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Modification of tryptophan residue in ribonuclease T1 with 2-hydroxy-5-nitrobenzyl bromide
347
SHORT COMMUNICATIONS
and completely ineffective with trypsin. Table I lists the polyamines in the order of their effectiveness with ribonuclease. It should be noted (Table I) that the combined presence of the two polyamines, butyl- and propyldiamine did not provide any more stability than that obtained from each individual substance. These results suggest a direct polyamine-enzyme interaction rather than a solvent-solute effect. The nature of this attraction is not known, however, electrostatic forces are a consideration, since the molar protective effect decreases linearly with the charge of the amine.
Mount Holyoke College, Department of Biological Sciences, South Hadley, Mass. (U.S.A.) I
2 3 4 5
JACOB GOLDSTEIN
J. HERMANS AND H. A. SHERAGA, J. Am. Chem. Soc., 83 (1961) 3283. A. GINSBURG AND W. R. CARROL, Biochemistry, 4 (1965) 2159. P. H. VON HIPPEL AND K. Y. WONG, J. Biol. Chem., 240 (1965) 3909. M. KUNTZ, J. Biol. Chem., 164 (1946) 563 . D. SHUGAR, Biochim. Biophys. Acta, 8 (1952) 302.
Received October 24th, 1968 Revised manuscript received January 27th, 1969 Biochim. Biophys. Acta, 181 (1969) 345-347
BBA 33151
Modification of tryptophan residue in ribonuclease T z with 2-hyd roxy-5-nit robenzyl bromide The primary structure of Takadiastase ribonuclease T 1 (ribonucleate guaninenucleotido-2'-transferase (cyclizing), EC 2.7.7.26) which has a different substrate specificity from that of bovine pancreatic ribonuclease A (ribonucleate pyrimidinenucleotido-2'-transferase (cyclizing), EC 2.7.7.16) has been elucidated by TAKAHASHI1. In contrast with ribonuclease A, which is known to contain methionine residues and no tryptophan residue, ribonuclease T 1 was found to have no methionine residue, one tryptophan residue and a lower content of basic amino acids such as histidine, lysine and arginine than ribonuclease A. The active center of the ribonuclease T 1 has been investigated by means of chemical modification and kinetics studies of the enzymatic reaction 2-e. YAMAGATA et al3 have reported that the photooxidation of ribonuclease T 1 in the presence of methylene blue caused a marked inactivation of the enzyme and nearly complete inactivation occurred when approx. I mole of histidine residue out of the total of 3 was photooxidized. At that stage about half of the tryptophan residue (0. 5 mole) remained unchanged. These authors have suggested that at least one histidine residue in the molecule would be essential for its catalytic activity, but from the observations they made, the participation of the tryptophan residue in the activity could not be excluded. Biochim. Biophys. Acta, 181 (1969) 347-35 °
348
SHORT COMMUNICATIONS
On the other hand, since the tryptophan residue in the native ribonuclease T 1 was only weakly attacked by N-bromosuccinimide, it was suggested that this residue might be buried in the protein molecule and inaccessible to this reagent 7. TAKAHASHI et al. 5, by carboxymethylation with a-bromoacetate, identified the glutamic acid residue at position 58 as a part of the active site of ribonuclease T 1. We now report the reaction between 2-hydroxy-5-nitrobenzyl bromide, a tryptophan-specific reagent recently reported b y KOSHLAND et al.8, 9, and the tryptophan residue at position 59 of ribonuclease T1 in order to elucidate the function of this amino acid residue in the enzymatic activity. Ribonuclease T 1 (I rag) was dissolved in 0.55 ml of 0.05 M glycine-HC1 buffer (pH 2.8) containing an appropriate amount of urea, and the solution stood for I h
,
e
1.0
.
•
~oo~
•
8o .~ .>__ 60 I~
=o~L~ =-
E
5e~ E ~ ~
a2F
Jo
o 6
I
4
o
4o ~
;g
20 o E I
6
Urea (M)
Fig. I. U p t a k e of 2 - h y d r o x y - 5 - n i t r o b e n z y l b r o m i d e b y r i b o n u c l e a s e T x w i t h i n c r e a s i n g concent r a t i o n of urea. The r e a c t i o n was c a r r i e d o u t in 0.0 5 M gl yc i ne -H C 1 buffer (pH 2.8) c o n t a i n i n g a n a p p r o p r i a t e a m o u n t of urea. R a t i o of t h e r e a g e n t to r i b o n u c l e a s e w a s 5 ° : I. O---O, n u m b e r of 2hydroxy-5-nitrobenzyl groups introduced; © - - O , percent activity remaining.
at 37 °, then 0.05 ml of a solution of 2-hydroxy-5-nitrobenzyl bromide (5o-fold molar excess) in acetone was added to the reaction mixture. The reaction was carried out for I h, and excess reagent was then removed by dialysis of the reaction mixture against distilled water. The dialysate was concentrated to dryness by evaporation. The number of 2-hydroxy-5-nitrobenzyl groups introduced into the enzyme protein was calculated from the absorbance at 412 m/z of the dried product dissolved in o.I M K O H solution, using the molar extinction coefficient of 18900 (ref. 8). The activity of ribonuclease T 1 derivatives was determined with RNA as substrate at pH 8.0 after appropriately diluting the reaction mixture. Fig. i shows the results of these investigations. In the buffer solution containing more than 2 M urea, almost one mole of 2-hydroxy-5-nitrobenzyl bromide reacted with one mole of ribonuclease T1, and after the reaction the modified enzyme retained approx. 30% of the original enzymatic activity. The introduction of approx. 0.3 mole of the 2-hydroxy-5-nitrobenzyl group at zero urea concentration might have been caused b y the presence of approx. 8% acetone in the reaction mixture, since it was used as the solvent of the reagent and seemed to effect the modification by loosening the protein structure. As the p H - a c t i v i t y profile of 2-hydroxy-5-nitrobenzylated ribonuclease T~ (Fig. 2) indicated t h a t the p H optimum of the modified enzyme was identical with that of the native ribonuclease T1, the possibility that the decrease of the activity was caused b y alteration of the p H optimum by the modification was excluded. Biochim. Biophys. Acta, 181 (1969) 347-35 °
349
SHORT cOMMUNICATIONS
0.8
0.6
oE 0.4 0.2 "5 <
0
I
50
I
6.0
1
]
7.0 8£) pN
I
9.0
Fig. 2. p H - A c t i v i t y profile of o. i M c i t r a t e (pH 5.o-6.5), a n d s u r e d u s i n g R N A as s u b s t r a t e b e n z y l r i b o n u c l e a s e T1; O - - O ,
2 - h y d r o x y - 5 - n i t r o b e n z y l a t e d r i b o n u c l e a s e T 1. Buffers us e d were o.I M Tris-HC1 (pH 7.2-9.0). R i b o n u c l e a s e T1 a c t i v i t y w a s me a b y t h e m e t h o d x° p r e v i o u s l y r e p o r t e d . 0 - - 0 , 2 - H y d r o x y - 5 - n i t r o ribonuclease T v
In Table I the results of analysis of the tryptophan residue in the native and the modified ribonuclease T 1 are given. This table indicates that b y reaction of 2-hydroxy-5-nitrobenzyl bromide with ribonuclease T v one mole of tryptophan residue in the enzyme protein disappeared with the introduction simultaneously of almost one mole of 2-hydroxy-5-nitrobenzyl group into the enzyme. Therefore, it is clear that 2-hydroxy-5-nitrobenzyl bromide combined with the tryptophan residue at position 59. Finally we investigated the influence of the above-described modification of the tryptophan residue on the effect of the carboxymethylation with iodoacetate of the TABLE I ANALYSIS OF THE TRYPTOPHAN RESIDUE AND 2-HYDROXY-5-NITROBENZYL GROUP IN RIBONUCLEASE T1 BEFORE AND AFTER TH]~ MODIFICATION 2 - H y d r o x y - 5 - n i t r o b e n z y l a t i o n w a s c a r r i e d o u t in o.o 5 M g l y c i n e - H C 1 (pH 2.8) c o n t a i n i n g 4 M urea. Molar r a t i o of r e a g e n t to r i b o n u c l e a s e T 1 was 5o:1. T r y p t o p h a n w a s d e t e r m i n e d b y t h e m e t h o d u s i n g p - d i m e t h y l a r n i n o b e n z a l d e h y d en.
Tryptophan residue in 2-Hydroxy-5-nitrobenzyl ribonuclease T 1 group in ribonuclease T l (mole~mole ribonuclease 71) (mole/mole ribonuclease T1) Native
Modified
Native
Modified
I.I0
0.I
0.0
0.90
glutamic acid residue at the neighboring position 58, which has been identified as a part of the active site of the native enzyme 5. The results of the experiment shown in Fig. 3 revealed that the enzymatic activity of ribonuclease T 1 which remained after the modification with 2-hydroxy-5nitrobenzylation did not disappear rapidly but was only slightly decreased by the subsequent carboxymethylation. The results might be explained by an assumed decrease of the reactivity of glutamic acid residue at position 58 toward iodoacetate due to a steric hindrance, Biochim. Biophys. Acta, 181 (1969) 347-35 o
SHORT COMMUNICATIONS
350
r 80
.~ 60 g 40
•
F '°°
'c5 20
Sol lime of corboxymethylot[on(h)
Fig. 3. Inactivation of 2-hydroxy-5-nitrobenzylated ribonuclease T 1 with iodoacetate. Ribonuclease T 1 (i mg) was dissolved in 0.55 ml of 0.005 M glycine-HC1 buffer (pH 2.8) containing 2. i8 M urea and incubated for i h at 37 ° and to the mixture 5°/~1 of acetone solution of 2-hydroxy- 5nitrobenzyl bromide (5o-fold molar excess) was added. The mixture was incubated at 37 ° for an additional hour. The mixture used in the control experiment contained only acetone instead of the acetone solution of 2-hydroxy-5-nitrobenzyl bromide. To these mixtures, 3 mg of iodoacetate in o. 4 ml of o.I M acetate buffer (pH 5.0) was added and carboxymethylation was carried out at 37 °. After the addition of iodoacetate, 2o-/~1 aliquots were withdrawn at appropriate time intervals and diluted with i.o ml of water; ribonuclease Tx activity was measured using RNA as substrate. Q - - O , ribonuclease T 1 in o.I M acetate buffer (pH 5.0); O - - © , control; (~--(]~, modified ribonuclease T 1.
a n e l e c t r i c a l effect o n t h e 7 - c a r b o x y g r o u p o f t h e g l u t a m i c a c i d r e s i d u e , a n d / o r a conformational change of the active site of the enzyme, which was the result of the introduction of the 2-hydroxy-5-nitrobenzyl group into the tryptophan residue at p o s i t i o n 59, p r o v i d e d t h a t t h e i o d o a c e t a t e still r e a c t e d s p e c i f i c a l l y w i t h t h e g l u t a m i c acid residue at position 58 of the ribonuclease T 1 which was previously modified with 2-hydroxy-5-nitrobenzyl bromide and that this glutamic acid residue was involved in t h e a c t i v e s i t e o f t h e m o d i f i e d e n z y m e . W e w i s h t o t h a n k S a n k y o Co. f o r g e n e r o u s l y s u p p l y i n g t h e T a k a d i a s t a s e Sankyo.
Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo ( J a p a n )
TADAO TERAO TYUNOSIN UKITA
K. TAKAHASHI, J. Biol. Chem., 240 (1965) 4117 • S. YAMAGATA, K. TAKAHASHI AND F. EGAMI, J. Biochem. Tokyo, 52 (1962) 261. S. SAIGUSA, K. TAKAHASHI AND F. EGAMI, Symp. Enzyme Chem. Tokyo, 12 (196o) 22. H. KASAI, K. TAKAHASHI AND T. ANDO, Symp. Enzyme Chem. Tokushima, 17 (1965) 77. K. TAKAHASHI, W. H. STEIN AND S. MOORE, J. Biol. Chem., 242 (1967) 4682. M. IRIE, J. Biochem. Tokyo, 61 (1967) 55 o. K. TAKAHASHI, J. Biochem. Tokyo, 52 (1962) 72. D. E. KOSHLAND, JR., Y. D. KARHANIS AND H. G. LATHARN, dr. Am. Chem. Soc., 86 (I964) 1448. 9 T. E. BARMAN AND D. E. KOSHLAND, JR., J. Biol. Chem., 242 (1967) 5771. io T. TERAO AND T. UKITA, Biochim. Biophys. Acta, 149 (1967) 613. II J. R. SPIES ANn D. C. CHAMBERS, Anal. Chem., 21 (1949) 1249. I 2 3 4 5 6 7 8
R e c e i v e d J a n u a r y 2 n d , 1969
Biochim. Biophys. Acta, 181 (1969) 3 4 7 - 3 5 °
SHORT COMMUNICATIONS
and completely ineffective with trypsin. Table I lists the polyamines in the order of their effectiveness with ribonuclease. It should be noted (Table I) that the combined presence of the two polyamines, butyl- and propyldiamine did not provide any more stability than that obtained from each individual substance. These results suggest a direct polyamine-enzyme interaction rather than a solvent-solute effect. The nature of this attraction is not known, however, electrostatic forces are a consideration, since the molar protective effect decreases linearly with the charge of the amine.
Mount Holyoke College, Department of Biological Sciences, South Hadley, Mass. (U.S.A.) I
2 3 4 5
JACOB GOLDSTEIN
J. HERMANS AND H. A. SHERAGA, J. Am. Chem. Soc., 83 (1961) 3283. A. GINSBURG AND W. R. CARROL, Biochemistry, 4 (1965) 2159. P. H. VON HIPPEL AND K. Y. WONG, J. Biol. Chem., 240 (1965) 3909. M. KUNTZ, J. Biol. Chem., 164 (1946) 563 . D. SHUGAR, Biochim. Biophys. Acta, 8 (1952) 302.
Received October 24th, 1968 Revised manuscript received January 27th, 1969 Biochim. Biophys. Acta, 181 (1969) 345-347
BBA 33151
Modification of tryptophan residue in ribonuclease T z with 2-hyd roxy-5-nit robenzyl bromide The primary structure of Takadiastase ribonuclease T 1 (ribonucleate guaninenucleotido-2'-transferase (cyclizing), EC 2.7.7.26) which has a different substrate specificity from that of bovine pancreatic ribonuclease A (ribonucleate pyrimidinenucleotido-2'-transferase (cyclizing), EC 2.7.7.16) has been elucidated by TAKAHASHI1. In contrast with ribonuclease A, which is known to contain methionine residues and no tryptophan residue, ribonuclease T 1 was found to have no methionine residue, one tryptophan residue and a lower content of basic amino acids such as histidine, lysine and arginine than ribonuclease A. The active center of the ribonuclease T 1 has been investigated by means of chemical modification and kinetics studies of the enzymatic reaction 2-e. YAMAGATA et al3 have reported that the photooxidation of ribonuclease T 1 in the presence of methylene blue caused a marked inactivation of the enzyme and nearly complete inactivation occurred when approx. I mole of histidine residue out of the total of 3 was photooxidized. At that stage about half of the tryptophan residue (0. 5 mole) remained unchanged. These authors have suggested that at least one histidine residue in the molecule would be essential for its catalytic activity, but from the observations they made, the participation of the tryptophan residue in the activity could not be excluded. Biochim. Biophys. Acta, 181 (1969) 347-35 °
348
SHORT COMMUNICATIONS
On the other hand, since the tryptophan residue in the native ribonuclease T 1 was only weakly attacked by N-bromosuccinimide, it was suggested that this residue might be buried in the protein molecule and inaccessible to this reagent 7. TAKAHASHI et al. 5, by carboxymethylation with a-bromoacetate, identified the glutamic acid residue at position 58 as a part of the active site of ribonuclease T 1. We now report the reaction between 2-hydroxy-5-nitrobenzyl bromide, a tryptophan-specific reagent recently reported b y KOSHLAND et al.8, 9, and the tryptophan residue at position 59 of ribonuclease T1 in order to elucidate the function of this amino acid residue in the enzymatic activity. Ribonuclease T 1 (I rag) was dissolved in 0.55 ml of 0.05 M glycine-HC1 buffer (pH 2.8) containing an appropriate amount of urea, and the solution stood for I h
,
e
1.0
.
•
~oo~
•
8o .~ .>__ 60 I~
=o~L~ =-
E
5e~ E ~ ~
a2F
Jo
o 6
I
4
o
4o ~
;g
20 o E I
6
Urea (M)
Fig. I. U p t a k e of 2 - h y d r o x y - 5 - n i t r o b e n z y l b r o m i d e b y r i b o n u c l e a s e T x w i t h i n c r e a s i n g concent r a t i o n of urea. The r e a c t i o n was c a r r i e d o u t in 0.0 5 M gl yc i ne -H C 1 buffer (pH 2.8) c o n t a i n i n g a n a p p r o p r i a t e a m o u n t of urea. R a t i o of t h e r e a g e n t to r i b o n u c l e a s e w a s 5 ° : I. O---O, n u m b e r of 2hydroxy-5-nitrobenzyl groups introduced; © - - O , percent activity remaining.
at 37 °, then 0.05 ml of a solution of 2-hydroxy-5-nitrobenzyl bromide (5o-fold molar excess) in acetone was added to the reaction mixture. The reaction was carried out for I h, and excess reagent was then removed by dialysis of the reaction mixture against distilled water. The dialysate was concentrated to dryness by evaporation. The number of 2-hydroxy-5-nitrobenzyl groups introduced into the enzyme protein was calculated from the absorbance at 412 m/z of the dried product dissolved in o.I M K O H solution, using the molar extinction coefficient of 18900 (ref. 8). The activity of ribonuclease T 1 derivatives was determined with RNA as substrate at pH 8.0 after appropriately diluting the reaction mixture. Fig. i shows the results of these investigations. In the buffer solution containing more than 2 M urea, almost one mole of 2-hydroxy-5-nitrobenzyl bromide reacted with one mole of ribonuclease T1, and after the reaction the modified enzyme retained approx. 30% of the original enzymatic activity. The introduction of approx. 0.3 mole of the 2-hydroxy-5-nitrobenzyl group at zero urea concentration might have been caused b y the presence of approx. 8% acetone in the reaction mixture, since it was used as the solvent of the reagent and seemed to effect the modification by loosening the protein structure. As the p H - a c t i v i t y profile of 2-hydroxy-5-nitrobenzylated ribonuclease T~ (Fig. 2) indicated t h a t the p H optimum of the modified enzyme was identical with that of the native ribonuclease T1, the possibility that the decrease of the activity was caused b y alteration of the p H optimum by the modification was excluded. Biochim. Biophys. Acta, 181 (1969) 347-35 °
349
SHORT cOMMUNICATIONS
0.8
0.6
oE 0.4 0.2 "5 <
0
I
50
I
6.0
1
]
7.0 8£) pN
I
9.0
Fig. 2. p H - A c t i v i t y profile of o. i M c i t r a t e (pH 5.o-6.5), a n d s u r e d u s i n g R N A as s u b s t r a t e b e n z y l r i b o n u c l e a s e T1; O - - O ,
2 - h y d r o x y - 5 - n i t r o b e n z y l a t e d r i b o n u c l e a s e T 1. Buffers us e d were o.I M Tris-HC1 (pH 7.2-9.0). R i b o n u c l e a s e T1 a c t i v i t y w a s me a b y t h e m e t h o d x° p r e v i o u s l y r e p o r t e d . 0 - - 0 , 2 - H y d r o x y - 5 - n i t r o ribonuclease T v
In Table I the results of analysis of the tryptophan residue in the native and the modified ribonuclease T 1 are given. This table indicates that b y reaction of 2-hydroxy-5-nitrobenzyl bromide with ribonuclease T v one mole of tryptophan residue in the enzyme protein disappeared with the introduction simultaneously of almost one mole of 2-hydroxy-5-nitrobenzyl group into the enzyme. Therefore, it is clear that 2-hydroxy-5-nitrobenzyl bromide combined with the tryptophan residue at position 59. Finally we investigated the influence of the above-described modification of the tryptophan residue on the effect of the carboxymethylation with iodoacetate of the TABLE I ANALYSIS OF THE TRYPTOPHAN RESIDUE AND 2-HYDROXY-5-NITROBENZYL GROUP IN RIBONUCLEASE T1 BEFORE AND AFTER TH]~ MODIFICATION 2 - H y d r o x y - 5 - n i t r o b e n z y l a t i o n w a s c a r r i e d o u t in o.o 5 M g l y c i n e - H C 1 (pH 2.8) c o n t a i n i n g 4 M urea. Molar r a t i o of r e a g e n t to r i b o n u c l e a s e T 1 was 5o:1. T r y p t o p h a n w a s d e t e r m i n e d b y t h e m e t h o d u s i n g p - d i m e t h y l a r n i n o b e n z a l d e h y d en.
Tryptophan residue in 2-Hydroxy-5-nitrobenzyl ribonuclease T 1 group in ribonuclease T l (mole~mole ribonuclease 71) (mole/mole ribonuclease T1) Native
Modified
Native
Modified
I.I0
0.I
0.0
0.90
glutamic acid residue at the neighboring position 58, which has been identified as a part of the active site of the native enzyme 5. The results of the experiment shown in Fig. 3 revealed that the enzymatic activity of ribonuclease T 1 which remained after the modification with 2-hydroxy-5nitrobenzylation did not disappear rapidly but was only slightly decreased by the subsequent carboxymethylation. The results might be explained by an assumed decrease of the reactivity of glutamic acid residue at position 58 toward iodoacetate due to a steric hindrance, Biochim. Biophys. Acta, 181 (1969) 347-35 o
SHORT COMMUNICATIONS
350
r 80
.~ 60 g 40
•
F '°°
'c5 20
Sol lime of corboxymethylot[on(h)
Fig. 3. Inactivation of 2-hydroxy-5-nitrobenzylated ribonuclease T 1 with iodoacetate. Ribonuclease T 1 (i mg) was dissolved in 0.55 ml of 0.005 M glycine-HC1 buffer (pH 2.8) containing 2. i8 M urea and incubated for i h at 37 ° and to the mixture 5°/~1 of acetone solution of 2-hydroxy- 5nitrobenzyl bromide (5o-fold molar excess) was added. The mixture was incubated at 37 ° for an additional hour. The mixture used in the control experiment contained only acetone instead of the acetone solution of 2-hydroxy-5-nitrobenzyl bromide. To these mixtures, 3 mg of iodoacetate in o. 4 ml of o.I M acetate buffer (pH 5.0) was added and carboxymethylation was carried out at 37 °. After the addition of iodoacetate, 2o-/~1 aliquots were withdrawn at appropriate time intervals and diluted with i.o ml of water; ribonuclease Tx activity was measured using RNA as substrate. Q - - O , ribonuclease T 1 in o.I M acetate buffer (pH 5.0); O - - © , control; (~--(]~, modified ribonuclease T 1.
a n e l e c t r i c a l effect o n t h e 7 - c a r b o x y g r o u p o f t h e g l u t a m i c a c i d r e s i d u e , a n d / o r a conformational change of the active site of the enzyme, which was the result of the introduction of the 2-hydroxy-5-nitrobenzyl group into the tryptophan residue at p o s i t i o n 59, p r o v i d e d t h a t t h e i o d o a c e t a t e still r e a c t e d s p e c i f i c a l l y w i t h t h e g l u t a m i c acid residue at position 58 of the ribonuclease T 1 which was previously modified with 2-hydroxy-5-nitrobenzyl bromide and that this glutamic acid residue was involved in t h e a c t i v e s i t e o f t h e m o d i f i e d e n z y m e . W e w i s h t o t h a n k S a n k y o Co. f o r g e n e r o u s l y s u p p l y i n g t h e T a k a d i a s t a s e Sankyo.
Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo ( J a p a n )
TADAO TERAO TYUNOSIN UKITA
K. TAKAHASHI, J. Biol. Chem., 240 (1965) 4117 • S. YAMAGATA, K. TAKAHASHI AND F. EGAMI, J. Biochem. Tokyo, 52 (1962) 261. S. SAIGUSA, K. TAKAHASHI AND F. EGAMI, Symp. Enzyme Chem. Tokyo, 12 (196o) 22. H. KASAI, K. TAKAHASHI AND T. ANDO, Symp. Enzyme Chem. Tokushima, 17 (1965) 77. K. TAKAHASHI, W. H. STEIN AND S. MOORE, J. Biol. Chem., 242 (1967) 4682. M. IRIE, J. Biochem. Tokyo, 61 (1967) 55 o. K. TAKAHASHI, J. Biochem. Tokyo, 52 (1962) 72. D. E. KOSHLAND, JR., Y. D. KARHANIS AND H. G. LATHARN, dr. Am. Chem. Soc., 86 (I964) 1448. 9 T. E. BARMAN AND D. E. KOSHLAND, JR., J. Biol. Chem., 242 (1967) 5771. io T. TERAO AND T. UKITA, Biochim. Biophys. Acta, 149 (1967) 613. II J. R. SPIES ANn D. C. CHAMBERS, Anal. Chem., 21 (1949) 1249. I 2 3 4 5 6 7 8
R e c e i v e d J a n u a r y 2 n d , 1969
Biochim. Biophys. Acta, 181 (1969) 3 4 7 - 3 5 °