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Purification of tetanus toxin by multi-membrane electrodecantation
v o I . . 1 6 (1955)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
291
Purification of tetanus toxin by multi-membrane electroclecantation I t is desirable t h a t a toxoid, to be used for p r o p h y l a x i s against h u m a n and animal diseases, should be as tmre as possible. Effective purification will reduce the incidence of reactions a t t r i b u t a b l e to impurities in the toxoids and will minimize the n u m b e r of undesirable antigens which m a y be p r e s e n t in relatively cruder p r e p a r a t i o n s , and which m a y affect the antigenic response evoked in their recipients. The toxin p r o d u c e d by CI. telani m a y be considerably purified by m e a n s of m u l t i - m e m b r a n e eiectrodecantation (M.M.E.D.) I. The m e t h o d has a n u m b e r of a d v a n t a g e s such as the mild conditions to which the toxin is exposed (relatively low t e m p e r a t u r e ; p H ' s near n e u t r a l i t y ; absence of denaturing solvents), the high yield, the r a p i d i t y of the process and the relative simplicity of the a p p a r a t u s . In a typical e x p e r i m e n t C/. tetani (G.S. 761) was g r o w n in a cellophane bag, such as was used by POLSOX AND STERNE in the p r e p a r a t i o n of C,l. b o t u l i n u s toxin ~, using a H o g ' s Stomach-Veal infusion m e d i u m a. There was no v e r y m a r k e d i m p r o v e m e n t in the q u a n t i t y of toxin p r o d u c e d as c o n t r a s t e d to t h a t obtained with g r o w t h in the m e d i u m itself. However, the cellophane bag technique had the a d v a n t a g e t h a t the only macro-molecular material p r e s e n t was produced by the Cl. letani and none was derived from the medium. Filtrates containing i o 5 or more M.L.D. (for 2o g mice) per ml were obtained. I n the preliminary investigation, which is r e p o r t e d here, the toxin was purified by the removal of those c o n s t i t u e n t s w i t h electrophoretic mobility at the iso-electric p o i n t of the toxin (pH 5.i) 4. The c o n t i n u o u s flow, 3-cell model of M.M.E.D. a p p a r a t u s was used for this purpose. I t was operated in the horizontal position since p a p e r electrophoresis of the crude toxin showed t h a t there were b o t h positively- and negatively-charged c o n s t i t u e n t s at p H 5.1Three litres of the crude t o x i n - c o n t a i n i n g filtrate were dialysed against an acetate buffer (pH -- 5.1 : ionic s t r e n g t h = o.o5) , containing Mg +~ , for 48 hours, the buffer h a v i n g been renewed at the end of 24 hours. A considerable portion of the p i g m e n t e d material, p r e s e n t in the original filtrate, diffused into the dialysate. The toxin was passed t h r o u g h the a p p a r a t u s at a p p r o x i m a t e l y lOO m l / h w i t h a voltage gradient of a p p r o x i m a t e l y 4 volts/cm. The c u r r e n t was reversed twice during the e x p e r i m e n t in order to minimize a n y a c c u m u l a t i o n of acid or alkali in either electrode comp a r t m e n t . The p H was s h o w n to have r e m a i n e d c o n s t a n t t h r o u g h o u t . All the p i g m e n t e d material m i g r a t e d and collected at the b o t t o m of the cells with the electrophoretically mobile c o n t a m i n a n t s . The e x t e n t of the purification was estimated b o t h by paper electrophoresis and the m e a s u r e m e n t of toxicity, expressed as M.I,.D./mg of protein nitrogen. P a p e r electrophoresis was carried o u t in veronal buffer at p H 8.6 and ionic s t r e n g t h o.o 5. The e l e c t r o p h o r e t o g r a m showed a s t a t i o n a r y band, 2 double b a n d s m o v i n g t o w a r d s the anode and a single b a n d m o v i n g t o w a r d s the cathode. By c u t t i n g u p the p a p e r strip, eluting and determining the toxicity, it w a s found t h a t the activity was associated with the slower c o m p o n e n t of the slower double band. This is in accordance w i t h the electrophoretic mobility, since it h a d the same speed as the /~-globulin of a h u m a n s e r u m u n d e r the same e x p e r i m e n t a l conditions. The p a p e r electrop h o r e t o g r a m of t h e purified t o x i n showed only the single t o x i n - c o n t a i n i n g b a n d w i t h a continuous, light, diffuse stain b e t w e e n it and the point of application on the paper. However, free electrophoresis indicated the presence of a non-charged c o m p o n e n t (the v e r y low protein concentration p r e v e n t e d effective analysis b y free electrophoresis, even using the Scale Method). Based on the toxicity, expressed as the n u m b e r of M.L.D. (for mice) per milligram of protein nitrogen (precipitated b y trichloracetic acid), there was a p p r o x i m a t e l y an 8-fold purification b y a single r u n t h r o u g h the M.M.E.D. a p p a r a t u s (see Table I). TABLE I PURIFICATION
Substance
Original t o x i n Purified toxin
Toxicity M.L.E./ml
OF TOXIN
mgN /ml
Toxicity * M.L.D./mgN
8.104
0.0035
2. 3 • io ~
7' i o 4
0.o0037
1.9. IO8
* The high toxicity figures per milligram of p r o t e i n nitrogen m a y be due to incomplete precipit a t i o n ~. However, t h e y serve as a c o m p a r i s o n to d e m o n s t r a t e the e x t e n t of the purification. The toxicity of t h e solution before and after M.M.E.D. was virtually the same, allowing for dilution b y the buffer originally p r e s e n t in tile a p p a r a t u s . The proteins, which c o n c e n t r a t e d in the b o t t o m of t h e three cells, contained progressively less toxin, v a r y i n g f r o m one-third to one-eighth of t h a t p r e s e n t in the original solution.
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SHORT COMMUNICATIONS, PRELIMINARY N()TES
VOL. ] 6 ( I 0 5 5 )
I t has also been possible to d e m o n s t r a t e t h a t t h e c rude T A B I , E [I t o x i n (even from d i l u t e solutions) m a y be q u a n t i t a t i v e l y r e c o v e r e d in a s m a l l e r v o l u m e in t h e M.M.E.D. a p p a r a t u s , o p e r a t e d a t a p H CONCENTRATIONOF TOXIN r e m o v e d from t h e iso-electric p o i n t of t h e t o x i n (e.g. p H 7.o). Using a sing le s e p a r a t i o n cell ( c a p a c i t y 5 o o ml) as a b a t c h process, 95 o/o .'%~bstancc Toxicityml " M.L.I). or m o r e of t h e t o x i n h a s been c o n c e n t r a t e d in 5 ° m l a t t h e b o t t o m of t h e cell w i t h i n 4 h o u r s w i t h a p o t e n t i a l g r a d i e n t of a p p r o x i m a t e l y 4 v o l t s / c m . T h e final c o n c e n t r a t i o n in t h e t o p of t h e cell Original 2. i o 4 w a s o n e - h u n d r e d t h of t h a t p r e s e n t o r i g i n a l l y (see T a b l e 11). Bottom fraction 2. ~o ~ F u r t h e r w o r k u s i n g t h e M.M.E.D. to p u r i f y t e t a n u s t o x i n Top f r a c t i o n 2. lo z from b o t h t h e G.S. 76I a n d t h e " H a r v a r d " s t r a i n s of CI. tetani (the l a t t e r p r o d u c i n g s i g n i f i c a n t l y more t o x i n u n d e r t h e c o n d i t i o n s of t h e s e experinaents) is in progress. The effect of tile p u r i f i c a t i o n Oll s uc h p r o p e r t i e s as flocculation, a n t i g e n i c i t y , etc. will also be i n v e s t i g a t e d . T h e a u t h o r wishes to t h a n k Dr. POLSON for his a s s i s t a n c e a n d P r o f e s s o r VAN DEN END~; for his i n t e r e s t in t h i s work. JOHN F. LARGIER
South ,4/rican C o u n c i l / o r Scientific and Induslrial Research, am/ University o/ Cape Tou,~ Virus Research Unit, Department o/ Pathology; and Department o~ Chemistry, University o/(?ape T o w n (South :t/riea)
1 A. POLSON, Biochim. Biophys. Acla, i i (I953) 315 . 2 A . P O L S O N AND .A.I. ,~TERNE, Nature, I58 (1946) 238. a E. ]~{. TAYLOR, .]. [mmunol., 5 ° (1945) 385 . 4 L. PILLEMER, R. G. \VITTLER, J. BURRELL ANI) I). GROSSBERG, J . E x p . 3Ied., 88 (1948) 205. s SV. KOCH AND D. KAPLAN, .]. Immum)l., 7 ° (1953) ~R e c e i v e d D e c e m b e r 7th, 1954
Conversion of o-xylose to D-xylulose phosphate by extracts of
Pseudomonas hydrophila* P h o s p h o r y l a t i o n studies, in which D-xylose w a s t h e p e n t o s e s uppl i e d, h a v e be e n r e p o r t e d wittl cell-free e x f r a c t s of Pseudomonas hydrophila 1, Lactobacillus pe~losus 2 a n d Aerobacter cloacae "~. The p r o d u c t of p h o s p h o r y l a t i o n w a s first d e s c r i b e d only as a n a c i d - s t a b l e p e n t o s e p h o s p h a t O . L a t e r , u s i n g L. pentosus, f,AMPEN 4 i d e n t i f i e d I ) - r i b o s e - 5 - p h o s p h a t e as t h e chief p r o d u c t of t h i s s y s t e m , s t a t i n g , h o w e v e r , t h a t r/-xylose or I)-xyhllose p h o s p h a t e s could n o t be d e t e c t e d . R e c e n t w o r k ha s s h o w n t h a t t h e cell-free e x t r a c t s used in p h o s p h o r y l a t i o n s t u d i e s of P. hydrophila a n d L. penlosus c o n t a i n e d n o t o n l y a k i n a s e b u t also a specific x y l o s e isomeraseS,S, 7. It, therefore, b e c a m e u n c e r t a i n w h e t h e r D-xylose or D-xylulose w a s t h e a c t u a l s u b s t r a t e in t h e p h o s p h o r y l a t i o n . A t t e m p t s to i d e n t i f y t h e p r o d u c t of t h e p h o s p h o r y l a t i o n h a v e now r e v e a l e d t h e pre s e nc e of a t l e a s t four a c i d - s t a b l e p h o s p h a t e esters. This c o m m u n i c a t i o n describes t h e i d e n t i f i c a t i o n of t h e first e s t e r f o r m e d in t h e s e q u e n c e of r e a c t i o n s now k n o w n t o t a k e place. T h e e n z y m e p r e p a r a t i o n used w a s a fresh sonic e x t r a c t of P. hydrophila 7. N u c l e o p r o t e i n w a s r e m o v e d w i t h MnCI2 s a n d nucleic acids a n d o t h e r e x t r a n e o u s p r o t e i n w i t h p r o t a m i n e s u l p h a t e 9.n). This p r o c e d u r e w a s used to p r e v e n t t h e possible f o r m a t i o n of p e n t o s e p h o s p h a t e b v e n z y m i c h y d r o l y s i s of nucleic acids or of n u c l e o p r o t e i n . After c e n t r i f u g a t i o n (~4,ooo r.p.m.), tile s u p e r n a t a n t s o l u t i o n was d i a l y z e d a g a i n s t w e a k p h o s p h a t e a n d t h e n a g a i n s t w e a k b i c a r b o n a t e buffers (pH 7.4 7-5) for a t o t a l of 48 h. The r e s u l t i n g p r o d u c t w a s used for all e x p e r i m e n t a l work. P h o s p h o r y l a t i o n e x p e r i m e n t s were c a r r i e d o u t in a n a t m o s p h e r e of N 2 CO 2 in 15 \ V a r b u r g vessels (27 ° C), each c o n t a i n i n g o.o2 3~/ N a H C O a, o.oi 3// MgC12, o.o26 2~I ATP, 1.8 ml e n z y m e p r e p a r a t i o n a n d o.o18 3 i D-xylose. C o n d i t i o n s chosen w e re s uc h t h a t no free s u g a r s r e m a i n e d a t t h e t e r m i n a t i o n of t h e e x p e r i m e n t s , as j u d g e d b y p a p e r c h r o m a t o g r a p h y of t h e p r o d u c t s . 1)hos p h o r y l a t i o n r a t e s were m e a s u r e d b y t h e m e t h o d of COLOWICK AND KALCKAR11. A f t e r 90 m i n u t e s , t h e c o n t e n t s of all vessels were c o m b i n e d (48 ml), 0.6 m l g l a c i a l a c e t i c acid w a s a d d e d a n d t h e s o l u t i o n w a s a l l o w e d to p a s s s l o w l y (1 1. 5 m l / m i n ) t h r o u g h a l a rge c o l u m n of I R i 2 o (H +) ion e x c h a n g e resin followed b y s e v e r a l w a s h i n g s w i t h deionized w a t e r . P r o t e i n f l o c c u l a t e d o u t a t t h e t op of t h e c o l u m n . T h e effluent (pH a b o u t 2.8) was c o l l e c t e d a t o 4 ° C c o n c e n t r a t e d in vacuo a t 3 °0 C 12 t o a v o l u m e of 5 ° ml, a n d t r e a t e d w i t h b a r i u m h y d r o x i d e . A f t e r r e m o v a l of t h e a d e n o s i n e p h o s p h a t e s a t p H 8.2, t h e p H was q u i c k l y b r o u g h t to 6.8. [;our v o l u m e s of e t h a n o l were t h e n a d d e d t o p r e c i p i t a t e t h e b a r i u m s a l t s of t h e s u g a r p h o s p h a t e s . The p r e c i p i t a t e w a s t h o r o u g h l y w a s h e d a n d dried. * I s s u e d as N.R.C. No. 3483 .
SHORT COMMUNICATIONS, PRELIMINARY NOTES
291
Purification of tetanus toxin by multi-membrane electroclecantation I t is desirable t h a t a toxoid, to be used for p r o p h y l a x i s against h u m a n and animal diseases, should be as tmre as possible. Effective purification will reduce the incidence of reactions a t t r i b u t a b l e to impurities in the toxoids and will minimize the n u m b e r of undesirable antigens which m a y be p r e s e n t in relatively cruder p r e p a r a t i o n s , and which m a y affect the antigenic response evoked in their recipients. The toxin p r o d u c e d by CI. telani m a y be considerably purified by m e a n s of m u l t i - m e m b r a n e eiectrodecantation (M.M.E.D.) I. The m e t h o d has a n u m b e r of a d v a n t a g e s such as the mild conditions to which the toxin is exposed (relatively low t e m p e r a t u r e ; p H ' s near n e u t r a l i t y ; absence of denaturing solvents), the high yield, the r a p i d i t y of the process and the relative simplicity of the a p p a r a t u s . In a typical e x p e r i m e n t C/. tetani (G.S. 761) was g r o w n in a cellophane bag, such as was used by POLSOX AND STERNE in the p r e p a r a t i o n of C,l. b o t u l i n u s toxin ~, using a H o g ' s Stomach-Veal infusion m e d i u m a. There was no v e r y m a r k e d i m p r o v e m e n t in the q u a n t i t y of toxin p r o d u c e d as c o n t r a s t e d to t h a t obtained with g r o w t h in the m e d i u m itself. However, the cellophane bag technique had the a d v a n t a g e t h a t the only macro-molecular material p r e s e n t was produced by the Cl. letani and none was derived from the medium. Filtrates containing i o 5 or more M.L.D. (for 2o g mice) per ml were obtained. I n the preliminary investigation, which is r e p o r t e d here, the toxin was purified by the removal of those c o n s t i t u e n t s w i t h electrophoretic mobility at the iso-electric p o i n t of the toxin (pH 5.i) 4. The c o n t i n u o u s flow, 3-cell model of M.M.E.D. a p p a r a t u s was used for this purpose. I t was operated in the horizontal position since p a p e r electrophoresis of the crude toxin showed t h a t there were b o t h positively- and negatively-charged c o n s t i t u e n t s at p H 5.1Three litres of the crude t o x i n - c o n t a i n i n g filtrate were dialysed against an acetate buffer (pH -- 5.1 : ionic s t r e n g t h = o.o5) , containing Mg +~ , for 48 hours, the buffer h a v i n g been renewed at the end of 24 hours. A considerable portion of the p i g m e n t e d material, p r e s e n t in the original filtrate, diffused into the dialysate. The toxin was passed t h r o u g h the a p p a r a t u s at a p p r o x i m a t e l y lOO m l / h w i t h a voltage gradient of a p p r o x i m a t e l y 4 volts/cm. The c u r r e n t was reversed twice during the e x p e r i m e n t in order to minimize a n y a c c u m u l a t i o n of acid or alkali in either electrode comp a r t m e n t . The p H was s h o w n to have r e m a i n e d c o n s t a n t t h r o u g h o u t . All the p i g m e n t e d material m i g r a t e d and collected at the b o t t o m of the cells with the electrophoretically mobile c o n t a m i n a n t s . The e x t e n t of the purification was estimated b o t h by paper electrophoresis and the m e a s u r e m e n t of toxicity, expressed as M.I,.D./mg of protein nitrogen. P a p e r electrophoresis was carried o u t in veronal buffer at p H 8.6 and ionic s t r e n g t h o.o 5. The e l e c t r o p h o r e t o g r a m showed a s t a t i o n a r y band, 2 double b a n d s m o v i n g t o w a r d s the anode and a single b a n d m o v i n g t o w a r d s the cathode. By c u t t i n g u p the p a p e r strip, eluting and determining the toxicity, it w a s found t h a t the activity was associated with the slower c o m p o n e n t of the slower double band. This is in accordance w i t h the electrophoretic mobility, since it h a d the same speed as the /~-globulin of a h u m a n s e r u m u n d e r the same e x p e r i m e n t a l conditions. The p a p e r electrop h o r e t o g r a m of t h e purified t o x i n showed only the single t o x i n - c o n t a i n i n g b a n d w i t h a continuous, light, diffuse stain b e t w e e n it and the point of application on the paper. However, free electrophoresis indicated the presence of a non-charged c o m p o n e n t (the v e r y low protein concentration p r e v e n t e d effective analysis b y free electrophoresis, even using the Scale Method). Based on the toxicity, expressed as the n u m b e r of M.L.D. (for mice) per milligram of protein nitrogen (precipitated b y trichloracetic acid), there was a p p r o x i m a t e l y an 8-fold purification b y a single r u n t h r o u g h the M.M.E.D. a p p a r a t u s (see Table I). TABLE I PURIFICATION
Substance
Original t o x i n Purified toxin
Toxicity M.L.E./ml
OF TOXIN
mgN /ml
Toxicity * M.L.D./mgN
8.104
0.0035
2. 3 • io ~
7' i o 4
0.o0037
1.9. IO8
* The high toxicity figures per milligram of p r o t e i n nitrogen m a y be due to incomplete precipit a t i o n ~. However, t h e y serve as a c o m p a r i s o n to d e m o n s t r a t e the e x t e n t of the purification. The toxicity of t h e solution before and after M.M.E.D. was virtually the same, allowing for dilution b y the buffer originally p r e s e n t in tile a p p a r a t u s . The proteins, which c o n c e n t r a t e d in the b o t t o m of t h e three cells, contained progressively less toxin, v a r y i n g f r o m one-third to one-eighth of t h a t p r e s e n t in the original solution.
29:
SHORT COMMUNICATIONS, PRELIMINARY N()TES
VOL. ] 6 ( I 0 5 5 )
I t has also been possible to d e m o n s t r a t e t h a t t h e c rude T A B I , E [I t o x i n (even from d i l u t e solutions) m a y be q u a n t i t a t i v e l y r e c o v e r e d in a s m a l l e r v o l u m e in t h e M.M.E.D. a p p a r a t u s , o p e r a t e d a t a p H CONCENTRATIONOF TOXIN r e m o v e d from t h e iso-electric p o i n t of t h e t o x i n (e.g. p H 7.o). Using a sing le s e p a r a t i o n cell ( c a p a c i t y 5 o o ml) as a b a t c h process, 95 o/o .'%~bstancc Toxicityml " M.L.I). or m o r e of t h e t o x i n h a s been c o n c e n t r a t e d in 5 ° m l a t t h e b o t t o m of t h e cell w i t h i n 4 h o u r s w i t h a p o t e n t i a l g r a d i e n t of a p p r o x i m a t e l y 4 v o l t s / c m . T h e final c o n c e n t r a t i o n in t h e t o p of t h e cell Original 2. i o 4 w a s o n e - h u n d r e d t h of t h a t p r e s e n t o r i g i n a l l y (see T a b l e 11). Bottom fraction 2. ~o ~ F u r t h e r w o r k u s i n g t h e M.M.E.D. to p u r i f y t e t a n u s t o x i n Top f r a c t i o n 2. lo z from b o t h t h e G.S. 76I a n d t h e " H a r v a r d " s t r a i n s of CI. tetani (the l a t t e r p r o d u c i n g s i g n i f i c a n t l y more t o x i n u n d e r t h e c o n d i t i o n s of t h e s e experinaents) is in progress. The effect of tile p u r i f i c a t i o n Oll s uc h p r o p e r t i e s as flocculation, a n t i g e n i c i t y , etc. will also be i n v e s t i g a t e d . T h e a u t h o r wishes to t h a n k Dr. POLSON for his a s s i s t a n c e a n d P r o f e s s o r VAN DEN END~; for his i n t e r e s t in t h i s work. JOHN F. LARGIER
South ,4/rican C o u n c i l / o r Scientific and Induslrial Research, am/ University o/ Cape Tou,~ Virus Research Unit, Department o/ Pathology; and Department o~ Chemistry, University o/(?ape T o w n (South :t/riea)
1 A. POLSON, Biochim. Biophys. Acla, i i (I953) 315 . 2 A . P O L S O N AND .A.I. ,~TERNE, Nature, I58 (1946) 238. a E. ]~{. TAYLOR, .]. [mmunol., 5 ° (1945) 385 . 4 L. PILLEMER, R. G. \VITTLER, J. BURRELL ANI) I). GROSSBERG, J . E x p . 3Ied., 88 (1948) 205. s SV. KOCH AND D. KAPLAN, .]. Immum)l., 7 ° (1953) ~R e c e i v e d D e c e m b e r 7th, 1954
Conversion of o-xylose to D-xylulose phosphate by extracts of
Pseudomonas hydrophila* P h o s p h o r y l a t i o n studies, in which D-xylose w a s t h e p e n t o s e s uppl i e d, h a v e be e n r e p o r t e d wittl cell-free e x f r a c t s of Pseudomonas hydrophila 1, Lactobacillus pe~losus 2 a n d Aerobacter cloacae "~. The p r o d u c t of p h o s p h o r y l a t i o n w a s first d e s c r i b e d only as a n a c i d - s t a b l e p e n t o s e p h o s p h a t O . L a t e r , u s i n g L. pentosus, f,AMPEN 4 i d e n t i f i e d I ) - r i b o s e - 5 - p h o s p h a t e as t h e chief p r o d u c t of t h i s s y s t e m , s t a t i n g , h o w e v e r , t h a t r/-xylose or I)-xyhllose p h o s p h a t e s could n o t be d e t e c t e d . R e c e n t w o r k ha s s h o w n t h a t t h e cell-free e x t r a c t s used in p h o s p h o r y l a t i o n s t u d i e s of P. hydrophila a n d L. penlosus c o n t a i n e d n o t o n l y a k i n a s e b u t also a specific x y l o s e isomeraseS,S, 7. It, therefore, b e c a m e u n c e r t a i n w h e t h e r D-xylose or D-xylulose w a s t h e a c t u a l s u b s t r a t e in t h e p h o s p h o r y l a t i o n . A t t e m p t s to i d e n t i f y t h e p r o d u c t of t h e p h o s p h o r y l a t i o n h a v e now r e v e a l e d t h e pre s e nc e of a t l e a s t four a c i d - s t a b l e p h o s p h a t e esters. This c o m m u n i c a t i o n describes t h e i d e n t i f i c a t i o n of t h e first e s t e r f o r m e d in t h e s e q u e n c e of r e a c t i o n s now k n o w n t o t a k e place. T h e e n z y m e p r e p a r a t i o n used w a s a fresh sonic e x t r a c t of P. hydrophila 7. N u c l e o p r o t e i n w a s r e m o v e d w i t h MnCI2 s a n d nucleic acids a n d o t h e r e x t r a n e o u s p r o t e i n w i t h p r o t a m i n e s u l p h a t e 9.n). This p r o c e d u r e w a s used to p r e v e n t t h e possible f o r m a t i o n of p e n t o s e p h o s p h a t e b v e n z y m i c h y d r o l y s i s of nucleic acids or of n u c l e o p r o t e i n . After c e n t r i f u g a t i o n (~4,ooo r.p.m.), tile s u p e r n a t a n t s o l u t i o n was d i a l y z e d a g a i n s t w e a k p h o s p h a t e a n d t h e n a g a i n s t w e a k b i c a r b o n a t e buffers (pH 7.4 7-5) for a t o t a l of 48 h. The r e s u l t i n g p r o d u c t w a s used for all e x p e r i m e n t a l work. P h o s p h o r y l a t i o n e x p e r i m e n t s were c a r r i e d o u t in a n a t m o s p h e r e of N 2 CO 2 in 15 \ V a r b u r g vessels (27 ° C), each c o n t a i n i n g o.o2 3~/ N a H C O a, o.oi 3// MgC12, o.o26 2~I ATP, 1.8 ml e n z y m e p r e p a r a t i o n a n d o.o18 3 i D-xylose. C o n d i t i o n s chosen w e re s uc h t h a t no free s u g a r s r e m a i n e d a t t h e t e r m i n a t i o n of t h e e x p e r i m e n t s , as j u d g e d b y p a p e r c h r o m a t o g r a p h y of t h e p r o d u c t s . 1)hos p h o r y l a t i o n r a t e s were m e a s u r e d b y t h e m e t h o d of COLOWICK AND KALCKAR11. A f t e r 90 m i n u t e s , t h e c o n t e n t s of all vessels were c o m b i n e d (48 ml), 0.6 m l g l a c i a l a c e t i c acid w a s a d d e d a n d t h e s o l u t i o n w a s a l l o w e d to p a s s s l o w l y (1 1. 5 m l / m i n ) t h r o u g h a l a rge c o l u m n of I R i 2 o (H +) ion e x c h a n g e resin followed b y s e v e r a l w a s h i n g s w i t h deionized w a t e r . P r o t e i n f l o c c u l a t e d o u t a t t h e t op of t h e c o l u m n . T h e effluent (pH a b o u t 2.8) was c o l l e c t e d a t o 4 ° C c o n c e n t r a t e d in vacuo a t 3 °0 C 12 t o a v o l u m e of 5 ° ml, a n d t r e a t e d w i t h b a r i u m h y d r o x i d e . A f t e r r e m o v a l of t h e a d e n o s i n e p h o s p h a t e s a t p H 8.2, t h e p H was q u i c k l y b r o u g h t to 6.8. [;our v o l u m e s of e t h a n o l were t h e n a d d e d t o p r e c i p i t a t e t h e b a r i u m s a l t s of t h e s u g a r p h o s p h a t e s . The p r e c i p i t a t e w a s t h o r o u g h l y w a s h e d a n d dried. * I s s u e d as N.R.C. No. 3483 .