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Complexing of riboflavin with the enolate form of barbituric acid
582
BIOCHIMICA ET BIOPHYSICA ACTA
m~A 46 264 (:()MPLEXING OF R I B O F L A V I N W I T H T H E ENOLATE FORM OF B A R B I T U R I C ACII) MALKA G()R()DETSKY, R U T H F. \ V E [ N E R ANt) \ V I L H E L M R. F R I S E L I ,
Department of Biochemistry, College of Medicine and Denlistry of New Jersey, Newark, N.J. o7zo 3
(u.x.A.) (Received September 2lst, i971)
SUMMARY
Ribottavin and the enolate form of barbituric acid form a complex at pH 7.80 as demonstrated by the appearance of two new absorption bands at 4oo and 49o nm. The association constant for the complex determined from a Benesi-Hildebrand analysis of absorbances at 49 ° n m is 3o M -1. The K evaluated from fluorescence quenching at various barbiturate: riboflavin ratios is 33 M-1. The complexing of the barbiturate and riboflavin is accompanied by an inhibition of the photo-oxidation of sarcosine to fornlaldehyde. Under the same conditions, the barbiturate enhances the rate of photo-oxidation of NADH. The 5,5-disubstituted barbiturates do not give a new absorption band with riboflavin and do not inhibit the photo-oxidation of sarcosine, indicating that an enolate configuration of the fi-diketone system is involved in conlplexing with riboflavin.
Previous studies in our laboratory had demonstrated that sarcosiue is photooxidized in the presence of catalytic amounts of riboflavin, FMN, or FAD1:
H~
§
H3C-N-CH/-COOH
H ~ § + Oz + HzO -~ H 3 C - N H + H C H O + CO2 + H202
It has been found subsequently that sodium barbiturate inlfibits the reaction and forms a complex with riboflavin. In the experiments described below, the difference absorption spectra were determined in I-cm cells with a Cary 17 spectrophotometer and the fluorescence measurements were made with a Hitachi spectrophotofluorimeter. The photo-oxidation of sarcosine with riboflavin was carried out in quartz cuvettes placed I I cm from a long-wave ultraviolet lamp (Model X 3o, Ultra-Violet Products, Inc.). Formaldehyde was determined by the chromotropie acid procedure "~. All reagents were of the highest quality available commercially. The amytal was a generous gift of Dr. Otto K. Behrens of the Lilly Research Laboratories. The difference spectra of mixtures of sodium barbiturate and riboflavin, nteasured against riboflavin as the reference, are presented in Fig. I. Two new absorption bands were observed at 4oo and 49 ° rim. However, only tlte absorption at 49 ° nnl could be analyzed by the BENEsI--HILDEBRANI)plot a. It should be noted l~iochim. Biophys. Acla, 256 (1972) 582-585
COMPLEXING OF RIBOFLAVIN WITH BARBITURATE
583
t h a t s o d i u m b a r b i t u r a t e itself has no absorption at this wavelength. Under the same conditions, no new a b s o r p t i o n was o b s e r v e d with the 5,5-disubstituted derivatives, barbital and amytal.
A
I
i
I
l
I
i
I
I
0
-'1
-2
-3
-4 ,
400
500
I
600
L[nrn]
Fig. I. Difference spectra of riboflavin plus sodium barbiturate versus riboflavin. The concentrations of the reactants ill O.075 M potassium phosphate, pH 7.80, were: riboflavin, 1.5' Io -~ M, and barbiturate, (a) C?; (b) 3.75.io -2M; (c) 5.o2.1o 2M; (d) 7.5-1o --0 M. The association c o n s t a n t for the complex, d e t e r m i n e d b y the d a t a shown in Fig. 2, was found to be 3 ° M -1. The e q u i l i b r i u m c o n s t a n t was also e v a l u a t e d b y m e a s u r i n g the degree of fluorescence quenching at various concentrations of b a r b i t u r a t e 4 (Fig. 3). The value of K o b t a i n e d b y this m e t h o d was 33 M-1. Complexing of the b a r b i t u r a t e a n d riboflavin is a c c o m p a n i e d b y an inhibition of the p h o t o - o x i d a t i o n of sarcosine to f o r m a l d e h y d e , as d e m o n s t r a t e d b y the d a t a in Fig. 4. B a r b i t a l a n d a m y t a l do not inhibit. I t m a y be a d d e d t h a t u n d e r the conditions of our reactions, b a r b i t u r i c acid enhances the r a t e of o x i d a t i o n of N A D H 5 as first o b s e r v e d b y GIUDITTA AND VITALE--NEUGEBAUER 6. I n the same molar ratios of flavin to b a r b i t u r a t e used for the analyses described in Figs. i a n d 2, new a b s o r p t i o n b a n d s were also d e t e c t e d w i t h riboflavin a n d 5 , 5 - d i m e t h y l - I , 3 - c y c l o h e x a n e d i o n e ( " d i m e d o n " ) (49o nm), resorcinol (49 o nm), a c e t y l a c e t o n e (488 nm), a n d phenol (488 nm). These results, t o g e t h e r with the i n a b i l i t y Biochim. Biophys. Acta, 256 (1972) 582-585
M. G O R T ) D E T S K Y 6'#a~/.
58~ I
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)
i
I
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M
O
r~ n,"
I
~
1
10
20
3O
1/[BA]
0
I
I
I
I
1
2
3
/*
[BA]
F i g . 2. T h e B E N E S I - H I L D E B R A N D p l o t a f o r t h e c o m p l e x of r i b o f l a v i n a n d b a r b i t u r a t e . T h e c o n c e n t r a t i o n of r i b o f l a v i n [ R f I w a s 1.2. ~o ¢ M a n d t h a t of t h e b a r b i t u r a t e EBAJ w a s v a r i e d f r o m 1 . 8 5 . i o 2 t o 7 . 5 ' i o 2 M. A p p r o p r i a t e a m o u n t s of s o d i u m a c e t a t e w e r e a d d e d t o t h e r e t e r e n c c solution to compensate for the barbiturate. F i g . 3. Q u e n c h i n g of f l u o r e s c e n c e of r i b o f l a v i n b y b a r b i t u r a t e . T h e c o n c e n t r a t i o n of r i b o f l a v i n w a s 5 . O - l O -6 M a n d t h a t of t h e b a r b i t u r a t e EBA] w a s v a r i e d f r o m o . 5 ' l o 2 t o 4 - 5 ' i o 2M. lo/I is t h e r a t i o of f l u o r e s c e n c e i n t e n s i t i e s b e f o r e a n d a f t e r a d d i t i o n of q u e n c h e r . S o d i u m a c e t a t e w a s a d d e d t o t h e r e f e r e n c e s o l u t i o n in a m o u n t s e q u i v a l e n t t o t h e b a r b i t u r a t e .
I
I
I
I
0.6
0.4
t
o
aP C~
Ig E
÷
l
0.2
0 =
I 1
I I 2 3 t [hours]
I 4
I 5
F i g . 4. E f f e c t of b a r b i t u r a t e , b a r b i t a l , a n d a m y t a l o n t h e p h o t o - o x i d a t i o n of s a r c o s i n e w i t h r i b o f l a v i n . T h e c o n c e n t r a t i o n s of r e a c t a n t s , all in o . o 7 5 M p o t a s s i u m p h o s p h a t e , p H 7 . 8 0 , w e r e ( Q ) r i b o f l a v i n , i . o . i o -~ M a n d s a r c o s i n e , 2 . o . i o -a M ; ( • ) s a m e , p l u s b a r b i t u r a t e ; ( + ) b a r b i t a l ; ( V ) A m y t a l ; ( © ) s o d i u m a c e t a t e , e a c h i . o . IO -a M.
Biochim. Biophys. Acta, 2 5 6 ( i 9 7 2 ) 5 8 2 - 5 8 5
COI~IPLI~XING OF I(IBOFLAVIN WITtf BARBITURATE
585
of t h e 5,5-dialkyl b a r b i t u r a t e s t o react, i n d i c a t e t h a t an e n o l a t e c o n f i g u r a t i o n ot t h e / 5 - d i k e t o n e s y s t e m is p r o b a b l y i n v o l v e d in c o m p l e x i n g w i t h riboflavin. R i b o f l a v i n a n d its d e r i v a t i v e s can c o m p l e x w i t h a v a r i e t y of N - h e t e r o c y c l i c c o m p o u n d s , i n c l u d i n g indoles a n d p u r i n e s 4,~-1°. E a r l i e r studies in our l a b o r a t o r y h a d d e m o n s t r a t e d also t h a t a d e n i n e d e r i v a t i v e s i n h i b i t t h e p h o t o - o x i d a t i o n of sarcosine b y r i b o f l a v i n k T h e r e a c t i o n of b a r b i t u r a t e r e p o r t e d in this p a p e r p r o v i d e s an a d d i t i o n a l e x a m p l e of t h e d i v e r s i t y of h e t e r o c y c l i c c o m p o u n d s c a p a b l e of c o m p l e x i n g w i t h flavins. T h i s w o r k was s u p p o r t e d in p a r t b y a G r a n t f r o m t h e U.S. P u b l i c H e a l t h S e r v i c e (AM-oo969-I6). REFERENCES CHUNG, AND C. C-. MACKENZIE,J. Biol. Chem., 234 (1959) 1297. W. R. FRISELL AND C. G. MACKENZIE,J. Biol. Chem., 207 (1954) 709. H. A. BENESI AND J. HILOEBRAND, J. Am. Chem. Soc., 71 (1949) 2703. G. WEBER, Biochem. J., 47 (195 °) 114. \~7. R. FEISELL AND C. G. MACKENZIE, Proc. Natl. Acad. ,';ci. U.S., 45 (1959) I568. A. (;IUDITTA AND A. VITALE- NEUGEBAUER,Biochim. Biophys. Acta, 11o (1965) 32. G. \VEBER, Nature, 18o (1957) 14o9. G. WEBER, in E. C. SLATER, Flavins and Flavoproteins, Elsevier, Amsterdam, 1966, p. 15. F. J. BULLOCK, in M. FLORKIN AND E. H. STOTZ, Comprehensive Biochemistry, Vol. 22, Elsevier, Amsterdam, 1967, pp. 1 lO-27. J. \v. WILSON, Bioche~istry, 5 (1966) 1352.
i W . R . FRISELL, C. W .
2 3 4 5 6
7
8 9 io
Biochim. Biophys. Acta, 256 (t972) 582-585
586
BIOCHIMICA ET BIOPIIYSICA
ACTA
VOLUME CODING FOR BBA-BIOENERGETICS B B A is p u b l i s h e d a c c o r d i n g t o a v o l u m e - n u n l b e r i n g s c h e m e t h a t e m b r a c e s all s e c t i o n s (71 t h e j o u r n a l : f o r 1972 t h e s c h e m e c o v e r i n g t h e v o l u m e s 29-5--287 is t o b e h m n d o n t h e i n s i d e cover of this issue. The seven individual sections are distinguished by a eolour code. In addition t o t h e c o l o u r c o d e e a c h s e c t i o n is g i v e n its o w n s e q u e n t i a l v o l u m e n m n b e r s . T h i s s y s t e m r u n s p a r a l l e l t o t h e o v e r a l l B B A s c h e m e : f o r t h e BmENERCETICS s e c t i o n t h e c o r r e s p o n d e n c e is i n d i c a t e d in t h e T a b l e b e l o w . T h i s i s s u e is t h e r e f o r e m O C m M m A ET mOPHYSlCA ACTa, Vol. 256/2 o r B B A aIOEN~RGETlCS B I 6 / 2 .
Parallel wdume coding for BBA-t3ioenergetics Biochimica et Biophysica Acta Volume No.
Bioenergetics Volume No.
Biochimica et Biophysica Acta Volume No.
Bioenergetics Volume No.
Vol. Vot. \%1. Vol. Vol. Vol. Vol. Vol. Vol. Vol.
B 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 t3 9 BIo
Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. \"ol.
BII BI2 BI 3 BI 4 BI 5 BI6 B17 BIB Bi 9
I3t 143 153 162 172 18o 189 197 205 216
-=
: "
....
(~967 / (1967) (1968) (1968 ) (1969) (1969) (1969) ( I 9 7 o) (197°) (197o)
223 : 226 234 245 : 253 2 5 6 --: 267 - 275 28.3
( I 9 7 o' (I97I) ( I 9 7 I) (I971) (1971) (1972) (1972 ) (1972) (1972)
A s u b s c r i p t i o n t o t h e B1OENERGETICS s e c t i o n of B B A f o r I 9 7 2 (4 v o l u m e s ) is Dfl. 2 8 8 . 4 0 (according to their B numbers) are a v a i l a b l e : r a t e s will b e s u p p l i e d o n r e q u e s t .
plus Dfl. "28.oo for p o s t a g e a n d h a n d l i n g . B a c k v o l u m e s
BIOCHIMICA ET BIOPHYSICA ACTA
m~A 46 264 (:()MPLEXING OF R I B O F L A V I N W I T H T H E ENOLATE FORM OF B A R B I T U R I C ACII) MALKA G()R()DETSKY, R U T H F. \ V E [ N E R ANt) \ V I L H E L M R. F R I S E L I ,
Department of Biochemistry, College of Medicine and Denlistry of New Jersey, Newark, N.J. o7zo 3
(u.x.A.) (Received September 2lst, i971)
SUMMARY
Ribottavin and the enolate form of barbituric acid form a complex at pH 7.80 as demonstrated by the appearance of two new absorption bands at 4oo and 49o nm. The association constant for the complex determined from a Benesi-Hildebrand analysis of absorbances at 49 ° n m is 3o M -1. The K evaluated from fluorescence quenching at various barbiturate: riboflavin ratios is 33 M-1. The complexing of the barbiturate and riboflavin is accompanied by an inhibition of the photo-oxidation of sarcosine to fornlaldehyde. Under the same conditions, the barbiturate enhances the rate of photo-oxidation of NADH. The 5,5-disubstituted barbiturates do not give a new absorption band with riboflavin and do not inhibit the photo-oxidation of sarcosine, indicating that an enolate configuration of the fi-diketone system is involved in conlplexing with riboflavin.
Previous studies in our laboratory had demonstrated that sarcosiue is photooxidized in the presence of catalytic amounts of riboflavin, FMN, or FAD1:
H~
§
H3C-N-CH/-COOH
H ~ § + Oz + HzO -~ H 3 C - N H + H C H O + CO2 + H202
It has been found subsequently that sodium barbiturate inlfibits the reaction and forms a complex with riboflavin. In the experiments described below, the difference absorption spectra were determined in I-cm cells with a Cary 17 spectrophotometer and the fluorescence measurements were made with a Hitachi spectrophotofluorimeter. The photo-oxidation of sarcosine with riboflavin was carried out in quartz cuvettes placed I I cm from a long-wave ultraviolet lamp (Model X 3o, Ultra-Violet Products, Inc.). Formaldehyde was determined by the chromotropie acid procedure "~. All reagents were of the highest quality available commercially. The amytal was a generous gift of Dr. Otto K. Behrens of the Lilly Research Laboratories. The difference spectra of mixtures of sodium barbiturate and riboflavin, nteasured against riboflavin as the reference, are presented in Fig. I. Two new absorption bands were observed at 4oo and 49 ° rim. However, only tlte absorption at 49 ° nnl could be analyzed by the BENEsI--HILDEBRANI)plot a. It should be noted l~iochim. Biophys. Acla, 256 (1972) 582-585
COMPLEXING OF RIBOFLAVIN WITH BARBITURATE
583
t h a t s o d i u m b a r b i t u r a t e itself has no absorption at this wavelength. Under the same conditions, no new a b s o r p t i o n was o b s e r v e d with the 5,5-disubstituted derivatives, barbital and amytal.
A
I
i
I
l
I
i
I
I
0
-'1
-2
-3
-4 ,
400
500
I
600
L[nrn]
Fig. I. Difference spectra of riboflavin plus sodium barbiturate versus riboflavin. The concentrations of the reactants ill O.075 M potassium phosphate, pH 7.80, were: riboflavin, 1.5' Io -~ M, and barbiturate, (a) C?; (b) 3.75.io -2M; (c) 5.o2.1o 2M; (d) 7.5-1o --0 M. The association c o n s t a n t for the complex, d e t e r m i n e d b y the d a t a shown in Fig. 2, was found to be 3 ° M -1. The e q u i l i b r i u m c o n s t a n t was also e v a l u a t e d b y m e a s u r i n g the degree of fluorescence quenching at various concentrations of b a r b i t u r a t e 4 (Fig. 3). The value of K o b t a i n e d b y this m e t h o d was 33 M-1. Complexing of the b a r b i t u r a t e a n d riboflavin is a c c o m p a n i e d b y an inhibition of the p h o t o - o x i d a t i o n of sarcosine to f o r m a l d e h y d e , as d e m o n s t r a t e d b y the d a t a in Fig. 4. B a r b i t a l a n d a m y t a l do not inhibit. I t m a y be a d d e d t h a t u n d e r the conditions of our reactions, b a r b i t u r i c acid enhances the r a t e of o x i d a t i o n of N A D H 5 as first o b s e r v e d b y GIUDITTA AND VITALE--NEUGEBAUER 6. I n the same molar ratios of flavin to b a r b i t u r a t e used for the analyses described in Figs. i a n d 2, new a b s o r p t i o n b a n d s were also d e t e c t e d w i t h riboflavin a n d 5 , 5 - d i m e t h y l - I , 3 - c y c l o h e x a n e d i o n e ( " d i m e d o n " ) (49o nm), resorcinol (49 o nm), a c e t y l a c e t o n e (488 nm), a n d phenol (488 nm). These results, t o g e t h e r with the i n a b i l i t y Biochim. Biophys. Acta, 256 (1972) 582-585
M. G O R T ) D E T S K Y 6'#a~/.
58~ I
I
I
)
i
I
_xl0 -3
M
O
r~ n,"
I
~
1
10
20
3O
1/[BA]
0
I
I
I
I
1
2
3
/*
[BA]
F i g . 2. T h e B E N E S I - H I L D E B R A N D p l o t a f o r t h e c o m p l e x of r i b o f l a v i n a n d b a r b i t u r a t e . T h e c o n c e n t r a t i o n of r i b o f l a v i n [ R f I w a s 1.2. ~o ¢ M a n d t h a t of t h e b a r b i t u r a t e EBAJ w a s v a r i e d f r o m 1 . 8 5 . i o 2 t o 7 . 5 ' i o 2 M. A p p r o p r i a t e a m o u n t s of s o d i u m a c e t a t e w e r e a d d e d t o t h e r e t e r e n c c solution to compensate for the barbiturate. F i g . 3. Q u e n c h i n g of f l u o r e s c e n c e of r i b o f l a v i n b y b a r b i t u r a t e . T h e c o n c e n t r a t i o n of r i b o f l a v i n w a s 5 . O - l O -6 M a n d t h a t of t h e b a r b i t u r a t e EBA] w a s v a r i e d f r o m o . 5 ' l o 2 t o 4 - 5 ' i o 2M. lo/I is t h e r a t i o of f l u o r e s c e n c e i n t e n s i t i e s b e f o r e a n d a f t e r a d d i t i o n of q u e n c h e r . S o d i u m a c e t a t e w a s a d d e d t o t h e r e f e r e n c e s o l u t i o n in a m o u n t s e q u i v a l e n t t o t h e b a r b i t u r a t e .
I
I
I
I
0.6
0.4
t
o
aP C~
Ig E
÷
l
0.2
0 =
I 1
I I 2 3 t [hours]
I 4
I 5
F i g . 4. E f f e c t of b a r b i t u r a t e , b a r b i t a l , a n d a m y t a l o n t h e p h o t o - o x i d a t i o n of s a r c o s i n e w i t h r i b o f l a v i n . T h e c o n c e n t r a t i o n s of r e a c t a n t s , all in o . o 7 5 M p o t a s s i u m p h o s p h a t e , p H 7 . 8 0 , w e r e ( Q ) r i b o f l a v i n , i . o . i o -~ M a n d s a r c o s i n e , 2 . o . i o -a M ; ( • ) s a m e , p l u s b a r b i t u r a t e ; ( + ) b a r b i t a l ; ( V ) A m y t a l ; ( © ) s o d i u m a c e t a t e , e a c h i . o . IO -a M.
Biochim. Biophys. Acta, 2 5 6 ( i 9 7 2 ) 5 8 2 - 5 8 5
COI~IPLI~XING OF I(IBOFLAVIN WITtf BARBITURATE
585
of t h e 5,5-dialkyl b a r b i t u r a t e s t o react, i n d i c a t e t h a t an e n o l a t e c o n f i g u r a t i o n ot t h e / 5 - d i k e t o n e s y s t e m is p r o b a b l y i n v o l v e d in c o m p l e x i n g w i t h riboflavin. R i b o f l a v i n a n d its d e r i v a t i v e s can c o m p l e x w i t h a v a r i e t y of N - h e t e r o c y c l i c c o m p o u n d s , i n c l u d i n g indoles a n d p u r i n e s 4,~-1°. E a r l i e r studies in our l a b o r a t o r y h a d d e m o n s t r a t e d also t h a t a d e n i n e d e r i v a t i v e s i n h i b i t t h e p h o t o - o x i d a t i o n of sarcosine b y r i b o f l a v i n k T h e r e a c t i o n of b a r b i t u r a t e r e p o r t e d in this p a p e r p r o v i d e s an a d d i t i o n a l e x a m p l e of t h e d i v e r s i t y of h e t e r o c y c l i c c o m p o u n d s c a p a b l e of c o m p l e x i n g w i t h flavins. T h i s w o r k was s u p p o r t e d in p a r t b y a G r a n t f r o m t h e U.S. P u b l i c H e a l t h S e r v i c e (AM-oo969-I6). REFERENCES CHUNG, AND C. C-. MACKENZIE,J. Biol. Chem., 234 (1959) 1297. W. R. FRISELL AND C. G. MACKENZIE,J. Biol. Chem., 207 (1954) 709. H. A. BENESI AND J. HILOEBRAND, J. Am. Chem. Soc., 71 (1949) 2703. G. WEBER, Biochem. J., 47 (195 °) 114. \~7. R. FEISELL AND C. G. MACKENZIE, Proc. Natl. Acad. ,';ci. U.S., 45 (1959) I568. A. (;IUDITTA AND A. VITALE- NEUGEBAUER,Biochim. Biophys. Acta, 11o (1965) 32. G. \VEBER, Nature, 18o (1957) 14o9. G. WEBER, in E. C. SLATER, Flavins and Flavoproteins, Elsevier, Amsterdam, 1966, p. 15. F. J. BULLOCK, in M. FLORKIN AND E. H. STOTZ, Comprehensive Biochemistry, Vol. 22, Elsevier, Amsterdam, 1967, pp. 1 lO-27. J. \v. WILSON, Bioche~istry, 5 (1966) 1352.
i W . R . FRISELL, C. W .
2 3 4 5 6
7
8 9 io
Biochim. Biophys. Acta, 256 (t972) 582-585
586
BIOCHIMICA ET BIOPIIYSICA
ACTA
VOLUME CODING FOR BBA-BIOENERGETICS B B A is p u b l i s h e d a c c o r d i n g t o a v o l u m e - n u n l b e r i n g s c h e m e t h a t e m b r a c e s all s e c t i o n s (71 t h e j o u r n a l : f o r 1972 t h e s c h e m e c o v e r i n g t h e v o l u m e s 29-5--287 is t o b e h m n d o n t h e i n s i d e cover of this issue. The seven individual sections are distinguished by a eolour code. In addition t o t h e c o l o u r c o d e e a c h s e c t i o n is g i v e n its o w n s e q u e n t i a l v o l u m e n m n b e r s . T h i s s y s t e m r u n s p a r a l l e l t o t h e o v e r a l l B B A s c h e m e : f o r t h e BmENERCETICS s e c t i o n t h e c o r r e s p o n d e n c e is i n d i c a t e d in t h e T a b l e b e l o w . T h i s i s s u e is t h e r e f o r e m O C m M m A ET mOPHYSlCA ACTa, Vol. 256/2 o r B B A aIOEN~RGETlCS B I 6 / 2 .
Parallel wdume coding for BBA-t3ioenergetics Biochimica et Biophysica Acta Volume No.
Bioenergetics Volume No.
Biochimica et Biophysica Acta Volume No.
Bioenergetics Volume No.
Vol. Vot. \%1. Vol. Vol. Vol. Vol. Vol. Vol. Vol.
B 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 t3 9 BIo
Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. \"ol.
BII BI2 BI 3 BI 4 BI 5 BI6 B17 BIB Bi 9
I3t 143 153 162 172 18o 189 197 205 216
-=
: "
....
(~967 / (1967) (1968) (1968 ) (1969) (1969) (1969) ( I 9 7 o) (197°) (197o)
223 : 226 234 245 : 253 2 5 6 --: 267 - 275 28.3
( I 9 7 o' (I97I) ( I 9 7 I) (I971) (1971) (1972) (1972 ) (1972) (1972)
A s u b s c r i p t i o n t o t h e B1OENERGETICS s e c t i o n of B B A f o r I 9 7 2 (4 v o l u m e s ) is Dfl. 2 8 8 . 4 0 (according to their B numbers) are a v a i l a b l e : r a t e s will b e s u p p l i e d o n r e q u e s t .
plus Dfl. "28.oo for p o s t a g e a n d h a n d l i n g . B a c k v o l u m e s