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The interaction of glycoxylate with cysteine and its application to the assay of isocitritase and of transaminases involving glyoxylate
262
BIOCHIMICA ET BIOPHYSICA ACTA
T H E I N T E R A C T I O N OF G L Y O X Y L A T E W I T H CYSTEINE AND ITS APPLICATION TO T H E ASSAY OF ISOCITRITASE AND OF TRANSAMINASES INVOLVING G L Y O X Y L A T E N. A. N. RAO AND T. RAMAKRISHNAN Pharmacology Laboratory, Indian Institute o/Science, Bangalore (India)
(Received July I7th, 1961) SUMMARY An interesting interaction between glyoxylate and cysteine takes place in phosphate buffer (pH 7.o) to form a product which is resistant to hydrolysis at ordinary temperatures. The reaction product is broken up by acid hydrolysis at elevated temperatures under controlled conditions, giving a quantitative yield of glyoxylate. Other keto acids, such as ~-ketoglutarate, pyruvate and oxaloacetate, do not interact with cysteine under similar conditions. Methods based on these findings are described for (a) direct estimation of other keto acids in the presence of glyoxylate, and (b) assay of isocitritase and glyoxylate transaminase. INTRODUCTION The reaction between cysteine and certain keto compounds was reported by SCHUBERT1,2, who showed that aldehydes and cysteine react to form a condensation product, while a ketonic compound like pyruvate yields, with cysteine, an addition compound. RATNER AND CLARKE3 studied in detail the conditions under which formaldehyde condenses with cysteine to yield thiazolidine 4-carboxylic acid, which is stable towards acid and alkali. RACKER4, however, showed that the product of reaction between methyl glyoxal and another sulphydryl compound, i.e. glutathione, is a straight-chain addition compound; he and KRIMSli¥ 5 postulated that a similar reaction takes place between glyceraldehyde phosphate and the bound glutathione of glyceraldehyde phosphate dehydrogenase. No information, however, is available on the successful quantitative recovery of the aldehyde from the aldehyde-cysteine complex, or on the reaction between cysteine and glyoxylate, which recently has been found to be an important intermediate in the metabolism of carbohydrates, fats and amino acids. In this paper we report that cysteine and glyoxylate react to form a product which can be hydrolysed to give the original reactants under specified conditions and that this reaction can be profitably applied to assays of isocitritase and of transaminases involving glyoxylate. RESULTS It has been found that glyoxylate reacts quantitatively with cysteine at pH 7.o when these two compounds are incubated together in an optimum molar ratio of 1:3 ° at Biochim. Biophys. Acta, 58 (1962) 262-265
ASSAY OF ISOCITRITASE AND GLYOXYLATE TRANSAMINASE
26 3
37 ° for 15 min. At the end of the incubation period, no glyoxylate is left as such to react with 2,4-dinitrophenylhydrazine. The temperature and duration of incubation are critical, but the p H m a y be varied from 7.0 to 8.o without any effect on the reaction. Either phosphate buffer or veronal buffer m a y be used, but in the presence of Tris buffer, the reaction does not continue to completion, presumably because a Schiff's base is formed with glyoxylate. Under the same conditions, other acids like pyruvate, oxaloacetate and ~-ketoglutarate do not react with cysteine and can be quantitatively recovered as their hydrazones. These results are presented in Table I. The glyoxylate which has reacted with cysteine can be recovered quantitatively if it is autoclaved with 2,4-dinitrophenylhydrazine in 2 N hydrochloric acid at 15 pounds pressure for 15 min under the controlled conditions described below and is then immediately cooled. Under these conditions there is no decomposition of the glyoxylate phenylhydrazone, as compared with controls kept at room temperature for the same TABLE
I
PERCENTAGE RECOVERY OF ~°KETO ACIDS ADDED TO THE REACTION MIXTURE AND INCUBATED AT 37 ° FOR 15 MIN T h e r e a c t i o n m i x t u r e c o n t a i n e d , i n a t o t a l v o l u m e of 2.0 ml, 0.25 p m o l e of k e t o acid, 25.0 p m o l e s of p h o s p h a t e buffer of p H 7.o a n d 7.5 p m o l e s of c y s t e i n e h y d r o c h l o r i d e . A f t e r i n c u b a t i o n , i ni l of a o . i % s o l u t i o n of 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e in 2 N HC1 w a s a d d e d a n d t h e k e t o a c i d e s t i m a t e d u s i n g a K l e t t p h o t o e l e c t r i c c o l o r i m e t e r p r o v i d e d w i t h a filter h a v i n g m a x i m a l t r a n s m i t t a n c e a t 420 m p .
c~-Keto acid
Water only
Phosphae buffer pH 7.0
Glyoxylate Pyruvate ,e-Ketoglutarate Oxaloacetate
ioo.o ioo.o ioo.o ioo.o
99.9 ioo.o lOO.1 ioo.4
Phospha.e Cysteine buffer and hydro-chloride c y s t e i n e
63.7 99.9 99.8 99.7
o.o 99.3 99.5 ioo.o
Phospha2e buffer, cysteineand glyoxylate o.o 99.8 IOO.5 1oi.2
TABLE II RECOVERY OF GLYOXYLATE ADDED XO REACTION MIXTURE AND INCUBATED AT 37 ° FOR 15 MIN T h e r e a c t i o n m i x t u r e c o n t a i n e d i n a t o t a l V o l u m e of 2.0 ml, i o . o p m o l e s of p h o s p h a t e b n f f e r of p H 7.0 for e v e r y o. i o p m o l e of g l y o x y l a t e a n d 3 . o / * m o l e s of c y s t e i n e h y d r o c h l o r i d e a n d I to m l vf o . I % 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e i n 2 N HC1. A f t e r a u t o c l a v i n g for 15 ra i n a t 15 lbs. pre s s ure , t h e s a m p l e s w e r e cooled u n d e r t h e t a p , k e p t a t r o o m t e m p e r a t u r e for 5 rain a n d t h e k e t o a c i d e s t i m a t e d u s i n g a K l e t t p h o t o e l e c t r i c c o l o r i m e t e r p r o v i d e d w i t h a filter h a v i n g a m a x i m a l t ra ns ~ m i t t a n c e a t 420 rap.
Glyoxylate added
( pmolvs)
i 2 3 4 5
o.io 0.20 o.3o o.4o o.5o
Cysteine added ( l~moles)
3.o 6.o 9.o 12.o 15-o
Glyozylat*recovered I~moles Before auJoclaping o o o o o
L fret
auJocla~ng
o.Io o.2o o.29 0.39 o.46
B i o c h i m . Biophys. Acta, 58 (I962) 262 -265
264
N. A. N. RAO, T. RAMAKRISHNAN
period, presumably because the phenylhydrazone is formed only after the mixture is cooled to room temperature. Table I I gives the data on the recovery of glyoxylate. The resistance of the cysteine-glyoxylate complex to hydrolysis at ordinary temperatures has been used for the evolution of a method for the analysis of the following two enzymes: Isocitritase
OLSOI~8 has shown that cysteine, added to activate isocitritase traps part of the glyoxylate formed during incubation, so that the recoveries of glyoxylate are lower than those obtained with 2,4-dinitrophenylhydrazone, because the reaction between glyoxylate and cysteine will even take place in an atmosphere of nitrogen. DIXON AND KORNBERG7 devised a procedure for isoeitritase assay which consists essentially of trapping the glyoxylate formed in situ with phenylhydrazine at p H 6.8 and measuring the formation of glyoxylate phenylhydrazone. This assay requires the use of a Cary recording spectrophotometer. However, because it has been shown that the o p t i m u m p H of isocitritase isolated from Pseudomonas aeruginosa is 7.6, this method of assay m a y not detect the m a x i m u m amount of glyoxylate which could be formed from isocitritase when the enzyme shows a p H optimum on the alkaline side. This disadvantage is overcome b y the following modified procedure. The isocitritase reaction is carried out as follows: The reaction mixture, which contains 20 /~moles of DL-isocitrate, 5 /,moles of cysteine hydrochloride, 5/~moles of magnesium sulphate, 200 ~moles of phosphate buffer of p H 7.6 and 0.2 ml of enzyme in a total volume of 1. 9 ml, is incubated at the desired temperature for 15 rain and the reaction is stopped b y the addition of o.i ml of lOO% (w/v) trichloroacetic acid. Aliquots of the protein-free supernatant, containing 0.25-0.50 /zmole of glyoxylate, as well as of the blank in which the reaction has been stopped at zero time, are taken in large p y r e x test tubes and I.O ml of o.I % dinitrophenylhydrazine in 2 N HCI is added. The total volume in each tube is then made up to 3 ml with distilled water and the tubes are covered with aluminium foil or other suitable material. The blank and experimental samples are then placed in a small autoclave which has been previously kept steaming, and the pressure is brought up to 15 pounds as rapidly as possible and is maintained at this pressure for 15 min. At the end of this time, the pressure release valve is opened slightly, so that the pressure falls to zero within 5-7 rain. The tubes are then immediately removed, quickly cooled in running water to room temperature and allowed to stand at room temperature for 5 rain before the glyoxylate is estimated b y the method of FRIEOMANN9. Using this procedure, we have assayed the isocitritase of Pseudomonas fluorescens A3.I2, which, as SMITH et al. 1° have shown, possesses the enzyme. We obtained a glyoxylate recovery of 1.86/zmoles/mg protein after autoclaving, as compared with a recovery of o.86 ~mole/mg protein before autoclaving. When Tris was used to replace phosphate as the buffer in the reaction mixture, the recoveries of glyoxylate were x.86 /zmoles and 1.28 /~moles respectively. In both instances, therefore, autoclaving ;gives a higher recovery of glyoxylate, a fact which indicates that a part of the glyoxylate formed has reacted upon cysteine present in the reaction mixture. That glyoxylate was the only keto acid formed in every instance was shown by paper chromatography .of the dinitrophenylhydrazone b y the method of CAVALLINIet al. n, using an authentic sample of glyoxylate dinitrophenylhydrazone as marker. Biochim. Biophys. Acta, 58 (1962) 262-265
ASSAY OF ISOCITRITASE AND GLYOXYLATE TRANSAMINASE
265
Glyoxylate transaminases
CAMPBELL12reported that glyoxylate transaminates enzymically with a number of amino acids, with the formation of glycine and the corresponding keto acid. Thus, alanine yields pyruvate, aspartate gives oxaloacetate and glutamate forms a-ketoglutarate. FRIEDMANN'S9 method has been the standard procedure for the assay of transaminases in which a monocarboxylic keto acid reacts with an amino acid to give a dicarboxylic acid or vice versa. It consists of conducting a duplicate assay, one for extracting most of the monocarboxylic acid dinitrophenylhydrazone and another for extracting most of the dicarboxylic acid dinitrophenylhydrazone, and the subsequent application of a correction factor to obtain the value of each. Using this method, SASTRY AND RAMAKRISHNAN13 have standardized a procedure for estimating ,¢-ketoglutarate in the presence of glyoxylate, but no similar standardisation has been developed for estimating the other dicarboxylic keto acids in the presence of glyoxylate. Further, pyruvate formed b y transamination of alanine with glyoxylate cannot be assayed by this procedure, because both are monocarboxylic acids. The modified method described above eliminates the need for a duplicate assay and permits the direct estimation of the a-ketoglutarate, oxaloacetate or pyruvate formed during transamination in the presence of glyoxylate. The transaminase reaction involving glyoxylate and any of the amino acids is allowed to proceed for the required time in phosphate buffer. To an aliquot of the deproteinised reaction mixture, which contains approx. 0.5 /~mole of glyoxylate, 15 ~moles of cysteine hydrochloride are added and the solution is then neutralised with 0.5 N sodium hydroxide. 50 ~moles of phosphate buffer, p H 7.5, are then added and the mixture is incubated for 15 min at 37 °. After the incubation, the new keto acid formed which does not react with cysteine is determined by FRIEDMANN'S method as the 2,4-dinitrophenylhydrazone. I t has been shown that the presence of up to IOO gg of pyridoxal phosphate in the reaction mixture does not interfere with the determination. ACKNOWLEDGEMENTS
The authors thank Dr. M. SIRSI for his keen interest in their work. One of them (N.A.N.R.) is indebted to the National Institute of Sciences of India for the award of a research fellowship to him. REFERENCES 1 M. P. SCHtlBERT, J. Biol. Chem., i i i (1935) 671. 2 M. P. SCHUBERT, J. Biol. Chem., 114 /I936) 341. 3 S. RATNER AND H. T. CLARKE, J. Am. Chem. Soc., 59 (1937) 2oo. 4 E. RACKER, J. Biol. Chem., 19o (1951) 685. 5 E. RACKER AND I. KRIMSKY, J. Biol. Chem., 198. (1952) 731. s j . A. OLSON, J. Biol. Chem., 234 (1959) 5. 7 G. H. DIXON AND H. L. KORNBERG, Biochem. J., 72 (1959) 3P. 8 R. A. SMITH AND I. C. GUNSALUS, J. Biol. Chem., 229 (1957) 3o5 . 9 T. E. FRIEDMANN, Methods in Enzymology, Vol. 3, A c a d e m i c Press, Inc., N e w Y ork, 1957, p. 414 • 10 R. A. SMITH AND I. C. GUNSALUS, Nature, 175 (1955) 77411 n . CAVALLINI, N. FRONTALI AND G. TOUCHI, Nature, 164 (1949) 782. 12 L. L. CAMPBELL, J. Bacteriol., 71 (1956) 81. 18 L. V. S. SASTRY AND T. RAMAKRISHNAN, J. Sci. Ind. Research (India), 2oC (1961) 277.
Biochim. Biophys. ,4cta, 58 (1962) 2 6 2 - 2 6 5
BIOCHIMICA ET BIOPHYSICA ACTA
T H E I N T E R A C T I O N OF G L Y O X Y L A T E W I T H CYSTEINE AND ITS APPLICATION TO T H E ASSAY OF ISOCITRITASE AND OF TRANSAMINASES INVOLVING G L Y O X Y L A T E N. A. N. RAO AND T. RAMAKRISHNAN Pharmacology Laboratory, Indian Institute o/Science, Bangalore (India)
(Received July I7th, 1961) SUMMARY An interesting interaction between glyoxylate and cysteine takes place in phosphate buffer (pH 7.o) to form a product which is resistant to hydrolysis at ordinary temperatures. The reaction product is broken up by acid hydrolysis at elevated temperatures under controlled conditions, giving a quantitative yield of glyoxylate. Other keto acids, such as ~-ketoglutarate, pyruvate and oxaloacetate, do not interact with cysteine under similar conditions. Methods based on these findings are described for (a) direct estimation of other keto acids in the presence of glyoxylate, and (b) assay of isocitritase and glyoxylate transaminase. INTRODUCTION The reaction between cysteine and certain keto compounds was reported by SCHUBERT1,2, who showed that aldehydes and cysteine react to form a condensation product, while a ketonic compound like pyruvate yields, with cysteine, an addition compound. RATNER AND CLARKE3 studied in detail the conditions under which formaldehyde condenses with cysteine to yield thiazolidine 4-carboxylic acid, which is stable towards acid and alkali. RACKER4, however, showed that the product of reaction between methyl glyoxal and another sulphydryl compound, i.e. glutathione, is a straight-chain addition compound; he and KRIMSli¥ 5 postulated that a similar reaction takes place between glyceraldehyde phosphate and the bound glutathione of glyceraldehyde phosphate dehydrogenase. No information, however, is available on the successful quantitative recovery of the aldehyde from the aldehyde-cysteine complex, or on the reaction between cysteine and glyoxylate, which recently has been found to be an important intermediate in the metabolism of carbohydrates, fats and amino acids. In this paper we report that cysteine and glyoxylate react to form a product which can be hydrolysed to give the original reactants under specified conditions and that this reaction can be profitably applied to assays of isocitritase and of transaminases involving glyoxylate. RESULTS It has been found that glyoxylate reacts quantitatively with cysteine at pH 7.o when these two compounds are incubated together in an optimum molar ratio of 1:3 ° at Biochim. Biophys. Acta, 58 (1962) 262-265
ASSAY OF ISOCITRITASE AND GLYOXYLATE TRANSAMINASE
26 3
37 ° for 15 min. At the end of the incubation period, no glyoxylate is left as such to react with 2,4-dinitrophenylhydrazine. The temperature and duration of incubation are critical, but the p H m a y be varied from 7.0 to 8.o without any effect on the reaction. Either phosphate buffer or veronal buffer m a y be used, but in the presence of Tris buffer, the reaction does not continue to completion, presumably because a Schiff's base is formed with glyoxylate. Under the same conditions, other acids like pyruvate, oxaloacetate and ~-ketoglutarate do not react with cysteine and can be quantitatively recovered as their hydrazones. These results are presented in Table I. The glyoxylate which has reacted with cysteine can be recovered quantitatively if it is autoclaved with 2,4-dinitrophenylhydrazine in 2 N hydrochloric acid at 15 pounds pressure for 15 min under the controlled conditions described below and is then immediately cooled. Under these conditions there is no decomposition of the glyoxylate phenylhydrazone, as compared with controls kept at room temperature for the same TABLE
I
PERCENTAGE RECOVERY OF ~°KETO ACIDS ADDED TO THE REACTION MIXTURE AND INCUBATED AT 37 ° FOR 15 MIN T h e r e a c t i o n m i x t u r e c o n t a i n e d , i n a t o t a l v o l u m e of 2.0 ml, 0.25 p m o l e of k e t o acid, 25.0 p m o l e s of p h o s p h a t e buffer of p H 7.o a n d 7.5 p m o l e s of c y s t e i n e h y d r o c h l o r i d e . A f t e r i n c u b a t i o n , i ni l of a o . i % s o l u t i o n of 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e in 2 N HC1 w a s a d d e d a n d t h e k e t o a c i d e s t i m a t e d u s i n g a K l e t t p h o t o e l e c t r i c c o l o r i m e t e r p r o v i d e d w i t h a filter h a v i n g m a x i m a l t r a n s m i t t a n c e a t 420 m p .
c~-Keto acid
Water only
Phosphae buffer pH 7.0
Glyoxylate Pyruvate ,e-Ketoglutarate Oxaloacetate
ioo.o ioo.o ioo.o ioo.o
99.9 ioo.o lOO.1 ioo.4
Phospha.e Cysteine buffer and hydro-chloride c y s t e i n e
63.7 99.9 99.8 99.7
o.o 99.3 99.5 ioo.o
Phospha2e buffer, cysteineand glyoxylate o.o 99.8 IOO.5 1oi.2
TABLE II RECOVERY OF GLYOXYLATE ADDED XO REACTION MIXTURE AND INCUBATED AT 37 ° FOR 15 MIN T h e r e a c t i o n m i x t u r e c o n t a i n e d i n a t o t a l V o l u m e of 2.0 ml, i o . o p m o l e s of p h o s p h a t e b n f f e r of p H 7.0 for e v e r y o. i o p m o l e of g l y o x y l a t e a n d 3 . o / * m o l e s of c y s t e i n e h y d r o c h l o r i d e a n d I to m l vf o . I % 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e i n 2 N HC1. A f t e r a u t o c l a v i n g for 15 ra i n a t 15 lbs. pre s s ure , t h e s a m p l e s w e r e cooled u n d e r t h e t a p , k e p t a t r o o m t e m p e r a t u r e for 5 rain a n d t h e k e t o a c i d e s t i m a t e d u s i n g a K l e t t p h o t o e l e c t r i c c o l o r i m e t e r p r o v i d e d w i t h a filter h a v i n g a m a x i m a l t ra ns ~ m i t t a n c e a t 420 rap.
Glyoxylate added
( pmolvs)
i 2 3 4 5
o.io 0.20 o.3o o.4o o.5o
Cysteine added ( l~moles)
3.o 6.o 9.o 12.o 15-o
Glyozylat*recovered I~moles Before auJoclaping o o o o o
L fret
auJocla~ng
o.Io o.2o o.29 0.39 o.46
B i o c h i m . Biophys. Acta, 58 (I962) 262 -265
264
N. A. N. RAO, T. RAMAKRISHNAN
period, presumably because the phenylhydrazone is formed only after the mixture is cooled to room temperature. Table I I gives the data on the recovery of glyoxylate. The resistance of the cysteine-glyoxylate complex to hydrolysis at ordinary temperatures has been used for the evolution of a method for the analysis of the following two enzymes: Isocitritase
OLSOI~8 has shown that cysteine, added to activate isocitritase traps part of the glyoxylate formed during incubation, so that the recoveries of glyoxylate are lower than those obtained with 2,4-dinitrophenylhydrazone, because the reaction between glyoxylate and cysteine will even take place in an atmosphere of nitrogen. DIXON AND KORNBERG7 devised a procedure for isoeitritase assay which consists essentially of trapping the glyoxylate formed in situ with phenylhydrazine at p H 6.8 and measuring the formation of glyoxylate phenylhydrazone. This assay requires the use of a Cary recording spectrophotometer. However, because it has been shown that the o p t i m u m p H of isocitritase isolated from Pseudomonas aeruginosa is 7.6, this method of assay m a y not detect the m a x i m u m amount of glyoxylate which could be formed from isocitritase when the enzyme shows a p H optimum on the alkaline side. This disadvantage is overcome b y the following modified procedure. The isocitritase reaction is carried out as follows: The reaction mixture, which contains 20 /~moles of DL-isocitrate, 5 /,moles of cysteine hydrochloride, 5/~moles of magnesium sulphate, 200 ~moles of phosphate buffer of p H 7.6 and 0.2 ml of enzyme in a total volume of 1. 9 ml, is incubated at the desired temperature for 15 rain and the reaction is stopped b y the addition of o.i ml of lOO% (w/v) trichloroacetic acid. Aliquots of the protein-free supernatant, containing 0.25-0.50 /zmole of glyoxylate, as well as of the blank in which the reaction has been stopped at zero time, are taken in large p y r e x test tubes and I.O ml of o.I % dinitrophenylhydrazine in 2 N HCI is added. The total volume in each tube is then made up to 3 ml with distilled water and the tubes are covered with aluminium foil or other suitable material. The blank and experimental samples are then placed in a small autoclave which has been previously kept steaming, and the pressure is brought up to 15 pounds as rapidly as possible and is maintained at this pressure for 15 min. At the end of this time, the pressure release valve is opened slightly, so that the pressure falls to zero within 5-7 rain. The tubes are then immediately removed, quickly cooled in running water to room temperature and allowed to stand at room temperature for 5 rain before the glyoxylate is estimated b y the method of FRIEOMANN9. Using this procedure, we have assayed the isocitritase of Pseudomonas fluorescens A3.I2, which, as SMITH et al. 1° have shown, possesses the enzyme. We obtained a glyoxylate recovery of 1.86/zmoles/mg protein after autoclaving, as compared with a recovery of o.86 ~mole/mg protein before autoclaving. When Tris was used to replace phosphate as the buffer in the reaction mixture, the recoveries of glyoxylate were x.86 /zmoles and 1.28 /~moles respectively. In both instances, therefore, autoclaving ;gives a higher recovery of glyoxylate, a fact which indicates that a part of the glyoxylate formed has reacted upon cysteine present in the reaction mixture. That glyoxylate was the only keto acid formed in every instance was shown by paper chromatography .of the dinitrophenylhydrazone b y the method of CAVALLINIet al. n, using an authentic sample of glyoxylate dinitrophenylhydrazone as marker. Biochim. Biophys. Acta, 58 (1962) 262-265
ASSAY OF ISOCITRITASE AND GLYOXYLATE TRANSAMINASE
265
Glyoxylate transaminases
CAMPBELL12reported that glyoxylate transaminates enzymically with a number of amino acids, with the formation of glycine and the corresponding keto acid. Thus, alanine yields pyruvate, aspartate gives oxaloacetate and glutamate forms a-ketoglutarate. FRIEDMANN'S9 method has been the standard procedure for the assay of transaminases in which a monocarboxylic keto acid reacts with an amino acid to give a dicarboxylic acid or vice versa. It consists of conducting a duplicate assay, one for extracting most of the monocarboxylic acid dinitrophenylhydrazone and another for extracting most of the dicarboxylic acid dinitrophenylhydrazone, and the subsequent application of a correction factor to obtain the value of each. Using this method, SASTRY AND RAMAKRISHNAN13 have standardized a procedure for estimating ,¢-ketoglutarate in the presence of glyoxylate, but no similar standardisation has been developed for estimating the other dicarboxylic keto acids in the presence of glyoxylate. Further, pyruvate formed b y transamination of alanine with glyoxylate cannot be assayed by this procedure, because both are monocarboxylic acids. The modified method described above eliminates the need for a duplicate assay and permits the direct estimation of the a-ketoglutarate, oxaloacetate or pyruvate formed during transamination in the presence of glyoxylate. The transaminase reaction involving glyoxylate and any of the amino acids is allowed to proceed for the required time in phosphate buffer. To an aliquot of the deproteinised reaction mixture, which contains approx. 0.5 /~mole of glyoxylate, 15 ~moles of cysteine hydrochloride are added and the solution is then neutralised with 0.5 N sodium hydroxide. 50 ~moles of phosphate buffer, p H 7.5, are then added and the mixture is incubated for 15 min at 37 °. After the incubation, the new keto acid formed which does not react with cysteine is determined by FRIEDMANN'S method as the 2,4-dinitrophenylhydrazone. I t has been shown that the presence of up to IOO gg of pyridoxal phosphate in the reaction mixture does not interfere with the determination. ACKNOWLEDGEMENTS
The authors thank Dr. M. SIRSI for his keen interest in their work. One of them (N.A.N.R.) is indebted to the National Institute of Sciences of India for the award of a research fellowship to him. REFERENCES 1 M. P. SCHtlBERT, J. Biol. Chem., i i i (1935) 671. 2 M. P. SCHUBERT, J. Biol. Chem., 114 /I936) 341. 3 S. RATNER AND H. T. CLARKE, J. Am. Chem. Soc., 59 (1937) 2oo. 4 E. RACKER, J. Biol. Chem., 19o (1951) 685. 5 E. RACKER AND I. KRIMSKY, J. Biol. Chem., 198. (1952) 731. s j . A. OLSON, J. Biol. Chem., 234 (1959) 5. 7 G. H. DIXON AND H. L. KORNBERG, Biochem. J., 72 (1959) 3P. 8 R. A. SMITH AND I. C. GUNSALUS, J. Biol. Chem., 229 (1957) 3o5 . 9 T. E. FRIEDMANN, Methods in Enzymology, Vol. 3, A c a d e m i c Press, Inc., N e w Y ork, 1957, p. 414 • 10 R. A. SMITH AND I. C. GUNSALUS, Nature, 175 (1955) 77411 n . CAVALLINI, N. FRONTALI AND G. TOUCHI, Nature, 164 (1949) 782. 12 L. L. CAMPBELL, J. Bacteriol., 71 (1956) 81. 18 L. V. S. SASTRY AND T. RAMAKRISHNAN, J. Sci. Ind. Research (India), 2oC (1961) 277.
Biochim. Biophys. ,4cta, 58 (1962) 2 6 2 - 2 6 5