ANALYTICAL

BIOCHEMISTRY

53,332-336

(1973)

A Rapid Micromethod
for Determination
of FMN and FAD in Mixtures

A rapid fluorometric method for the determination

of FMN and FAD
concentrations in mixtures of the two compounds is described. The method
is appIicabIe to measurement of ffavin concentrations in the nanomolar
range. It can be readily used for determination
of the flavin composition
of purified flavoproteins.

Flavin
adenine dinucleotide
(FAD)
and riboflavin-5-phosphate
(FMN) are the two prosthetic groups commonly found in flavoproteins.
Mixtures of FMN and FAD are generally found in all tissues, in subcellular organelles, and occasionally even in the same purified protein (14).
Currently available assays to determine (1) whether the prosthetic group
of a flavoprotein is FMN, FAD, or both, and (2) the flavocoenzyme
content, are time-consuming,
generally involving hydrolysis of FAD to
FMN (5)) conversion to lumiflavin (6)) or physical separation of the two
flavins (7), with estimation of the FAD content by the differences in
fluorescence observed with untreated and treated aliquots of the original
flavin solution. The method described in this communication
provides a
rapid quantitative
assay for FAD and FMN in mixtures of the two compounds. The method is based on the markedly different behavior of
FMN and FAD when the fluorescence of each compound is examined as
a function of pH (8,9). The procedure utilizes only reagents which are
commonly available. The sensitivity of the assay is dependent upon the
fluorometer used, but we have easily measured concentrations of FAD
and FMN in mixtures containing a total flavin concentration of 5 X
1O-DM. Typically, FMN and FAD concentrations in the UP8 M range are
used.
FMN was purchased from Sigma and purified by chromatography
on
DEAE-cellulose
according to the procedure of Swoboda and Massey
(10). FAD was purchased from Schwarz/Mann and reported to be 97.6%
pure. Each compound was analyzed by thin-layer
chromatography
on
silica gel G with a pyridine: acetic acid: water (19:2: 79) development
system (11). The FMN sample showed a single fluorescent spot, while
the FAD showed a single spot with slight tailing. The chromatographic
system was such that it could be established with certainty that the
FMN sample was not significantly
contaminated
with either FAD or
332
Copyright @ 1973 by Academic Press, Inc.
All rights of reproduction in any form reserved.

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333

riboflavin, while the FAD sample was free of FMN; slight contamination
of the FAD with riboflavin could not be ruled out. Analysis of the FAD
sample by the method of Bessey et al. (8)) indicated that it contained
less than 2% FMN + riboflavin, using the fluorescence ratios reported by
these authors for the experimental conditions defined by them.
Escherichia c&i NADPH-sulfite
reductase was prepared by the method
of Siegel et al. (12). The preparation appeared to be homogeneous by
disc-gel electrophoresis, immunoelectrophoresis,
and sedimentation equilibrium techniques (12). Milk xanthine oxidase and chicken liver xanthine
dehydrogenase (both at least 90% pure by disc-gel electrophoresis) were
kindly provided by Dr. K. V. Rajagopalan of Duke University.
Unless otherwise indicated, all solutions were prepared with the following standard buffer: 0.1 M potassium phosphate, pH 7.70, containing
0.1 mM EDTA. Stock solutions of 1 X 10m5M FAD and FMN in standard buffer were prepared fresh daily, and were diluted to the appropriate
flavin concentration with standard buffer shortly before assay. Flavin
concentrations in the stock solutions were determined by measuring absorbance at 450 nm and utilizing reported extinction coefficients (1.22 X
lo4 M-l cm-l for FMN, and 1.13 X W4 M-l cm-l for FAD) (13,14). All
flavin solutions were protected from light by storage in brown glassware
or tubes covered with aluminum foil.
Protein samples were diluted with standard buffer so that the final
flavin concentration was 1 to 5 X 19* M in each sample. Aliquots of
3.0 ml for each sample were placed in aluminum foil-covered Corex centrifuge tubes, immersed in a boiling water bath for 3 min to remove
protein from the flavin, and cooled rapidly. Tubes were centrifuged at
20,OOOg for 10 min to remove denatured protein. From each sample, a
2-ml aliquot was transferred to a l-cm pathlength fluorometer cuvette.
Fluorescence emission was recorded at 535 nm; the exciting wavelength
was 450 nm. The temperature of the samples in the fluorometer was
maintained
at 23 rtr 1. After measurement of the fluorescence of the
sample in pH 7.7 buffer, 0.2 ml of l.ON HCl were added to the cuvette
(bringing the pH of the soIution down to 2.6), and the fluorescence determined again. This procedure was repeated on all samples and standards.
In dilute solutions, in which absorption of exciting light is negligible,
i.e., 10e7M or less for flavins, the fluorescence intensity of the solution is
proportional to the concentration [c] of the fluorescing species
F = k[cj,

(1)

where the constant lc applies only for a given set of experimental conditions. For mixtures of FMN and FAD, the fluorescence of dilute solutions is the sum of the fluorescence intensities for each compound:

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F = N[FMN]

+ D[FAD]

(2)
where N, the fluorescence const,ant for FMN,
and D, the fluorescence
constant for FAD, are functions of solution pH. If the fluorescence intensity of a given mixture of FMN and FAD is measured at pH 7.7 and
pH 2.6, as indicated in the previous paragraph, then

FY., = N,.dFMNl + DT.~FADI

(3)
F 2.6 = NM[FMN]
+ he[FAD].
The values of the constants LV~.~, D;.;, N,,,, and D,.,, can be determined

and

by measurement of standard flavin solutions. With measurements

of the
fluorescence intensity of the solution at the two pH values, and values
for the constants of Eq. (3)) the concentrations
of FAD and FMN in the
original mixture can be readily calculated.
Table 1 shows the relative fluorescence measurements
obtained for a
series of FMN
and FAD standard solutions. From these results, the
fluorescence constants reported in Table 2 can be calculated. The values
reported here were obtained with a Turner fluorometer
and could be
reproduced from week to week with great accuracy; the constants will
differ, of course, if a different instrument is used, or if measurements
are
made at a different temperature
than 23C. The results of fluorescence
measurements
on mixtures of FMN and FAD, the enzymes NADPHsulfite reductase
(an enzyme which contains equimolar quantities
of
FMN and FAD (12))) xanthine oxidase, and xanthine dehydrogenase
Relative

TABLE
1
Fluorescence
of Flavin

Standardsa

Relative
FAD
(nM)

FMN
Wf)

pH

56.8
28.5
14.3
0
0
0

0
0
0
78.3
39.2
19.6

0.0727
0.0353
0.0181
1.000
0.500
0.247

7.7

fluorescence
pH 2.6
0.323
0.163
0.0842
0.690
0.341
0.170

a Fluorescence
intensities
were determined
at 23 on a Turner
Spectra
210 spectrophotofluorometer
relative
to settings
of unit sensitivity,
emission
slit width
25 nm,
excitation
slit width
10 nm, and energy meter reading
of 54. Excitation
wavelength
was
450 nm, and emission
was measured
at 535 nm. Flavin
concentrations
were calculated
from the known
extinction
coefficients
of FMN
and FAD
at 450 nm (13,14).
Solutions
were in 0.1 M potassium
phosphate
buffer,
pH 7.70, containing
0.1 mM EDTA.
Each
measurement
represents
an average
of three independent
determinations.

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TABLE
2
Fluorescence
Constants
Measured
value
(IZ~-~ cm-r)

Constant

N7.7

1.27
1.26
8.76
5.79

07.7
N2.8
D2.6

(1 Fluorescence
constants
for FMN
and FAD (for
from the data in Table 1. Errors
represent
standard

*
+
+
+

.Ol
.02
.12
.07

x 107
X lo6
x lo6
x

definitions
deviations

106

see text) were determined

from the mean.

(FAD-containing
enzymes (15,16)), are shown in Table 3. The method
is rapid, taking only a few minutes after extraction of the flavin from the
protein and, as can be seen from the table, reasonably accurate. The
method is valid, of course, only if other methods, e.g., chromatography,
have limited the choice of flavins to FMN and FAD, since compounds
such as riboflavin, not normally found in flavoproteins, would mimic
FMN in this procedure (8). The method is not useful for covalently
bound flavin prosthetic groups. Weiner and Heppel (17) have recently
applied the procedure to identification
of FAD as the prosthetic group
of a bacterial glycerol-3-phosphate
dehydrogenase. Use of the technique
for measurement of residual flavin composition of xanthine dehydrogenase
and sulfite reductase enzymes treated to specifically remove one type of
nonequivalent
flavin prosthetic group has also been described (16,18).
Assay

Results

TABLE
for Enzymes

3
and Flavin

Mixturesa

Actual

Flavin

source

NADPH-sulfite
reductase
Xanthine
oxidase
Xanthine
dehydrogenase
Mixture:
FMN,
FAD
Mixture:
FMN,
FAD
Mixture:
FMN,
FAD

Enzyme
Wf)
6.1
9.3
9.6

FMN
(nM)
24
0
0
20
39
78

Measured
FAD
(n.W
24
19
19
57
28
14

FMN
bM)
24
1
1
21
40
81

FAD
(nM)
24
17
17
57
2x
17

a Fluorescence
measurements
were performed
as described
in the text, and the data
treated
with the fluorescence
constants
of Table 2. Concentrations
of the enzymes
and
standard
flavins were calculated
from the known
extinction
coefficients
for these materials (12-16).
All solutions
were in 0.1 M potassium
phosphate,
pH 7.70, containing
0.1 mM EDTA.

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ACKNOWLEDGMENTS
The work reported in this paper was supported in part by Grant AM-13469 from
the National Institutes
of Health. The authors thank Patricia Davis for able
technical assistance.
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EDWAFSD J. FAEDEB
LEWIS
M. bxmr~

Department
of Biochemistry
Duke
University
Medical
Center and the
Veterans
Administration
Hospital
Durham, North
Carolina
17706
Received
November
7, 1979; accepted
January

8,197S