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Chromatographic purification of isoenzymes of human α-amylases
362
SHORT ( OMM('NICATIONS
BBA 3 3 Z O 9
Chromatographic purification of isoenzymes of human ~-amylases W e have been s t u d y i n g the e l e c t r o p h o r e t i c p r o p e r t i e s of tile isoenzymes of h u m a n a - a m y l a s e s ((t-I,4-glucan 4-glucanohydrolase, EC 3.2. i. I) from pancreas, saliva, milk a, urine a n d o v a r i a n cyst fluid 2. On agar-gel electrophoresis eight isoenzymes m a y be recognised (Fig. I). W e decided to p u r i f y some of these isoenzymes as well as s t u d v t h e i r b e h a v i o u r on c h r o m a t o g r a p h i c columns c o n t a i n i n g v a r i o u s media.
®
i 53
oFT-
Kig. I. Electrophoretic mobilities of the isoenzymes of human a-amylas( in agar gel. I o.o5, Veronal buffer (pH 8.(~). lq, P2, P3 : pancreatic, $1, $2, S3 salivary, ()l, ()z : ovarian cyst fluid. The relative mobilities of serum ?z-globulins (y) and transfcrrin (ill) are shown alongside. H u m a n p a n c r e a t i c tissues were fresh a u t o p s y m a t e r i a l . T h e y were homogenised with twice their weight of ice-cold buffer in an A T O - M I X b l e n d o r run at " h i g h " speed for 2 rain. The buffer used is (I o.o5) T r i s . HC1-Tris (pH 8.6 at 4°) a c o n t a i n i n g 0.o2 M CaC12 to stabilise tile enzyme. The e x t r a c t was c e n t r i f u g e d at IO ooo r e v . / m i n for 2o rain at 4 °. The clear s u p e r n a t a n t was t y p e d as to i s o a m y l a s e c o n s t i t u t i o n b y agar-gel electrophoresis 2 a n d tile e n z y m e a c t i v i t y e s t i m a t e d b y an a m y l o c l a s t i c m e t h o d 4. A b o u t 80 ml of saliva was collected from 2 v o l u n t e e r s a n d o. 5 nil toluene was a d d e d as preservative. The saliva was s t o r e d in t h e cold overnight, d e c a n t e d from cellular a n d mucinous d e b r i s a n d c e n t r i f u g e d at 5ooo r e v . / m i n for IO rain. Tile clarified s a l i v a was p u t into Visking cellophane t u b i n g a n d d i a l y s e d against the Tris buffer for 24 h u n d e r m a g n e t i c stirring. P a r t i c u l a t e m a t t e r fornled d u r i n g t h e dialysis was again centrifuged off a n d t h e e n z y m e a c t i v i t y of the saliva e s t i m a t e d . Milk e x t r a c t e d b y b r e a s t - p u m p from six l a c t a t i n g m o t h e r s were pooled. This milk, a l n o u n t i n g to a b o u t 15o ml, was c e n t r i f u g e d in the cold at 1o ooo r e v . / m i n for 3o min. L i p i d m a t e r i a l f o r m e d a firm " b u t t o n " on t h e t o p of an opalescent lower laver. This l a y e r was d i a l y s e d a g a i n s t tile Tris buffer as described for saliva, a n d centrifuged again. R a n d o m urine specimens collected from 22 h e a l t h y staff m e m b e r s were filtered a n d 500 ml of tile urine pool was c o n c e n t r a t e d in Visking cellophane t u b i n g a g a i n s t p o l y e t h y l e n e glycol (Carbowax 4ooo, British D r u g Houses Ltd.), The c o n c e n t r a t e d Biochim. Biophvs. Hera, I~)S (1968) 362 365
363
SHORT COMMUNICATIONS
urine, about 1/~0oth the original volume, was then dialysed in the cold against the Tris buffer at a lower I of o.o175.
Purification of pancreatic, salivary and milk a-amylases DEAE-Sephadex A-5o columns, 2.5 cm × 24 cm, were equilibrated with the Tris buffer (I 0.05). Preliminary experiments showed that the enzyme could be eluted from the DEAE-Sephadex without altering the composition of the buffer. Neither increasing I to 0.5 with NaC1, nor decreasing the p H to 5 in the form of a linear gradient resulted in the further recovery of the enzyme. The D E A E columns were charged with 2 ml of the material to be fractionated which should contain about 15oo Somogyi units of enzyme activity. It is important not to overload the column. With a flow rate of 20 ml/h the effluent was collected in 3-ml fractions by siphon. Aliquots from each fraction were spotted on a starch substrate plate 5. After incubation at 37 ° for 20 min and development with ethanolic iodine, digested areas on the plate indicated which tubes contained amylase activity. The amounts of enzyme in these tubes were quantitatively determined. Agar-gel electrophoresis was carried out on selected tubes with amyloclastic activity to find out the order in which the isoenzymes were being eluted. All fractions with enzyme activity were combined and concentrated to a small volume by ultrafiltration. This was dialysed overnight against phosphate buffer used in the next stage. Columns of Sephadex G-75 2.5 cm × 30 cm, were equilibrated with o.I M phosphate buffer (pH 7.0). Enzyme concentrates obtained from the D E A E column were applied on these columns in I-ml portions. The detection of amyloclastic activity in the effluent is similar to that already described. The specific extinction E 1% cm = 23.3 at 280 m~u was given b y STEIN, H s l o AND FISCHER6 for crystalline salivary amylase. Using this factor the specific activities of the initial and final stages of purification of pancreatic and salivary amylases were calculated.
Purification of pancreatic and salivary amylases from urine In this experiment DEAE-cellulose was preferred to DEAE-Sephadex because the latter tended to shrink greatly during the application of the buffer with o.I M NaC1, making regeneration of the ion-exchange dextran difficult. A DEAE-cellulose (Whatman Powder D E 50) column (12 cm × 0.8 cm) was washed several times with (I o.o175 ) Tris buffer (pH 8.6 at 4°). Concentrated and dialysed urine was electrophoresed to establish its a-amylase isoenzyme structure. A o.5-ml sample volume was allowed to soak into the top layer of the column and collected in 2-ml fractions before elution was continued with the same buffer containing o.I M NaC1. The effluent was treated as described above. The a-amylases recovered before and after the step-wise elution were concentrated and identified b y agar-gel electrophoresis against the original urine. After several experiments it was possible to calibrate the column in terms of the number of fractions needed to recover most of the isoenzymes. Five recovery experiments were done. Each column m a y be used m a n y times, if re-equilibrated with the starting buffer, without losing its characteristics. Some of the experiments were done at room temperature (22 °) at which the buffer p H was 8.2. This had no effect on the quality of the isoenzyme separation. Biochim. Biophys. Acta, 168 (1968) 362-365
364
SHORT COMMUNICATIONS
3oo
P2 J\
J
-~, 2 0 0
P1
o~
I
$
[
i
t
!,
300
/
\ i
I I
~
/ i
Ji
f 0
,7 0
.-'(".'.'.. . . \
.-" I ........ '--,
\ \
... ,," \.
,' ........ . .... ~'"
7-0
-
."" -; ...,----40 Tube
.
', ". " ,
.........:,,.
6o
;"':.-:.-.>--
8o
number
Fig. 2. C h r o m a t o g r a p h i c separation of h u m a n a-amylases. Elution profiles of two types of pancreatic e x t r a c t s (containing P2, and P I + P2), saliva and milk on the same D E A E - S e p h a d e x A-5 o c o l u m n (2.5 cm × 24 cm) with (I 0.o5) Tris buffer (pH 8.6). E a c h of the four samples contained a b o u t I3OO Somogyi units of enzyme activity. (For isoenzyme notation, please see Fig. I.) ----, pancreas (PI @ P2); • . ., pancreas (P2); . . . . . . , saliva; . . . . , milk. Fig. 3. Agar-gel electrophoresis of isoenzymes of u r i n a r y ~z-amylase separated on a o.8 c m x ]2 cm DEAE-cellulose column, with Tris buffer (pH 8.2}. A, pancreatic isoenzymes obtained with the buffer at I o.o175; B, salivary isoenzymes obtained w i t h the same buffer containing o.i M NaC1; U, original concentrated urine. The cathode is above.
A common feature in the behaviour of the various a-amylases, whether with DEAE-Sephadex A-5o or Sephadex G-75, is the tendency to trail as they come off the column. Fig. 2 illustrates the elution profiles of pancreatic, salivary and milk amylases fractionated on the same DEAE-Sepbadex A-5o column. The order in which the isoenzymes were eluted was P I , P2, SI and $2. On the Sephadex G-75 column PI was eluted just before P2, and P2 before SI, with some overlap. The specific activity of salivary amylase after purification was 4 times that of the starting material. COLOWlCKAND KAPLAN7 found the specific activity of reerystallised human salivary amylase to be 8 times that of saliva. The specific activity of pancreatic amylase was 45 times that of the original extract. I t can be seen that the procedure outlined separates the urinary pancreatic isoenzymes, P2 and P3, from the salivary group SI and $2 {Fig. 3)- In five recovery experiments almost all the enzyme applied to the column was retrieved. The order in which the isoenzymes are eluted, t h a t is, PI, P2, SI, $2 appears to be in accord with their electrophoretic mobilities. One can exclude the likelihood that molecular seiving contributes to the results, as Sephadex A-5o has an exclusion molecular weight of IO ooo. However on the Sephadex G-75 column the order of elution is also sinfilar to that on DEAE-Sephadex A-5o. Two unrelated explanations m a y be offered for these results. One is that molecular size and shape favour the elution of PI, then P2 and SI. Minor differences of these parameters cannot be excluded b y the results of the present experiments. The other explanation favours the possibility that differences in affinity of the various isoenzymes for the gel-matrix, rich in a-I,6-glucosidic bonds, m a y be Biochim. Biophys. Acta, i68 (1968) 362-365
SHORT COMMUNICATIONS
365
decisiveS,9. a-Amylase is not entirely specific for a-I,4-glucosidic linkages 1°. Polyacrylamide gel beads, Bio-Gel P-6o (Bio-Rad Laboratories, Calif.) have a porosity comparable with that of Sephadex G-75. Elution from columns made with Bio-Gel P-6o fail to separate P I from P2 or P2 from SI (unpublished observations). Thus penetration of the gel matrix per se cannot account for the differential retardation. GELOTTE performed elution experiments with 0.02 M Tris buffer (pH 7) and showed that amylase was retarded on Sephadex G-75, the magnitude depending on the flow rate of the buffer. No retardation was seen with Sephadex G-25, which the enzyme would not be expected to penetrate 9. ONO, HIROMI AND YOSHIKAWA11 found that the Michaelis constant of B. subtilis a-amylase was unaffected by either the nature or concentration of the anions present in the buffer. The Michaelis constant is an expression of the affinity of the binding site of an enzyme for its substrate. This site, or sites, with which C1- and other anions react, differ from the catalytic centre(s) responsible for the actual scission of the a-I,4-bonds. It is probably the binding site that is involved in the amylase-Sephadex interaction. It appears significant that ONo and his associates discovered that the Michaelis constant for their enzyme was constant over the pH range 3.6-8. 4. GELOTTE9 similarly noted that the elution pattern of amylase was not affected by variation of pH from 5 to 8. One of us (S.E.A.) is grateful to the University of Singapore for an Overseas Training Scholarship.
Department of Chemical Pathology, Royal Postgraduate Medical School, London W. 12 (Great Britain) i 2 3 4 5 6 7 8 9 IO ii
S. E. Aw* J. R. HOBBS I. D. P. WOOTTON
S. E. A w AND J. R. HOBBS, Biochem. J., 99 (1966) I6P. S. E. Aw, J. R. HOBBS AND I. D. P. WOOTTON, Gut, 8 (1967) 402. C. LONG, Biochemists' Handbook, V a n N o s t r a n d , Toronto, 1961 , p. 33. I. D. P. WOOTTON, Micro-Analysis in Medical Biochemistry, 4 t h edition, Churchill, L o n d o n , 1964, p. lO6. S. E. Aw, Nature, 2o9 (1966) 298. E. A. STEIN, J. H s l u AND E. H. FISCHER, Biochemistry, 3 (1964) 56. S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, A c a d e m i c Press, N e w York, 1955, p. 15o. P. WILDING, Clin. Chim. Acta, 8 (1963) 918. B. GELOTTE, Acta Chem. Scand., 18 (1964) 1283. ]3. J. BINES AND W. J. WHELAN, Chem. Ind. London, 31 (196o) 997. S. ONO, K. HIROMI AND Y. YOSHIKAWA, Bull. Chem. Soc. Japan, 31 (1958) 957-
Received May 7th, 1968 * P r e s e n t a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , F a c u l t y of Medicine, S e p o y Lines, Singapore 3.
Biochim. Biophys. Acta, 168 (1968) 362-365
SHORT ( OMM('NICATIONS
BBA 3 3 Z O 9
Chromatographic purification of isoenzymes of human ~-amylases W e have been s t u d y i n g the e l e c t r o p h o r e t i c p r o p e r t i e s of tile isoenzymes of h u m a n a - a m y l a s e s ((t-I,4-glucan 4-glucanohydrolase, EC 3.2. i. I) from pancreas, saliva, milk a, urine a n d o v a r i a n cyst fluid 2. On agar-gel electrophoresis eight isoenzymes m a y be recognised (Fig. I). W e decided to p u r i f y some of these isoenzymes as well as s t u d v t h e i r b e h a v i o u r on c h r o m a t o g r a p h i c columns c o n t a i n i n g v a r i o u s media.
®
i 53
oFT-
Kig. I. Electrophoretic mobilities of the isoenzymes of human a-amylas( in agar gel. I o.o5, Veronal buffer (pH 8.(~). lq, P2, P3 : pancreatic, $1, $2, S3 salivary, ()l, ()z : ovarian cyst fluid. The relative mobilities of serum ?z-globulins (y) and transfcrrin (ill) are shown alongside. H u m a n p a n c r e a t i c tissues were fresh a u t o p s y m a t e r i a l . T h e y were homogenised with twice their weight of ice-cold buffer in an A T O - M I X b l e n d o r run at " h i g h " speed for 2 rain. The buffer used is (I o.o5) T r i s . HC1-Tris (pH 8.6 at 4°) a c o n t a i n i n g 0.o2 M CaC12 to stabilise tile enzyme. The e x t r a c t was c e n t r i f u g e d at IO ooo r e v . / m i n for 2o rain at 4 °. The clear s u p e r n a t a n t was t y p e d as to i s o a m y l a s e c o n s t i t u t i o n b y agar-gel electrophoresis 2 a n d tile e n z y m e a c t i v i t y e s t i m a t e d b y an a m y l o c l a s t i c m e t h o d 4. A b o u t 80 ml of saliva was collected from 2 v o l u n t e e r s a n d o. 5 nil toluene was a d d e d as preservative. The saliva was s t o r e d in t h e cold overnight, d e c a n t e d from cellular a n d mucinous d e b r i s a n d c e n t r i f u g e d at 5ooo r e v . / m i n for IO rain. Tile clarified s a l i v a was p u t into Visking cellophane t u b i n g a n d d i a l y s e d against the Tris buffer for 24 h u n d e r m a g n e t i c stirring. P a r t i c u l a t e m a t t e r fornled d u r i n g t h e dialysis was again centrifuged off a n d t h e e n z y m e a c t i v i t y of the saliva e s t i m a t e d . Milk e x t r a c t e d b y b r e a s t - p u m p from six l a c t a t i n g m o t h e r s were pooled. This milk, a l n o u n t i n g to a b o u t 15o ml, was c e n t r i f u g e d in the cold at 1o ooo r e v . / m i n for 3o min. L i p i d m a t e r i a l f o r m e d a firm " b u t t o n " on t h e t o p of an opalescent lower laver. This l a y e r was d i a l y s e d a g a i n s t tile Tris buffer as described for saliva, a n d centrifuged again. R a n d o m urine specimens collected from 22 h e a l t h y staff m e m b e r s were filtered a n d 500 ml of tile urine pool was c o n c e n t r a t e d in Visking cellophane t u b i n g a g a i n s t p o l y e t h y l e n e glycol (Carbowax 4ooo, British D r u g Houses Ltd.), The c o n c e n t r a t e d Biochim. Biophvs. Hera, I~)S (1968) 362 365
363
SHORT COMMUNICATIONS
urine, about 1/~0oth the original volume, was then dialysed in the cold against the Tris buffer at a lower I of o.o175.
Purification of pancreatic, salivary and milk a-amylases DEAE-Sephadex A-5o columns, 2.5 cm × 24 cm, were equilibrated with the Tris buffer (I 0.05). Preliminary experiments showed that the enzyme could be eluted from the DEAE-Sephadex without altering the composition of the buffer. Neither increasing I to 0.5 with NaC1, nor decreasing the p H to 5 in the form of a linear gradient resulted in the further recovery of the enzyme. The D E A E columns were charged with 2 ml of the material to be fractionated which should contain about 15oo Somogyi units of enzyme activity. It is important not to overload the column. With a flow rate of 20 ml/h the effluent was collected in 3-ml fractions by siphon. Aliquots from each fraction were spotted on a starch substrate plate 5. After incubation at 37 ° for 20 min and development with ethanolic iodine, digested areas on the plate indicated which tubes contained amylase activity. The amounts of enzyme in these tubes were quantitatively determined. Agar-gel electrophoresis was carried out on selected tubes with amyloclastic activity to find out the order in which the isoenzymes were being eluted. All fractions with enzyme activity were combined and concentrated to a small volume by ultrafiltration. This was dialysed overnight against phosphate buffer used in the next stage. Columns of Sephadex G-75 2.5 cm × 30 cm, were equilibrated with o.I M phosphate buffer (pH 7.0). Enzyme concentrates obtained from the D E A E column were applied on these columns in I-ml portions. The detection of amyloclastic activity in the effluent is similar to that already described. The specific extinction E 1% cm = 23.3 at 280 m~u was given b y STEIN, H s l o AND FISCHER6 for crystalline salivary amylase. Using this factor the specific activities of the initial and final stages of purification of pancreatic and salivary amylases were calculated.
Purification of pancreatic and salivary amylases from urine In this experiment DEAE-cellulose was preferred to DEAE-Sephadex because the latter tended to shrink greatly during the application of the buffer with o.I M NaC1, making regeneration of the ion-exchange dextran difficult. A DEAE-cellulose (Whatman Powder D E 50) column (12 cm × 0.8 cm) was washed several times with (I o.o175 ) Tris buffer (pH 8.6 at 4°). Concentrated and dialysed urine was electrophoresed to establish its a-amylase isoenzyme structure. A o.5-ml sample volume was allowed to soak into the top layer of the column and collected in 2-ml fractions before elution was continued with the same buffer containing o.I M NaC1. The effluent was treated as described above. The a-amylases recovered before and after the step-wise elution were concentrated and identified b y agar-gel electrophoresis against the original urine. After several experiments it was possible to calibrate the column in terms of the number of fractions needed to recover most of the isoenzymes. Five recovery experiments were done. Each column m a y be used m a n y times, if re-equilibrated with the starting buffer, without losing its characteristics. Some of the experiments were done at room temperature (22 °) at which the buffer p H was 8.2. This had no effect on the quality of the isoenzyme separation. Biochim. Biophys. Acta, 168 (1968) 362-365
364
SHORT COMMUNICATIONS
3oo
P2 J\
J
-~, 2 0 0
P1
o~
I
$
[
i
t
!,
300
/
\ i
I I
~
/ i
Ji
f 0
,7 0
.-'(".'.'.. . . \
.-" I ........ '--,
\ \
... ,," \.
,' ........ . .... ~'"
7-0
-
."" -; ...,----40 Tube
.
', ". " ,
.........:,,.
6o
;"':.-:.-.>--
8o
number
Fig. 2. C h r o m a t o g r a p h i c separation of h u m a n a-amylases. Elution profiles of two types of pancreatic e x t r a c t s (containing P2, and P I + P2), saliva and milk on the same D E A E - S e p h a d e x A-5 o c o l u m n (2.5 cm × 24 cm) with (I 0.o5) Tris buffer (pH 8.6). E a c h of the four samples contained a b o u t I3OO Somogyi units of enzyme activity. (For isoenzyme notation, please see Fig. I.) ----, pancreas (PI @ P2); • . ., pancreas (P2); . . . . . . , saliva; . . . . , milk. Fig. 3. Agar-gel electrophoresis of isoenzymes of u r i n a r y ~z-amylase separated on a o.8 c m x ]2 cm DEAE-cellulose column, with Tris buffer (pH 8.2}. A, pancreatic isoenzymes obtained with the buffer at I o.o175; B, salivary isoenzymes obtained w i t h the same buffer containing o.i M NaC1; U, original concentrated urine. The cathode is above.
A common feature in the behaviour of the various a-amylases, whether with DEAE-Sephadex A-5o or Sephadex G-75, is the tendency to trail as they come off the column. Fig. 2 illustrates the elution profiles of pancreatic, salivary and milk amylases fractionated on the same DEAE-Sepbadex A-5o column. The order in which the isoenzymes were eluted was P I , P2, SI and $2. On the Sephadex G-75 column PI was eluted just before P2, and P2 before SI, with some overlap. The specific activity of salivary amylase after purification was 4 times that of the starting material. COLOWlCKAND KAPLAN7 found the specific activity of reerystallised human salivary amylase to be 8 times that of saliva. The specific activity of pancreatic amylase was 45 times that of the original extract. I t can be seen that the procedure outlined separates the urinary pancreatic isoenzymes, P2 and P3, from the salivary group SI and $2 {Fig. 3)- In five recovery experiments almost all the enzyme applied to the column was retrieved. The order in which the isoenzymes are eluted, t h a t is, PI, P2, SI, $2 appears to be in accord with their electrophoretic mobilities. One can exclude the likelihood that molecular seiving contributes to the results, as Sephadex A-5o has an exclusion molecular weight of IO ooo. However on the Sephadex G-75 column the order of elution is also sinfilar to that on DEAE-Sephadex A-5o. Two unrelated explanations m a y be offered for these results. One is that molecular size and shape favour the elution of PI, then P2 and SI. Minor differences of these parameters cannot be excluded b y the results of the present experiments. The other explanation favours the possibility that differences in affinity of the various isoenzymes for the gel-matrix, rich in a-I,6-glucosidic bonds, m a y be Biochim. Biophys. Acta, i68 (1968) 362-365
SHORT COMMUNICATIONS
365
decisiveS,9. a-Amylase is not entirely specific for a-I,4-glucosidic linkages 1°. Polyacrylamide gel beads, Bio-Gel P-6o (Bio-Rad Laboratories, Calif.) have a porosity comparable with that of Sephadex G-75. Elution from columns made with Bio-Gel P-6o fail to separate P I from P2 or P2 from SI (unpublished observations). Thus penetration of the gel matrix per se cannot account for the differential retardation. GELOTTE performed elution experiments with 0.02 M Tris buffer (pH 7) and showed that amylase was retarded on Sephadex G-75, the magnitude depending on the flow rate of the buffer. No retardation was seen with Sephadex G-25, which the enzyme would not be expected to penetrate 9. ONO, HIROMI AND YOSHIKAWA11 found that the Michaelis constant of B. subtilis a-amylase was unaffected by either the nature or concentration of the anions present in the buffer. The Michaelis constant is an expression of the affinity of the binding site of an enzyme for its substrate. This site, or sites, with which C1- and other anions react, differ from the catalytic centre(s) responsible for the actual scission of the a-I,4-bonds. It is probably the binding site that is involved in the amylase-Sephadex interaction. It appears significant that ONo and his associates discovered that the Michaelis constant for their enzyme was constant over the pH range 3.6-8. 4. GELOTTE9 similarly noted that the elution pattern of amylase was not affected by variation of pH from 5 to 8. One of us (S.E.A.) is grateful to the University of Singapore for an Overseas Training Scholarship.
Department of Chemical Pathology, Royal Postgraduate Medical School, London W. 12 (Great Britain) i 2 3 4 5 6 7 8 9 IO ii
S. E. Aw* J. R. HOBBS I. D. P. WOOTTON
S. E. A w AND J. R. HOBBS, Biochem. J., 99 (1966) I6P. S. E. Aw, J. R. HOBBS AND I. D. P. WOOTTON, Gut, 8 (1967) 402. C. LONG, Biochemists' Handbook, V a n N o s t r a n d , Toronto, 1961 , p. 33. I. D. P. WOOTTON, Micro-Analysis in Medical Biochemistry, 4 t h edition, Churchill, L o n d o n , 1964, p. lO6. S. E. Aw, Nature, 2o9 (1966) 298. E. A. STEIN, J. H s l u AND E. H. FISCHER, Biochemistry, 3 (1964) 56. S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, A c a d e m i c Press, N e w York, 1955, p. 15o. P. WILDING, Clin. Chim. Acta, 8 (1963) 918. B. GELOTTE, Acta Chem. Scand., 18 (1964) 1283. ]3. J. BINES AND W. J. WHELAN, Chem. Ind. London, 31 (196o) 997. S. ONO, K. HIROMI AND Y. YOSHIKAWA, Bull. Chem. Soc. Japan, 31 (1958) 957-
Received May 7th, 1968 * P r e s e n t a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , F a c u l t y of Medicine, S e p o y Lines, Singapore 3.
Biochim. Biophys. Acta, 168 (1968) 362-365