ANALYTICAL

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

and Donald B. McCormick

Research Center, Emory

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.

FAD functions as a prosthetic group for numerous

flavoenzymes that catalyze metabolically
essential oxidation-reduction
reactions. For study of structure/
function relationships
within these proteins involving
interaction
of the coenzyme with the apoprotein,
it is
expedient to use highly purified FAD.
In biological systems, this coenzyme is synthesized
from FMN2 and ATP in a reaction catalyzed by FAD

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;

FMN and FAD were purchased from Sigma, Fluka,

and Boehringer-Mannheim.
ATP, dithiothreitol,
bovine serum albumin,
o-dianisidine,
peroxidase,
and
OGO3-2697/92

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

the absorbance at 254 nm. Pools corresponding to peaks

of absorbance were collected manually, concentrated to
dryness by rotary evaporation
at 4OC, and stored in
darkness at -20C.
UV-Visible

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

FMN used to assay FAD synthetase activity during

purification
of the enzyme was partially
purified by
DEAE-Sepharose
chromatography
essentially according to the procedure published by Moffatt and Khorana
(8) except that following the elution of riboflavin from
the column with distilled water, a gradient of lithium
chloride from 0.25 to 0.75 M was employed for separation of riboflavin monophosphates
from riboflavin 4,5cyclophosphate and riboflavin diphosphates. After elution of the shoulder enriched with 4-FMN,
which
precedes the FMN peak (9), the remainder of the peak
was collected. This dilute FMN pool was concentrated
by rotary evaporation to a final concentration
of 5 mM
and frozen in aliquots at -20C until used. FMN for
determination
of kinetic constants and for structural
analysis was further purified by reverse-phase HPLC
according to the method of Nielson et al. (9) to remove
contaminating
3-FMN
and 4-FMN. This procedure
improved the purity of 5-FMN obtained from DEAESepharose chromatography
from 89 to 99%.
Purification of Commercial FAD and Isolation
of Riboflavin 5-Pyrophosphate

FAD was purified and riboflavin

5-pyrophosphate
isolated from commercial FAD preparations by reversephase HPLC using a Shimadzu system composed of a
LPM-600 low pressure mixing proportioning
valve, LC
600 solvent delivery module, Rheodyne Model 7125 syringe loading sample injector, SPD-GA ultraviolet
spectrophotometric
detector, and a CR501 chromatopac recording integrator.
The system was equipped with a
Brown Lee RP 18 New Guard 7-pm guard column (1.5 x
3.2 mm) and a C,, Dynamax Macro HPLC column (21.4
X 250 mm). Fractionations
were performed using a linear gradient of 5 mM ammonium
acetate, pH 6.0, to 70%
methanol developed over 70 min at a flow rate of 5.0
ml/min after washing the sample into the column for 5
min with 5 mM ammonium
acetate, pH 6.0. Elution of
sample components from the column was detected by

Flavin solutions (50 pM in sodium phosphate buffer,

pH 7.0) were pipetted into quartz cuvettes and placed
approximately
10 cm from a Model UVGL-25 Mineralight. The solutions were irradiated at 366 or 254 nm
during aerobic incubation
at 22C for 24 h. Photodecomposition of the flavin compounds was monitored by
their decreasing absorbance at 445 nm. Aliquots of each
solution were taken at 30-min intervals and applied to
cellulose TLC plates for identification
of photolysis
products.
Periodate Oxidation

Solutions containing 7.7 pM flavin and 770 pM sodium

periodate were incubated at room temperature
in the
dark. After 3.5 h, aliquots of each solution were applied
to cellulose TLC plates for identification
of oxidation
products.
Peracetylation

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

Samples containing 25 nmol of flavin were prepared

as previously described and resuspended in 50 ~1 of 2 N
hydrochloric acid. The samples were incubated in a boil-

350

HARTMAN,

EDMONDSON,

ing water bath for 4 h. Aliquots taken at 60-min intervals were immediately
applied to cellulose TLC plates.

AND

MCCORMICK

the difference in properties

the bovine enzyme.3

between the rat enzyme and

Step 1: Homogenization and centrifugation.

Partially
thawed
bovine
liver
(about
2
kg)
was
minced
and
homog31P NMR Spectral Analysis
enized in 4 vol of cold 20 mM potassium phosphate
31P NMR spectra were measured at 81 MHz on an buffer, pH 5.5, containing 1 mM dithiothreitol,
0.05 mM
IBM/Bruker
WP 200 SP spectrometer
using lo-mm
EDTA, and 0.1 mM phenylmethylsulfonyl
fluoride usNMR tubes (Wilmad).
Riboflavin
5-pyrophosphate
ing a Waring commercial blender at low speed. Homoge(compound C) was 3 mM in distilled water. Chemical
nates were centrifuged at 12,000 rpm for 30 min at 4C
shift values are relative to an external standard of 85% using a Sorvall GSA rotor, and the supernatant was filphosphoric
acid. Spectral parameters
included: 1 W tered through four layers of cheesecloth.
broad-band proton decoupling, 4000 Hz spectral width,
Step 2: Ammonium sulfate fractionation.
The superand a 30 flip angle. Free induction decays were sub- natant was brought to 50% saturation by addition of
jected to a 3 Hz exponential
line broadening prior to finely ground ammonium
sulfate over 30 min with conFourier transformation;
50,000 acquisitions were accu- tinuous stirring. The resulting precipitate was collected
mulated.
by centrifugation
at 12,000 rpm for 30 min at 4C using
a Sorvall GSA rotor. After decanting the supernatants,
the pellets were stored at -20C overnight. For removal
Preparation of D-Amino-Acid Apooxidase
of ammonium
sulfate, the protein pellets were resus2 liters of the homogenizing
D-Amino-acid
apooxidase was prepared by dialysis of pended in approximately
buffer, pH 6.0, dialyzed (Spectra/Par
7 dialysis memoxidase against a solution of 0.1 M sodium pyrophosbrane, J4, CO: 25,000) for 8 h against 20 liters of the
phate, pH 8.5, containing 3 mM EDTA and 1 M potassame buffer with two changes, and then further dialyzed
sium bromide, followed by dialysis against 65 mM soovernight
against 20 liters of the homogenizing
buffer,
dium
pyrophosphate
buffer, pH 8.5, to remove
pH
7.5.
A
large
amount
of
contaminating
protein
is prepotassium bromide, according to the procedure pubcipitated
by
dialysis
at
pH
6.0.
This
does
not
renature
lished by Massey and Curti (10).
when the pH of the dialysis buffer is increased to 7.5.
The dialysate was then centrifuged at 12,000 rpm for 30
Enzyme Activity Assays
min at 4C using a Sorvall GSA rotor to remove precipitated
protein, and the supernatant filtered through four
The activity of D-amino-acid oxidase [EC 1.4.3.31 was
layers
of cheesecloth.
determined by measuring the increase in absorbance at
Step
3: DEAE-Sepharose chromatography.
Fast flow
252 nm resulting from the conversion of D-phenylglyDEAE-Sepharose
(1.5
liters)
equilibrated
with
the hotine to benzoyl formate at 37C as described by Fonda
mogenizing
buffer,
pH
7.5,
was
poured
into
a
3-liter,
and Anderson (11) at an enzyme concentration
of 5 1.181
course,
fritted-glass
funnel
and
the
buffer
was
removed
ml. The reaction velocity of glucose oxidase [EC 1.1.3.41
The partially dehywas measured by the increase in absorbance at 460 nm from the gel by vacuum filtration.
drated
gel
was
transferred
to
a
4-liter
beaker and immeresulting from the oxidation of o-dianisidine
in the perdiately
combined
with
the
dialysate
supernatant.
The
oxidase-coupled
reaction described by Worthington
(12) at an enzyme concentration
of 35 rig/ml. FAD syn- resulting suspension was stirred for 30 min using an
Eastern overhead stirrer and then transferred back into
thetase activity was assayed using the two-step procedure described by Oka and McCormick,
in which the the filter funnel. After removal of the buffer by gravity
was repeatedly (four to
synthetase is first incubated with FMN and ATP sub- filtration, the DEAE-Sepharose
strates, and the concentration
of FAD formed is subse- six times) combined with 2 liters of cold homogenizing
buffer, pH 7.5, stirred for 10 min, and filtered by gravity
quently quantitated
by conversion of D-amino-acid
until the absorbance of the filtrate at 280 nm no longer
apooxidase to its active holoenzyme form (2), except
decreased
significantly.
The gel was washed in the same
that assays were performed at pH 6.1 using 100 mM
manner
with
lo-15
liters
of homogenizing
buffer, pH
Pipes buffer rather than 50 mM potassium phosphate
7.5,
containing
0.1
M
sodium
chloride.
Suspended
in this
buffer, pH 7.1, and MgCl, was substituted for MgSO,.
same solution, the gel was poured into a 5 X loo-cm
column and further washed (2 to 3 liters) at a flow rate
Purification of FAD Synthetase from Bovine Liver
of 3 ml/min until no significant decrease in the absor-

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

bance of the eluate was observed using an Isco V4 uv-vis

absorbance detector at a sensitivity
of 0.05. For elution
of FAD synthetase, the column was washed with homogenizing buffer, pH 7.5, containing 0.2 M sodium chloride
(about 4 liters), and 20-ml fractions were collected using
an Isco Foxy fraction collector. The FAD synthetase
activity (fractions
39-78) was pooled, and after the addition of dithiothreitol
(0.15 g/liter), the enzyme solution was concentrated
to a volume of about 150 ml using
an Amicon stirred ultrafiltration
cell equipped with a
PM 30 Diaflo membrane (Amicon).
Step 4: Sepharose CL-6B gel filtration.
The enzyme
solution was applied to a Sepharose CL-6B column (5 X
130 cm) previously
equilibrated
in 50 InM Pipes buffer,
pH 6.1, containing
1 mM dithiothreitol
and 0.05 mM
EDTA. The enzyme was eluted with the same buffer
(about 4 liters) at a flow rate of 2 ml/min, and 20-ml
fractions were collected. The elution of protein from the
column was detected as described for DEAE-Sepharose
chromatography.
FAD synthetase
activity
(fractions
66-80) was pooled and after the addition of dithiothreito1 (0.15 g/liter), the enzyme solution was concentrated
to a volume of about 100 ml by ultrafiltration
as previously described.
Step 5: FMN-Sepharose
affinity
chromatography.
The concentrated
enzyme was applied to an FMN-Sepharose affinity column (2.5 X 40 cm) previously equilibrated in 50 InM Pipes (pH 6.1) containing
1 mM dithiothreitol and 0.05 mM EDTA. The column was washed
with the same buffer solution containing
0.3 M sodium
chloride (- 1 liter) at a flow rate of 0.7 ml/min until no
further
decrease in the absorbance
of the eluate was
detected at a sensitivity
of 2.0. To remove the remaining
nonspecifically
bound protein, the column was washed
with about 500 ml of buffer containing
0.3 M sodium
chloride until absorbance
of the eluate no longer decreased at a sensitivity
of 0.01. FAD synthetase
was
eluted by washing the column with the solution (-1
liter) of 50 mM Pipes, pH 6.1, containing
0.05 mM
EDTA, 1 mM dithiothreitol,
and 400 PM FMN. Fractions of 7 ml were collected. FAD synthetase
activity
(fractions
42-144 after start of FMN) was pooled, and
after the addition of dithiothreitol(O.15
g/liter), the enzyme solution was concentrated
to 0.4 U/ml by ultrafiltration as previously
described. Aliquots of the concentrated
enzyme were frozen
at -20C
to preserve
activity.
Typically these preparations
yielded 0.5 to 1 mg of
FAD synthetase
having a specific activity of 200-250
U/mg. The homogenity
of the protein was verified by
SDS-polyacrylamide
electrophoresis
as described
by
Laemmli
(14). Samples were electrophoresed
in 10%
isocratic polyacrylamide
gels with a 3% stacking gel using a Hoeffer Mightly
Small mini-gel electrophoresis
unit. Protein concentrations
were determined by a dye-

351

5-PYROPHOSPHATE

20

40

60

60

MINUTES
FIG.
1. Reverse-phase
mercial
FAD.

HLPC

separation

of compounds

binding method according to Bradford

vine serum albumin as standard.
Synthesis

in com-

(15), using bo-

of FMN-Sepharose

FMN-Sepharose synthesis was performed using the

modified method of Kazarinoff et al. (16) described by
Bowers-Komro et al. (13) except that CH Sepharose 4B
was prepared by cyanogen bromide coupling of 6amino-n-hexanoic acid to Sepharose 4B, according to
the procedure published by Cuatrecasas et al. (17)
rather than purchased. The ligand concentration of the
affinity gel was 1.2 pmol FMN/ml sepharose.
RESULTS

Determination of the Composition of Commercial FAD

Preparations
Commercial preparations of synthetic FAD from
Fluka, Boehringer-Mannheim,
and Sigma were fractionated by reverse-phase HPLC as describedunder Materials and Methods. Elution profiles for FAD from
Fluka and Boehringer-Mannheim
exhibited five peaks
of absorbance, while those for Sigma FAD exhibited six.
A typical pattern with 25 pmol of commercial FAD is
shown in Fig. 1. As shown in Table 1, each of the FAD
preparations analyzed was about 94% pure and contained similar amounts of four minor contaminants.
After rechromatography of compounds C, D, and E to
ensure their purity, absorption spectra of the five compounds isolated from commercial FAD were obtained.
Compounds eluting earlier than B exhibited two peaks
of absorbance at 214 and 262 nm, whereas the spectra of

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.

compounds B, C, D, and E consisted of the four maxima

of absorbance (m-225,270,375,450
nm) indicative of the
flavin isoalloxazine
structure. When these compounds
and appropriate standards were subjected to analysis by
TLC (Table 2), compounds B, C, D, and E also displayed the greenish yellow fluorescence typical of flavins under near-uv light. Compound A was not detected.
On the basis of its relative abundance, retention time,
mobility on TLC plates, and capacity to reactivate Dammo-acid apooxidase, D was identified as FAD. Compound E did not significantly
restore activity of the
apooxidase and exhibited
chromatographic
behavior
similar to that of FMN. Of considerable interest was the
finding that compound C could function as a cofactor
for this apoenzyme, albeit not as efficiently as FAD.
Thus, further studies were undertaken to determine the

R, Values

of Flavins on Cellulose Thin-Layer

Chromatography
Plates

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

Principal photolysis product (lumichrome)

from flavins
Riboflavin, 5-FMN, or RPP
Principal periodate oxidation product
(lo-formylmethylflavin)
from flavins
Riboflavin, 5-FMN, or RPP
Peracetylation products of flavins
Riboflavin
5-FMN
RPP
Acid hydrolysis products of flavins
5-FMN
RPP

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 C and to more fully evaluate its biological

Spectral and Chemical Characterization

5-Pyrophosphate (Compound C)

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).

31P NMR spectrum of compound C (riboflavin 5-pyrophos-

compound C, by the method of Nielson et al. (9) that

resolves 4-FMN from 5-FMN, exhibited 3 major peaks
eluting at the retention times of C, 5-FMN, and riboflavin. This result established the position of the pyrophosphate moiety at the 5-carbon of the ribityl chain
and confirmed the identity of the unknown flavin analog as riboflavin 5-pyrophosphate.
Evaluation of the Biological Activity
5-Pyrophosphute

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,

Reactivation of the Apoproteins of D-amino-acid Oxidase

and Glucose Oxidase by FAD and RPP
Relative K,,,

Compound
D-Amino-acid
apooxidase
FAD
RPP

Km

Vmax efficiency(%I*

Relative
activity

FMN was fixed at 0.5 mM while concentrations

of ATP
were varied between 5 and 100 PM. Though inhibition
was observed at 20 and 30 pM riboflavin 5-pyrophosphate, the type was unclear from Lineweaver-Burk
plots of the data; hence, a Ki value was not calculated.
DISCUSSION

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

Change in absorbance per minute.

* 100 + K,,, (RPP)IK,,, (FAD).
V,, (RPP)/V,,
(FAD).

cofactor for both FAD-requiring

enzymes. Comparison
of Km and V,, values for FAD and riboflavin 5-pyrophosphate indicates that both enzymes have a lower affinity for the flavin analog than for the natural cofactor,
and that the catalytic efficiency of the enzymes reconstituted with riboflavin 5-pyrophosphate
is decreased
relative to that of the enzymes reconstituted with FAD.
Given that FAD has been shown to be a strong product inhibitor of the reaction catalyzed by FAD synthetase, with Ki values of 0.75 pM against FMN and 1.3 pM
against ATP (23), and that riboflavin 5-pyrophosphate
protected this enzyme against modification
of active
site residues by arginyl reagents (24), it was of interest
to determine whether this compound could function as a
substrate or product inhibitor. Because the enzyme isolated from bovine liver differs in some respects from the
rat liver enzyme, K,,, values for the reaction substrates
were determined. Activity assays were performed as described under Materials
and Methods except that for
determination
of the K,,, for ATP, the concentration
of
FMN was fixed at 0.5 mM and concentrations
of ATP
varied between 5 and 100 pM. The Km for FMN was
determined with the ATP concentration
fixed at 0.5 mM
and concentrations
of FMN ranging from 7.5 to 200 pM.
Lineweaver-Burk
plots were used for data analysis and
Km values of 41 pM for ATP and 5.5 PM for FMN were
obtained. These results are consistent with those reported by Oka and McCormick
(2) and Bowers-Komro
et al. (13) for the rat liver enzyme. Using the same conditions as described for determination
of the K,,, for
FMN and fixed concentrations
of riboflavin
5-pyrophosphate ranging from 10 to 30 pM, the Ki for the flavin
analog against FMN was determined to be 9 pM and the
type of inhibition
competitive. An attempt was made to
determine
the Ki for riboflavin
5-pyrophosphate
against ATP using the same conditions as described for
the determination
of the Ki for riboflavin 5-pyrophosphate against FMN, except that the concentration
of

The results of this study show that presently available commercial

preparations
of synthetic FAD are
about 94% pure with the remaining 6% being composed
of four or five minor contaminants
that can be separated from FAD by a modification
of the HPLC procedure described by Light et al. (5) for separation of flavin
compounds.
FAD purified in this manner has been
shown to be 100% pure by rechromatography.
One of the four contaminants,
compound C (cf. Fig.
l), was identified as riboflavin 5-pyrophosphate.
This
flavin is of particular interest as it has now been shown
to exhibit biological activity as a weak cofactor for FADrequiring enzymes (cf. Table 4), one of which, viz. Damino-acid oxidase, is commonly used to assay FAD.
The finding that the binding efficiency for riboflavin
5-pyrophosphate
relative to the binding efficiency for
FAD is only 6% in the case of D-amino-acid
oxidase, or
9% in the case of glucose oxidase, supports previously
published
results indicating
the importance
of the
adenyl moiety of FAD for binding to these apoproteins
(26-26). However, since 35% of the catalytic activity of
the D-amino-acid
oxidase apoprotein
and 23% of the
activity of glucose apooxidase is restored by their reconstitution
with riboflavin 5-pyrophosphate,
it appears
that the adenyl moiety does not participate in the catalytic mechanisms of these enzymes but rather aids in
binding of the FAD cofactor and in maintaining
optimal
protein conformation
for catalysis and/or substrate
binding to occur. Further, reactivation of these apoproteins by riboflavin 5-pyrophosphate
establishes the requirement for binding of the pyrophosphate
moiety of
FAD for effective catalysis, since similar restoration of
activity by FMN does not occur (26,28). It has been
shown that binding of FAD to these apoenzymes proceeds in two stages. The first involves the rapid binding
of FAD to the apoenzyme, and the second is characterized by slow conformational
changes which correlate
with the appearance of catalytic activity (10,28,29).
Thus, it is conceivable that in addition to its role in FAD
binding, the pyrophosphate
moiety functions to induce
rearrangement
of the protein into its more compact and
catalytically
active conformation
by bridging and neutralizing positively charged active-site residues. This
mechanism for conformational
change has also been established in the case of aspartate aminotransferase
in
which the carboxyl groups of dianionic substrates or
substrate analogs bind two active-site arginine residues,
thus inducing the closed conformation
of the enzyme
(30-38).

SPECTROSCOPIC

STRUCTURE/FUNCTION

STUDIES

Results of this study also show that riboflavin 5-pyrophosphate

exhibits biological activity as an inhibitor
of FAD synthetase.
Inhibition
was observed
against
both substrates
and a Ki of 9 PM against FMN calculated. From the data obtained, it is clear that riboflavin
5-pyrophosphate
effectively
competes
for the FMN
binding site. The lesser degree of inhibition
observed
against ATP likely reflects binding of riboflavin
5pyrophosphate
at the FMN site, resulting in an inactive
ternary complex. Alternatively,
inhibition against ATP
may result from competition
of the pyrophosphate
moiety of the flavin analog with ATP for positively
charged active-site residues such as arginine (24), thus
decreasing the binding efficiency of ATP, or from a conformational
change induced by binding of riboflavin 5pyrophosphate,
which renders the ATP site less accessible to the substrate.
Further
studies are needed to
distinguish
between these possibilities.
However,
from
the data at hand it is clear that riboflavin 5-pyrophosphate will prove to be of value in elucidating the catalytic mechanism
of this enzyme and in the study of
structure/function
relationships
within flavoproteins.
ACKNOWLEDGMENTS

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RIBOFLAVIN

15. Bradford,

26.

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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