between D and E.
R, Values
Compounds
Flavin standards
2, 3,4, 5-Tetra-Oacetylriboflavin
lo-Formylmethylflavin
Lumichrome
Riboflavin
5-Flavin mononucleotide
Flavin adenine dinucleotide
Flavins isolated from
commercial FAD
B
C
D
E
Solvent
A
Solvent
Bb
Solvent
C
0.94
0.81
0.76=
0.60
0.49
0.31
0.21
0.45
0.33
0.88
0.64d
0.43
0.46
-
0.30
0.35
0.31
0.51
0.53
0.50
0.33
0.42
0.45
-
DBuOH/HOAc/H,O
(4/2/3).
b Five percent aqueous dibasic sodium phosphate.
c BuOH/EtOH/H,O
(50/15/35).
d Exhibited blue fluorescence.
Products
Treatments/compounds
TABLE
and Derived
on TLC
Solvent A
0.76b
0.81
0.94
0.79
0.67
0.50, 0.58
0.31, 0.50, 0.58
DBuOH/HOAc/H,O
(4/2/3).
b Exhibited blue fluorescence.
structure
activity.
of Riboflavin
The greenish yellow fluorescence of compound C suggested that it was a flavin. This was confirmed by the
uv-visible spectrum at pH 7.0. Four maxima at 224,269,
375, and 447 nm were observed, which indicated presence of a 7,8dimethylisoalloxazine
ring structure. The
ratio of absorbance at 260 to 450 nm was calculated to
be 2.1 to 2.2. This result indicated that C did not contain
an adenyl moiety, as does FAD, which has an A,,/A,,
ratio of 3.26 (8).
To determine whether compound C contained a lopolyol side chain in common with that of riboflavin and
FMN, the N-10 substituent group was photochemically
cleaved from each of the three flavins by irradiation
with uv light. TLC analysis of the photolysis products
from each compound revealed that the primary product
of aerobic photodecomposition
of all three flavins had a
mobility identical to that of the lumichrome
standard
and exhibited its blue fluorescence (Table 3). This result is consistent with the well known photolability
of
the flavin side chain (18).
Further studies were carried out to characterize the
N-10 substituent. TLC analyses revealed that periodate
oxidation of riboflavin, FMN, and compound C in each
case resulted in the formation of lo-formylmethylflavin
(Table 3). This indicated a polyhydroxy chain containing at last two vicinyl hydroxyl groups located at the 2and 3-positions in the side chain of compound C.
Additional evidence for the presence of a polyhydroxy
side chain was obtained from TLC analysis of products
from peracetylation
of riboflavin, FMN, and C (Table
3). Using solvent A [BuOH/HOAc/H,O,
(4/2/3)] the
SPECTROSCOPIC
STRUCTURE/FUNCTION
STUDIES
mobilities
of their peracetylation
products relative to
that of riboflavin (which generates the 2,3,4,5-tetra0-acetylriboflavin),
FMN, and C were increased by approximately
a third. When solvent C [BuOH/EtOH/
H,O (50/15/35)] was employed, the mobilities
of their
most highly acetylated products were increased most
for tetra-0-acetylriboflavin
and less for acetylated products of FMN and C. The faster migration of the riboflavin product relative to those from FMN and C is consistent with four hydroxyl groups present on the ribityl
side chain of riboflavin, while that of FMN contains
only three, since the 5-position is phosphorylated.
The
appearance of three products from peracetylation
of
both FMN and C, as well as the similarity of the difference in mobility
on TLC plates between these compounds and that of their peracetylation
products, suggested that the polyhydroxy
side chain of C also
contained three hydroxyl groups. When the peracetylation reactions were attempted without addition of the
perchloric acid catalyst, it appeared that FMN was hydrolyzed to riboflavin and compound C to FMN.
Partial acid hydrolysis of FMN and C at 100C in 2 N
HCl, in which riboflavin remains relatively stable, revealed that the rate of conversion of C to FMN exceeded
the rate of conversion of FMN to riboflavin (Table 3).
These findings confirmed a D-ribityl side chain and suggested the presence of a polyphosphate
moiety on the
terminal
5-position of C, since the phosphoric anhydride bond(s) of a polyphosphate
group would be expected to exhibit a higher degree of acid lability than a
phosphoric ester bond (19).
Taken together, the results from uv-visible spectral
analysis, photolysis,
periodate oxidation,
peracetylation, and acid hydrolysis of compound C, as well as its
fluorescence and chromatographic
behavior, indicated
that the structure of the unknown flavin was identical to
that of FMN, save for the presence of an acid labile
substituent group, most likely a pyrophosphate,
at the
5-position of the N-10 ribityl side chain.
31P NMR data provided unequivocal evidence for the
presence of the pyrophosphate
moiety in compound C.
As shown in Fig. 2, the 31P NMR spectrum showed an
upfield AB splitting pattern consistent with a pyrophosphate moiety with magnetically nonequivalent
phosphorus atoms. The chemical shift values for the most intense peaks (-9.3 and -10.3 ppm) were somewhat lower
field than those observed for FAD (-10.8 and -11.3
ppm) (20). This difference is probably a result of the
known stacking
of the adenine and flavin rings of
FAD in aqueous solution (21,22). The NMR data also
distinguished
riboflavin pyrophosphate
from riboflavin
diphosphates, since the latter would be expected to exhibit 31P NMR signals downfield from H,PO, and would
also exhibit a pH-dependent
chemical shift.
The chromatograph
obtained
from reverse-phase
HPLC analysis of the acid hydrolysis products from
OF RIBOFLAVIN
5-PYROPHOSPHATE
-7
-9
-11
13
PPM
FIG. 2.
phate).
of Riboflavin
Preliminary
results indicated that compound C, now
identified as riboflavin 5-pyrophosphate,
was a biologically active compound in that it restored the catalytic
activity of D-amino-acid apooxidase. To evaluate the efficiency of the flavin analog as a cofactor for the apoprotein compared with that of the natural coenzyme,
FAD, this apooxidase (5 pug) was incubated with FAD
(lo-100 nM) or riboflavin 5-pyrophosphate
(0.275-2.75
pM) in 900 ~1 of sodium pyrophosphate
buffer, pH 8.5,
for 10 min at 37C. Immediately
following the addition
of 100 ~1 of D-phenylglycine
solution (40 mM) to the
reaction, the activity of the enzyme was measured as
described under Materials and Methods. Similarly, the
efficiency of riboflavin 5-pyrophosphate
as a cofactor
for glucose oxidase was evaluated by incubation of glucose apooxidase (2 pg) with FAD (2.5-10 nM) or riboflavin 5-pyrophosphate
(50-250 nM) in 1 ml of 0.1 M potassium phosphate buffer, pH 7.0, containing 0.1% bovine
serum allumin for 16 h at room temperature.
Following
preincubation
of the apoenzyme with cofactor, activity
assays were performed as described under Materials
and Methods. The data obtained from these experiments were analyzed using Lineweaver-Burk
plots to
determine K,,, and V,,, values for each case. As shown in
Table 4, riboflavin 5-pyrophosphate
behaves as a weak
354
HARTMAN,
TABLE
EDMONDSON,
Compound
D-Amino-acid
apooxidase
FAD
RPP
Km
Vmax efficiency(%I*
Relative
activity
0.47
8.4
PM
FM
0.28
0.10
100
6
100
35
0.09
100
9
100
23
Glucoseapooxidase
FAD
RPP
AND MCCORMICK
45
nM
474nM
0.02
SPECTROSCOPIC
STRUCTURE/FUNCTION
STUDIES
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The authors
gratefully
acknowledge
the contribution
of Dr. Yukiko
Yamada,
who first purified
bovine liver FAD synthetase
to homogeneity. Insights
provided
from helpful
discussions
with Dr. Preetha
Ram
are also sincerely
appreciated.
Finally,
we thank Khaleelah
Muwwakkil for typing
this manuscript.
The work
was supported
by N.I.H.
Grants
DK 38940 and GM 29433.
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