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Pure crystalline flavine adenine dinucleotide
ARCHIVES
OF
BIOCHEMISTRY
AND
Pure Crystalline
BIOPHYSICS
76, 214-224 (1968)
Flavine Adenine
Dinucleotide
Paolo Cerletti and Noris Siliprandi From the Institute of Biological Chemistry of the University of Rome and National Research Council Unit on Enzyme Studiea, Rome, Italy Received January 18, 1958 INTRODUCTION
In recent years new interest has arisen in flavine adenine dinucleotide (FAD), and several workers (l-7) have attempted to obtain highly purified preparations of FAD by simpler methods than were first described by Warburg and Christian (8). The later stages of Warburg’s method, for which considerable skill is required, have been substituted by chromatographic procedures, and degrees of purity ranging from 60 to 92% have been obtained. Siliprandi and Bianchi (9) described a procedure based on cellulose-column electrophoresis and ion-exchange chromatography by which about 98% pure FAD is obtained. In subsequent work reported in the present paper, this method has been simplified, and absolutely pure FAD has been obtained and crystallized. EXPERIMENTAL
Methods Paper and column electrophoreaia procedures previously described (10, 11) were followed. The column must be repacked with new cellulose powder every 5-10 electrophoresis runs. Ion-Exchange Chromatography. Carboxylic acid exchanger (Amberlite IRC-50, 129-300 mesh) in the hydrogen form, packed in a 5 X 75 cm. column was used. Spectrophotometric measurements were made in a Beckman DU spectrophotometer, in 0.1 M phosphate pH 6.89 and phosphate-acetate-borate pH 2.01 and 12.28 buffers. For determining the absorption spectra, readings were made every 2 w in the regions of the maxima and every 5 ma elsewhere. The molecular extinction coefficient (c) of FAD at 450 rnN was assumed to be 11.3 X lo” mole-r cm.-’ (5). During the purification procedures a value of 3.28 was taken as representing the 214
FLAVINE
ADENINE
215
DINUCLEOTIDE
ratio between the extinction coefficients of pure FAD at 260 and 450 rmc (&a,~/&. Fluorimetric measurements were made in a Klett fluorimeter : lamp filter Corning glass 5543 and photocell filter Corning 3335. The pH range from 1.5 to 11.6 was covered with the following buffers: glycine pH from 1.5 to 3.5 and from 10 to 11.6; acetate from 4 to 5.5; phosphate from 6 to 7; borate from 8 to 9. &enzyme Activity. The activity of FAD as coenzyme for n-amino acid oxidase was determined according to Warburg and Christian (8) using an apoenayme prepared from pig kidney according to DeLuca et al. (12).
Preparation of FAD A. Five kilograms Titan0 baker’s yeast is extracted by the method previously described (9) as far as the precipitation of the crude Ag salt of FAD. B. The Ag precipitate is washed two times with 10 ml. of water, resuspended in 100 ml. of water, and dissolved by adding a slight excess of saturated KCl; the solution is brought to pH 3 by addition of 1 N HNOs and centrifuged. The precipitate is washed three times with 5 ml. water, the washings are combined to the clear brownish supernatant (final solution = 115 ml.) and brought to pH 4.9 by addition of solid NaHCOa . Analysis by paper electrophoresis of this solution shows a large yellow fluorescent spot corresponding to FAD, a smaller spot corresponding to flavine mononucleotide (FMN), and two spots with blue fluorescence due to unidentified compounds. The purity of this extract as determined by applying the figure of R260,,r0 found to the value of R260,460of pure FAD, does not exceed 8yo (see Table I). C. The solution obtained in B is concentrated in vacua at 30°C. to a volume of about 35 ml. and submitted to column electrophoresis on normal columns (4 X 40 cm.) (10) connected in series in the circuit or on a “multiple column” (11) ; 5-ml. aliquots are applied on each segment. Before the column is assembled the substance is displaced 4-5 cm. down each segment by allowing a sufficient amount of buffer to flow through each segment; the original brown-yellow zone splits into a faster moving brownish red zone and a slower yellow zone containing FAD and FMN. The current is then applied, and the electrophoresis and elution are performed as previously described (11). The electrophoresis is run in the dark, and the temperature of the buffer in which the column is dipped must not exceed 30°C. Under the influence of the electric field, FAD is separated from FMN while the brownish red zone moves the fastest and is eluted first (Fig. 1, peak A). The elution curves are shown in Fig. 1. Four peaks of unknown ultraviolet-absorbing compounds are obtained, two of which partially overlap with the peak correspondTABLE I Purity Degree of FAD at Different Stages of Preparation Calculated from the Ratios of Light Absorption at 260 and ,460rnk (Rz~& R m,m found
B c D
41 6.2 3.276
R ta,w) of pure FAD R ,a,,~ found
0.079 0.523 1
216
CERLETTI
AND SILIPRANDI
O---O
? c v)
260 mp 450 mp
2 3.0b ‘0
2Q 2.5 0 2.0 -
v 1.5 -
a
l.O-
0.5-
A ,p-0. -P
o-%50
I 100
D
,? 150
‘P
-au-
P-4
200
lx4 250
300
ml effluent FIG. 1. Separation of crude FAD by cellulose powder electrophoresis from other unidentified substances from yeast. (Acetate buffer pH 5.1, r/2 = 0.05, 40 ma. for 14 hr.
ing to FAD. This fraction, when examined by paper electrophoresis, reveals the presence of FAD as the only flavine compound and of two other spots corresponding to unidentified ultraviolet-absorbing substances. The purity of the fraction containing FAD averages 52% with an increase of 6.5 times as compared to stage B (see Table I). D. The combined FAD fractions obtained from the different segments are concentrated in vacua at 32%. to a volume of about 10 ml. and then applied to the top of a column containing activated Amberlite IRC 50, and eluted with water, the flow rate being adjusted to 20 ml./hr. Six-milliliter fractions are collected, and the absorbancy is determined at 260 and 450 mp. The elution curves are shown in Fig. 2. A single yellow fluorescent fraction is obtained, corresponding to pure FAD. Two minor peaks corresponding to ultraviolet-absorbing compounds are eluted immediately before and after the FAD fraction and partially overlap it. The contaminated tubes of FAD can however be completely purified by submitting them again on the ion exchanger. Samples were analyzed by paper electrophoresis and no other ultravioletabsorbing or fluorescent spot could be detected, apart from that corresponding to FAD. The R.JM)I~value of 3.28 consistently corresponded to FAD pure with respect to other flavines or other nucleotides.
217
FLAVINE ADENINE DINUCLEOTIDE
o----O
260 mp 450 m/A
V
FAD ,**\ d
:
‘? \
i
/
,/ 300
\ ‘9 ‘\ \
Id’
I 350
400
‘b \
I -
450
s;a
ml eflluent
FIG.
2.
Ion-exchange separation of pure FAD (Amberlite
IRC-50). Flow rate
20 ml./hr. Yield. A weight of 12.8 mg. of pure FAD was isolated from 5 kg. baker’s yeast. This yield is slightly superior to that obtained by the method previously described and almost identical with the yield of FAD obtained by other workers (5, 8).
The substance is fairly stable in distilled water or in the buffer used for electrophoresis and can be kept for several days at 0°C. in the dark. In all stages of the purification procedure contact with light should be avoided, FAD being highly photosensitive. RESULTS
Absorption Spectra of FAD Figure 3 shows the absorption spectra of FAD at pH 2.01 (phosphate-acetate-borate buffer (13), pH 6.98 (phosphate buffer), and pH 12.29 (phosphate-acetate-borate buffer). Three peaks are evident throughout the pH range already tested.
218
CERLETTI
AND
SILIPRANDI ---__
I
250
300
350
400
450
5ooA Wovelengthimp!
FIQ. 3. Absorption spectra of FAD in 0.1 M phosphate buffer pH 6.98 and 0.1 M phosphate-acetate-borate buffer pH 2.01 and pH 12.29.
The spectra are almost identical in neutral or acid solution with maxima at 264, 375, and 450 rnp. At alkaline pH the maximum in the ultraviolet region is found at 270 rnp, and the peak in the region of 360-380 rnp is displaced toward shorter wavelengths, having its maximum at 357 mp. The extinction coefficent of this maximum has a higher value than the coefficent of the corresponding maxima at pH 2.01 and 6.98, and it can be calculated to be E = 11.5 X 103 in terms of the known value of e at 450 rnp (11.3 X 103). The ratios of light absorbtion at different pH and wavelengths are given in Table II. When the spectra of FMN and riboflavine pyrophosphate (RPP) are compared with those of FAD, it is remarkable that the absorbancy of this latter substance (FAD) steadily increases in the region below TABLE II Ratios of Light Absorption of Pure FAD at Differmt PH
2.01 6.98 11.29
Wavelengths and pH
Rt6WU.l
Rnqrso
R?70/4M
RW/4M
R676,4W
Raw/m
3.49 3.276 3.55
3.67 3.34 3.89
3.38 3.09 3.92
0.704 0.655 1.01
0.81 0.75 0.797
4.41 4.24 4.32
FLAVINE
ADENINE
219
DINUCLEOTIDE
240 rnp while that of FMN and that of RPP show a maximum at 224 rnp and decrease at shorter wavelengths. It is not thought that these differences are due to spectrophotometric errors as they depend upon readings obtained in the same instrument and can be attributed to the adenylic acid (AMP) moiety of FAD. The decrease in light absorption of FAD at 450 rnp by a reducing agent has also been studied. Addition to FAD in 0.1 M buffer of 0.1 vol. of 10 % Na&$04 in 5 % NaHC03 produces a decrease of 90.7 % of the absorption intensity at pH 2.01, 91.3% at pH 6.98, and 90.2% at pH 12.29. It should be taken into account, however, that the pH of the alkaline and acid samples, tested after the addition of dithionite, was not more than 1 pH unit nearer to neutrality. Fluorescence of FAD The effect of pH on the fluorescence of FAD was investigated in 0.01 M buffer in the pH range from 1.5 to 11.6. As shown in Fig. 4, the fluorescence has a maximum at pH 2.9, falls between pH 3 and 5.5, and then has a constant value up to pH 8.5. The fluorescent efficiency almost disappears at higher pH. Addition of 0.1 vol. of 10% NazSz04 in 5 % NaHC03 completely destroys the fluorescence throughout the pH range examined.
3
4
5
6
7
8
9
10
11
12
PH FIQ. 4. Fluorescence of FAD at different pH. fluorescence a8 compared to a riboflavine standard.
Ordinates
give
the
percent
220
CERLETTI
AND
SILIPRANDI
Moreover, the fluorescence of FAD before and after acid hydrolysis has been studied under conditions in which complete hydrolysis of FAD to riboflavine is stated to occur by previous authors (2, 8). All samples compared contained identical saline concentration and were measured in 0.1 or 0.01 M phosphate buffer 7.0. The fluorescence before hydrolysis was always found to be 18% as compared to that after hydrolysis. Biological Activity The activity of our purest preparations as coensyme for n-amino acid oxidase has been determined with several different apoenzyme preparations. As a reference we used a commercial sample (Wakamoto Pharm. Co., Tokyo) 90% pure; its electrophoretic and spectrophotometric analyses agreed for a high purity. As seen in Fig. 5, assuming as 90 % the activity of this sample, the activity of our pure FAD was 101%. The apparent dissociation constant for FAD-protein, KFl, was also determined and a value of 2.2 X 1OV mole/l. was found.
WA .---.
10
6
20
30
40
50
Time in min. FIG. 5. Activity as coenayme of D-amino acid oxidase of FAD prepared with the present method (A) as compared to a commercial sample of 90% purity (B).
FLAVINE
ADENINE
Crystallizatim
DINUCLEOTIDE
221
of FAD
Crystallization was obtained from a solution purified by several passages on ion exchanger and concentrated to a volume of about 5 ml. and then left in the dark at 04°C. on a crystallization dish until evaporation was nearly complete. When examined under the microscope (Leitz, Ortholux with Ultropak) in reflected light, bright yellow hexagonal crystals appeared, many of them in the form of aggregates. The smaller crystals were often closely grouped together like a bunch of grapes. DISCUSSION
As all purification procedures so far described for FAD, the present method is, with minor modification, the same as that of Warburg and Christian (8) in its initial stages. Of the many procedures devised to substitute the later stages of Warburg’s method, the combination of ion exchange and electrophoresis previously described (9) is among the simplest and for the purity of the product the best. In this method, the procedure is simplified and makes pure FAD readily available for laboratory work. The hindering factor represented by the limited capacity of column electrophoresis has been overcome by the use of “multiple columns.” The dissolved Ag precipitate is used for electrophoresis without further extraction, and the elution from the ion exchanger directly yields pure FAD in aqueous solution. Yagi et al. (14) recently described the separation of flavines by ion exchange on Dowex 1. In their paper it is not stated whether their method can also be applied to products of extraction nor whether FAD can be freed from other nucleotides. In any case, the final product is obtained in rather concentrated saline solution. The extraction stages as far as the precipitation of the Ag salt of FAD do not act on the biological activity of the coenzyme, as was already known from the work of previous authors. The electrophoretic and ion-exchange techniques followed for the further purification also do not alter the coenzymic activity. In fact, preparations that had repeatedly undergone electrophoresis and chromatography did not show loss of activity. The criteria of purity adopted in this work seem most direct and effective. Elementary analysis of FAD was not taken into consideration for testing the purity of our preparation since it is of no value in as-
222
CERLETTI
AND
SILIPRANDI
certaining whether the molecule of FAD is intact; in fact both FAD and an equimolar mixture of FMN and AMP give the same analytical figures. A more direct approach to the estimation of flavines at the different stages of the purification has been done by previous authors who used paper chromatography and electrophoresis. For reasons given in a former paper (lo), we preferred paper electrophoresis. This method allows a ready identification of very small amounts of flavines and reveals many other nonflavine fluorescent substances and compounds absorbing in the ultraviolet region of the spectrum. Fluorescent inpurities are moreover easily detected by measuring the decrease in fluorescence due to addition of dithionite, and eventually a reliable estimate of the amount of substances that absorb at 450 rnp is obtained by measuring the reduction in light absorption at this wavelength after addition of dithionite. When FAD is the only substance absorbing at 450 rnp, the value of I” .. .c 1... . .. -z_.. 73
224
CERLETTI
AND
SILIPRANDI
by chromatography on carboxylic resin is described. The spectra and fluorescence of the pure coensyme are studied. REFERENCES 1. HELLERMAN,
8. 9. 10. 11. 12.
Chem. 163, 553 (1946). BESSEY, 0. A., LOWRY, 0. H., AND LOVE, R. H., J. Biol. Chem. 180,755 (1949). BURTON, K., Biochem. J. 46, 458 (1951). DIMANT, E., SANADI, D. R., AND HUENNEKENS, F. M., J. Am. Chem. Sot. 74, 5440 (1962). WEITBY, L. G., Biochem. J. 54, 437 (1953). WHITBY L. G., Biochem. et Biophys. Acta 16, 148 (1954). HUENNEICENS, F. M., AND FELTON, S. P., in “Methods in Enzymology” (Colowick and Kaplan, eds.), Vol. III, 950. Academic Press, New York, 1957. WARBURG, O., AND CHRISTIAN, W., Biochem. Z. 266, 150,377 (1938). SILIPRANDI, N., AND BIANCHI, P. Biochim. et Biophys. Acta 16, 424 (1954). CERLETTI, P., AND SILIPRANDI, N., Biochem. J. 61,324 (1955). CERLETTI, P., AND ROSSI-FANELLI, A., J. VitaminoE. (Osaka) 4, 71 (1958). DELUCA, C., WEBER, M. M., AND KAPLAN, N. O., J. Biol. Chem. 223. 561
13. 14. 15. 16.
PRIDEAUX, E. B. R., AND WARD, A. I., Proc. Roy Sot. (London) 92,463 (1916). YAGI, K., AKU~A, J., AND MATSUOKA, Y., Nature 176, 555 11955). WEBER, G., Biochem. J. 47, 115 (1950). CHRISTIE, S., KENNER, G. W., AND TODD, A. R., J. Chem. Sot. 1964,46.
2. 3. 4. 5. 6. 7.
L., LINDSAY,
A., AND BOVARNICK, M. R., J. Biol.
11956).
FLAVINE ADENINE DINUCLEOTIDE
223
however that at high saline concentration a value of 15 % is found. This figure seems more in accord with our value of 18%. It is worth noting also that Weber (15) found for FAD a fluorescence of 20 % as compared to that of riboflavine. The decrease in light absorption at 450 rnp on addition of dithionite is stated to be at least 95 % by Weber (15). Whitby gives a figure of 91%. However, he does not completely discount the hypothesis of an impurity to which the residual absorption might be due. That this is not true is seen from our results that refer to a pure product. The spectra drawn on pure FAD are almost identical with those of previous authors (5, 16). Also, the ratios of light absorption at different wavelengths are in support of the extinction coefficients calculated by previous authors. The value of Kp we found is in good agreement with values previously reported (1, 3, 5, 8, 12). Our results, referring to the pure compound show that data obtained by previous authors with preparations of high purity are substantially sound. Crystallization of FAD was claimed only by Warburg and Christian (8) who described the crystals of the Ba salt of FAD as “grapelikegrouped balls.” Huennekens and Felton (7) describe a semicrystalline uranyl FAD complex as clusters of regular beadlike bodies. Whitby (5) observed orange-yellow needles forming during concentration of a highly purified preparation of FAD. However, whereas no such individuals could be observed among our FAD crystals, Warburg’s “grapelike balls” and Huennekens’ beads are probably related to the groups of small hexagonal crystals we described in a former section. It is not surprising that so far crystallization could not be achieved even from highly purified preparations. The addition of organic solvents result,s in a partial breakdown of FAD, and even precipitation from an aqueous solution by concentration must be extremely slow in order to obtain the beginning of crystallization. ACKNOWLEDGMENT
The work was aided by grants from the Rockefeller which grateful acknowledgement is made.
Foundation,
to
SUMMARY
The preparation of pure, crystalline flavine adenine dinucleotide using electrophoresis on a column packed with cellulose powder followed
224
CERLETTI
AND
SILIPRANDI
by chromatography on carboxylic resin is described. The spectra and fluorescence of the pure coenzyme are studied. REFERENCES 1. HELLERMAN, L., LINDSAY, A,, AND BOVARNICH, M. R., J. Biol. Chem. 163, 553 (1946). 2. BESSEY, 0. A., LOWRY, 0. H., AND LOVE, R. H., J. Biol. Chem. 180,755 (1949). 3. BURTON, K., Biochem. J. 48, 458 (1951). 4. DIMANT, E., SANADI, D. R., AND HUENNEKENS, F. M., J. Am. Chem. Sot. 74, 5440 (1952). 5. WHITBY, L. G., Biochem. J. 64, 437 (1953). 6. WHITBY L. G., Biochem. et Biophys. Acta 16, 148 (1954). 7. HUENNEKENS, F. M., AND FELTON, S. P., in “Methods in Enzymology” (Colowick and Kaplan, eds.), Vol. III, 950. Academic Press, New York, 1957. 8. WARBURG, O., AND CHRISTIAN, W., Biochem. 2.298, 150,377 (1938). 9. SILIPRANDI, N., AND BIANCHI, P. Biochim. et Biophys. Acta 16, 424 (1954). 10. CERLETTI, P., AND SILIPRANDI, N., Bioch,em. J. 61, 324 (1955). 11. CERLETTI, P., AND ROSSI-FANELLI, A., J. Vitaminol. (Osaka) 4, 71 (1958). 12. DELUCA, C., WEBER, M. M., AND KAPLAN, N. O., J. Biol. Chem. 993, 561 (1956). 13. PRIDEAUX, E. B. R., AND WARD, A. I., Proc. Roy Sot. (London) 62,463 (1916). 14. YAGI, K., AKU~A, J., AND MATSUOKA, Y., Nature 176, 555 11955). 15. WEBER, G., Biochem. J. 47, 115 (1950). 16. CHRISTIE, S., KENNER, G. W., AND TODD, A. R., J. Chem. Sot. 1964,413.
OF
BIOCHEMISTRY
AND
Pure Crystalline
BIOPHYSICS
76, 214-224 (1968)
Flavine Adenine
Dinucleotide
Paolo Cerletti and Noris Siliprandi From the Institute of Biological Chemistry of the University of Rome and National Research Council Unit on Enzyme Studiea, Rome, Italy Received January 18, 1958 INTRODUCTION
In recent years new interest has arisen in flavine adenine dinucleotide (FAD), and several workers (l-7) have attempted to obtain highly purified preparations of FAD by simpler methods than were first described by Warburg and Christian (8). The later stages of Warburg’s method, for which considerable skill is required, have been substituted by chromatographic procedures, and degrees of purity ranging from 60 to 92% have been obtained. Siliprandi and Bianchi (9) described a procedure based on cellulose-column electrophoresis and ion-exchange chromatography by which about 98% pure FAD is obtained. In subsequent work reported in the present paper, this method has been simplified, and absolutely pure FAD has been obtained and crystallized. EXPERIMENTAL
Methods Paper and column electrophoreaia procedures previously described (10, 11) were followed. The column must be repacked with new cellulose powder every 5-10 electrophoresis runs. Ion-Exchange Chromatography. Carboxylic acid exchanger (Amberlite IRC-50, 129-300 mesh) in the hydrogen form, packed in a 5 X 75 cm. column was used. Spectrophotometric measurements were made in a Beckman DU spectrophotometer, in 0.1 M phosphate pH 6.89 and phosphate-acetate-borate pH 2.01 and 12.28 buffers. For determining the absorption spectra, readings were made every 2 w in the regions of the maxima and every 5 ma elsewhere. The molecular extinction coefficient (c) of FAD at 450 rnN was assumed to be 11.3 X lo” mole-r cm.-’ (5). During the purification procedures a value of 3.28 was taken as representing the 214
FLAVINE
ADENINE
215
DINUCLEOTIDE
ratio between the extinction coefficients of pure FAD at 260 and 450 rmc (&a,~/&. Fluorimetric measurements were made in a Klett fluorimeter : lamp filter Corning glass 5543 and photocell filter Corning 3335. The pH range from 1.5 to 11.6 was covered with the following buffers: glycine pH from 1.5 to 3.5 and from 10 to 11.6; acetate from 4 to 5.5; phosphate from 6 to 7; borate from 8 to 9. &enzyme Activity. The activity of FAD as coenzyme for n-amino acid oxidase was determined according to Warburg and Christian (8) using an apoenayme prepared from pig kidney according to DeLuca et al. (12).
Preparation of FAD A. Five kilograms Titan0 baker’s yeast is extracted by the method previously described (9) as far as the precipitation of the crude Ag salt of FAD. B. The Ag precipitate is washed two times with 10 ml. of water, resuspended in 100 ml. of water, and dissolved by adding a slight excess of saturated KCl; the solution is brought to pH 3 by addition of 1 N HNOs and centrifuged. The precipitate is washed three times with 5 ml. water, the washings are combined to the clear brownish supernatant (final solution = 115 ml.) and brought to pH 4.9 by addition of solid NaHCOa . Analysis by paper electrophoresis of this solution shows a large yellow fluorescent spot corresponding to FAD, a smaller spot corresponding to flavine mononucleotide (FMN), and two spots with blue fluorescence due to unidentified compounds. The purity of this extract as determined by applying the figure of R260,,r0 found to the value of R260,460of pure FAD, does not exceed 8yo (see Table I). C. The solution obtained in B is concentrated in vacua at 30°C. to a volume of about 35 ml. and submitted to column electrophoresis on normal columns (4 X 40 cm.) (10) connected in series in the circuit or on a “multiple column” (11) ; 5-ml. aliquots are applied on each segment. Before the column is assembled the substance is displaced 4-5 cm. down each segment by allowing a sufficient amount of buffer to flow through each segment; the original brown-yellow zone splits into a faster moving brownish red zone and a slower yellow zone containing FAD and FMN. The current is then applied, and the electrophoresis and elution are performed as previously described (11). The electrophoresis is run in the dark, and the temperature of the buffer in which the column is dipped must not exceed 30°C. Under the influence of the electric field, FAD is separated from FMN while the brownish red zone moves the fastest and is eluted first (Fig. 1, peak A). The elution curves are shown in Fig. 1. Four peaks of unknown ultraviolet-absorbing compounds are obtained, two of which partially overlap with the peak correspondTABLE I Purity Degree of FAD at Different Stages of Preparation Calculated from the Ratios of Light Absorption at 260 and ,460rnk (Rz~& R m,m found
B c D
41 6.2 3.276
R ta,w) of pure FAD R ,a,,~ found
0.079 0.523 1
216
CERLETTI
AND SILIPRANDI
O---O
? c v)
260 mp 450 mp
2 3.0b ‘0
2Q 2.5 0 2.0 -
v 1.5 -
a
l.O-
0.5-
A ,p-0. -P
o-%50
I 100
D
,? 150
‘P
-au-
P-4
200
lx4 250
300
ml effluent FIG. 1. Separation of crude FAD by cellulose powder electrophoresis from other unidentified substances from yeast. (Acetate buffer pH 5.1, r/2 = 0.05, 40 ma. for 14 hr.
ing to FAD. This fraction, when examined by paper electrophoresis, reveals the presence of FAD as the only flavine compound and of two other spots corresponding to unidentified ultraviolet-absorbing substances. The purity of the fraction containing FAD averages 52% with an increase of 6.5 times as compared to stage B (see Table I). D. The combined FAD fractions obtained from the different segments are concentrated in vacua at 32%. to a volume of about 10 ml. and then applied to the top of a column containing activated Amberlite IRC 50, and eluted with water, the flow rate being adjusted to 20 ml./hr. Six-milliliter fractions are collected, and the absorbancy is determined at 260 and 450 mp. The elution curves are shown in Fig. 2. A single yellow fluorescent fraction is obtained, corresponding to pure FAD. Two minor peaks corresponding to ultraviolet-absorbing compounds are eluted immediately before and after the FAD fraction and partially overlap it. The contaminated tubes of FAD can however be completely purified by submitting them again on the ion exchanger. Samples were analyzed by paper electrophoresis and no other ultravioletabsorbing or fluorescent spot could be detected, apart from that corresponding to FAD. The R.JM)I~value of 3.28 consistently corresponded to FAD pure with respect to other flavines or other nucleotides.
217
FLAVINE ADENINE DINUCLEOTIDE
o----O
260 mp 450 m/A
V
FAD ,**\ d
:
‘? \
i
/
,/ 300
\ ‘9 ‘\ \
Id’
I 350
400
‘b \
I -
450
s;a
ml eflluent
FIG.
2.
Ion-exchange separation of pure FAD (Amberlite
IRC-50). Flow rate
20 ml./hr. Yield. A weight of 12.8 mg. of pure FAD was isolated from 5 kg. baker’s yeast. This yield is slightly superior to that obtained by the method previously described and almost identical with the yield of FAD obtained by other workers (5, 8).
The substance is fairly stable in distilled water or in the buffer used for electrophoresis and can be kept for several days at 0°C. in the dark. In all stages of the purification procedure contact with light should be avoided, FAD being highly photosensitive. RESULTS
Absorption Spectra of FAD Figure 3 shows the absorption spectra of FAD at pH 2.01 (phosphate-acetate-borate buffer (13), pH 6.98 (phosphate buffer), and pH 12.29 (phosphate-acetate-borate buffer). Three peaks are evident throughout the pH range already tested.
218
CERLETTI
AND
SILIPRANDI ---__
I
250
300
350
400
450
5ooA Wovelengthimp!
FIQ. 3. Absorption spectra of FAD in 0.1 M phosphate buffer pH 6.98 and 0.1 M phosphate-acetate-borate buffer pH 2.01 and pH 12.29.
The spectra are almost identical in neutral or acid solution with maxima at 264, 375, and 450 rnp. At alkaline pH the maximum in the ultraviolet region is found at 270 rnp, and the peak in the region of 360-380 rnp is displaced toward shorter wavelengths, having its maximum at 357 mp. The extinction coefficent of this maximum has a higher value than the coefficent of the corresponding maxima at pH 2.01 and 6.98, and it can be calculated to be E = 11.5 X 103 in terms of the known value of e at 450 rnp (11.3 X 103). The ratios of light absorbtion at different pH and wavelengths are given in Table II. When the spectra of FMN and riboflavine pyrophosphate (RPP) are compared with those of FAD, it is remarkable that the absorbancy of this latter substance (FAD) steadily increases in the region below TABLE II Ratios of Light Absorption of Pure FAD at Differmt PH
2.01 6.98 11.29
Wavelengths and pH
Rt6WU.l
Rnqrso
R?70/4M
RW/4M
R676,4W
Raw/m
3.49 3.276 3.55
3.67 3.34 3.89
3.38 3.09 3.92
0.704 0.655 1.01
0.81 0.75 0.797
4.41 4.24 4.32
FLAVINE
ADENINE
219
DINUCLEOTIDE
240 rnp while that of FMN and that of RPP show a maximum at 224 rnp and decrease at shorter wavelengths. It is not thought that these differences are due to spectrophotometric errors as they depend upon readings obtained in the same instrument and can be attributed to the adenylic acid (AMP) moiety of FAD. The decrease in light absorption of FAD at 450 rnp by a reducing agent has also been studied. Addition to FAD in 0.1 M buffer of 0.1 vol. of 10 % Na&$04 in 5 % NaHC03 produces a decrease of 90.7 % of the absorption intensity at pH 2.01, 91.3% at pH 6.98, and 90.2% at pH 12.29. It should be taken into account, however, that the pH of the alkaline and acid samples, tested after the addition of dithionite, was not more than 1 pH unit nearer to neutrality. Fluorescence of FAD The effect of pH on the fluorescence of FAD was investigated in 0.01 M buffer in the pH range from 1.5 to 11.6. As shown in Fig. 4, the fluorescence has a maximum at pH 2.9, falls between pH 3 and 5.5, and then has a constant value up to pH 8.5. The fluorescent efficiency almost disappears at higher pH. Addition of 0.1 vol. of 10% NazSz04 in 5 % NaHC03 completely destroys the fluorescence throughout the pH range examined.
3
4
5
6
7
8
9
10
11
12
PH FIQ. 4. Fluorescence of FAD at different pH. fluorescence a8 compared to a riboflavine standard.
Ordinates
give
the
percent
220
CERLETTI
AND
SILIPRANDI
Moreover, the fluorescence of FAD before and after acid hydrolysis has been studied under conditions in which complete hydrolysis of FAD to riboflavine is stated to occur by previous authors (2, 8). All samples compared contained identical saline concentration and were measured in 0.1 or 0.01 M phosphate buffer 7.0. The fluorescence before hydrolysis was always found to be 18% as compared to that after hydrolysis. Biological Activity The activity of our purest preparations as coensyme for n-amino acid oxidase has been determined with several different apoenzyme preparations. As a reference we used a commercial sample (Wakamoto Pharm. Co., Tokyo) 90% pure; its electrophoretic and spectrophotometric analyses agreed for a high purity. As seen in Fig. 5, assuming as 90 % the activity of this sample, the activity of our pure FAD was 101%. The apparent dissociation constant for FAD-protein, KFl, was also determined and a value of 2.2 X 1OV mole/l. was found.
WA .---.
10
6
20
30
40
50
Time in min. FIG. 5. Activity as coenayme of D-amino acid oxidase of FAD prepared with the present method (A) as compared to a commercial sample of 90% purity (B).
FLAVINE
ADENINE
Crystallizatim
DINUCLEOTIDE
221
of FAD
Crystallization was obtained from a solution purified by several passages on ion exchanger and concentrated to a volume of about 5 ml. and then left in the dark at 04°C. on a crystallization dish until evaporation was nearly complete. When examined under the microscope (Leitz, Ortholux with Ultropak) in reflected light, bright yellow hexagonal crystals appeared, many of them in the form of aggregates. The smaller crystals were often closely grouped together like a bunch of grapes. DISCUSSION
As all purification procedures so far described for FAD, the present method is, with minor modification, the same as that of Warburg and Christian (8) in its initial stages. Of the many procedures devised to substitute the later stages of Warburg’s method, the combination of ion exchange and electrophoresis previously described (9) is among the simplest and for the purity of the product the best. In this method, the procedure is simplified and makes pure FAD readily available for laboratory work. The hindering factor represented by the limited capacity of column electrophoresis has been overcome by the use of “multiple columns.” The dissolved Ag precipitate is used for electrophoresis without further extraction, and the elution from the ion exchanger directly yields pure FAD in aqueous solution. Yagi et al. (14) recently described the separation of flavines by ion exchange on Dowex 1. In their paper it is not stated whether their method can also be applied to products of extraction nor whether FAD can be freed from other nucleotides. In any case, the final product is obtained in rather concentrated saline solution. The extraction stages as far as the precipitation of the Ag salt of FAD do not act on the biological activity of the coenzyme, as was already known from the work of previous authors. The electrophoretic and ion-exchange techniques followed for the further purification also do not alter the coenzymic activity. In fact, preparations that had repeatedly undergone electrophoresis and chromatography did not show loss of activity. The criteria of purity adopted in this work seem most direct and effective. Elementary analysis of FAD was not taken into consideration for testing the purity of our preparation since it is of no value in as-
222
CERLETTI
AND
SILIPRANDI
certaining whether the molecule of FAD is intact; in fact both FAD and an equimolar mixture of FMN and AMP give the same analytical figures. A more direct approach to the estimation of flavines at the different stages of the purification has been done by previous authors who used paper chromatography and electrophoresis. For reasons given in a former paper (lo), we preferred paper electrophoresis. This method allows a ready identification of very small amounts of flavines and reveals many other nonflavine fluorescent substances and compounds absorbing in the ultraviolet region of the spectrum. Fluorescent inpurities are moreover easily detected by measuring the decrease in fluorescence due to addition of dithionite, and eventually a reliable estimate of the amount of substances that absorb at 450 rnp is obtained by measuring the reduction in light absorption at this wavelength after addition of dithionite. When FAD is the only substance absorbing at 450 rnp, the value of I” .. .c 1... . .. -z_.. 73
224
CERLETTI
AND
SILIPRANDI
by chromatography on carboxylic resin is described. The spectra and fluorescence of the pure coensyme are studied. REFERENCES 1. HELLERMAN,
8. 9. 10. 11. 12.
Chem. 163, 553 (1946). BESSEY, 0. A., LOWRY, 0. H., AND LOVE, R. H., J. Biol. Chem. 180,755 (1949). BURTON, K., Biochem. J. 46, 458 (1951). DIMANT, E., SANADI, D. R., AND HUENNEKENS, F. M., J. Am. Chem. Sot. 74, 5440 (1962). WEITBY, L. G., Biochem. J. 54, 437 (1953). WHITBY L. G., Biochem. et Biophys. Acta 16, 148 (1954). HUENNEICENS, F. M., AND FELTON, S. P., in “Methods in Enzymology” (Colowick and Kaplan, eds.), Vol. III, 950. Academic Press, New York, 1957. WARBURG, O., AND CHRISTIAN, W., Biochem. Z. 266, 150,377 (1938). SILIPRANDI, N., AND BIANCHI, P. Biochim. et Biophys. Acta 16, 424 (1954). CERLETTI, P., AND SILIPRANDI, N., Biochem. J. 61,324 (1955). CERLETTI, P., AND ROSSI-FANELLI, A., J. VitaminoE. (Osaka) 4, 71 (1958). DELUCA, C., WEBER, M. M., AND KAPLAN, N. O., J. Biol. Chem. 223. 561
13. 14. 15. 16.
PRIDEAUX, E. B. R., AND WARD, A. I., Proc. Roy Sot. (London) 92,463 (1916). YAGI, K., AKU~A, J., AND MATSUOKA, Y., Nature 176, 555 11955). WEBER, G., Biochem. J. 47, 115 (1950). CHRISTIE, S., KENNER, G. W., AND TODD, A. R., J. Chem. Sot. 1964,46.
2. 3. 4. 5. 6. 7.
L., LINDSAY,
A., AND BOVARNICK, M. R., J. Biol.
11956).
FLAVINE ADENINE DINUCLEOTIDE
223
however that at high saline concentration a value of 15 % is found. This figure seems more in accord with our value of 18%. It is worth noting also that Weber (15) found for FAD a fluorescence of 20 % as compared to that of riboflavine. The decrease in light absorption at 450 rnp on addition of dithionite is stated to be at least 95 % by Weber (15). Whitby gives a figure of 91%. However, he does not completely discount the hypothesis of an impurity to which the residual absorption might be due. That this is not true is seen from our results that refer to a pure product. The spectra drawn on pure FAD are almost identical with those of previous authors (5, 16). Also, the ratios of light absorption at different wavelengths are in support of the extinction coefficients calculated by previous authors. The value of Kp we found is in good agreement with values previously reported (1, 3, 5, 8, 12). Our results, referring to the pure compound show that data obtained by previous authors with preparations of high purity are substantially sound. Crystallization of FAD was claimed only by Warburg and Christian (8) who described the crystals of the Ba salt of FAD as “grapelikegrouped balls.” Huennekens and Felton (7) describe a semicrystalline uranyl FAD complex as clusters of regular beadlike bodies. Whitby (5) observed orange-yellow needles forming during concentration of a highly purified preparation of FAD. However, whereas no such individuals could be observed among our FAD crystals, Warburg’s “grapelike balls” and Huennekens’ beads are probably related to the groups of small hexagonal crystals we described in a former section. It is not surprising that so far crystallization could not be achieved even from highly purified preparations. The addition of organic solvents result,s in a partial breakdown of FAD, and even precipitation from an aqueous solution by concentration must be extremely slow in order to obtain the beginning of crystallization. ACKNOWLEDGMENT
The work was aided by grants from the Rockefeller which grateful acknowledgement is made.
Foundation,
to
SUMMARY
The preparation of pure, crystalline flavine adenine dinucleotide using electrophoresis on a column packed with cellulose powder followed
224
CERLETTI
AND
SILIPRANDI
by chromatography on carboxylic resin is described. The spectra and fluorescence of the pure coenzyme are studied. REFERENCES 1. HELLERMAN, L., LINDSAY, A,, AND BOVARNICH, M. R., J. Biol. Chem. 163, 553 (1946). 2. BESSEY, 0. A., LOWRY, 0. H., AND LOVE, R. H., J. Biol. Chem. 180,755 (1949). 3. BURTON, K., Biochem. J. 48, 458 (1951). 4. DIMANT, E., SANADI, D. R., AND HUENNEKENS, F. M., J. Am. Chem. Sot. 74, 5440 (1952). 5. WHITBY, L. G., Biochem. J. 64, 437 (1953). 6. WHITBY L. G., Biochem. et Biophys. Acta 16, 148 (1954). 7. HUENNEKENS, F. M., AND FELTON, S. P., in “Methods in Enzymology” (Colowick and Kaplan, eds.), Vol. III, 950. Academic Press, New York, 1957. 8. WARBURG, O., AND CHRISTIAN, W., Biochem. 2.298, 150,377 (1938). 9. SILIPRANDI, N., AND BIANCHI, P. Biochim. et Biophys. Acta 16, 424 (1954). 10. CERLETTI, P., AND SILIPRANDI, N., Bioch,em. J. 61, 324 (1955). 11. CERLETTI, P., AND ROSSI-FANELLI, A., J. Vitaminol. (Osaka) 4, 71 (1958). 12. DELUCA, C., WEBER, M. M., AND KAPLAN, N. O., J. Biol. Chem. 993, 561 (1956). 13. PRIDEAUX, E. B. R., AND WARD, A. I., Proc. Roy Sot. (London) 62,463 (1916). 14. YAGI, K., AKU~A, J., AND MATSUOKA, Y., Nature 176, 555 11955). 15. WEBER, G., Biochem. J. 47, 115 (1950). 16. CHRISTIE, S., KENNER, G. W., AND TODD, A. R., J. Chem. Sot. 1964,413.