- Email: [email protected]
A convenient method for preparing crystalline catalase from rat liver
VOL. 1 2 (1953) A CONVENIENT
SHORT COMMUNICATIONS, PRELIMINARY NOTES METHOD
FOR
PREPARING
FROM
RAT
CRYSTALLINE
487 CATALASE
LIVER
by VIRA KENNEDY,
L L O Y D H. N E W M A N
AND F E L I X
FRIEDBERG
Department o[ Biochemistry, College o~ Medicine, Howard University, Washington, D.C. (U.S.A.)
T h e r e h a v e been p u b l i s h e d several m e t h o d s for t h e p r e p a r a t i o n of crystalline beef liver c a t a lasO a. No m e t h o d , however, is available for t h e p r e p a r a t i o n of t h e e n z y m e from o r g a n s of t h e r a t . Described below is a simple w a y suitable for o b t a i n i n g eatalase f r o m r a t liver. I t r e p r e s e n t s a m o d i fication of t h e p r o c e d u r e s u g g e s t e d b y TAUBER AND PETIT ~. A p p r o x i m a t e l y 2oo g r a m s of tissue are h o m o g e n i z e d in a \ V a r i n g b l e n d o r w i t h a n e q u a l a m o u n t of cold distilled water. T h e h o m o g e n a t e is cooled a n d o. 4 v o l u m e of cold a c e t o n e is a d d e d slowly w i t h stirring. T h e m i x t u r e is filtered a n d t h e filtrate o b t a i n e d is allowed to s t a n d u n t i l a red p r e c i p i t a t e a p p e a r s a r o u n d t h e walls of t h e container. T h e n t h e filtrate is c e n t r i f u g e d a n d t h e p r e c i p i t a t e s u s p e n d e d in t h e s m a l l e s t v o l u m e of o.I M p o t a s s i u m p h o s p h a t e buffer of p H 7.4. T h e r e s u l t i n g s u s p e n s i o n is c e n t r i f u g e d a n d t h e s u p e r n a t a n t placed into t h e refrigerator. Crystals f o r m on s t a n d i n g . A f t e r c e n t r i f u g a t i o n t h e y are r e s u s p e n d e d in I ml of p h o s p h a t e buffer. T h e s u p e r n a t a n t o b t a i n e d b y c e n t r i f u g a t i o n is placed into t h e refrigerator. I n it crystals will a p p e a r w h i c h d i s p l a y t h e c h a r a c teristic s h e e n of catalase. R e - e x t r a c t i o n of t h e v a r i o u s p r e c i p i t a t e s f o r m e d in t h e a b o v e p r o c e d u r e c a n be u s e d to i m p r o v e t h e yield. A b s o r p t i o n s p e c t r a of r a t liver c a t a l a s e in t h e u l t r a v i o l e t a n d visible r a n g e are given in Fig. 4.
REFERENCES 1 j . 13. SUMNER AND A. L. DOUNCE, J. Biol. Chem., 121 (1937) 417 • ,2 ~V. MOSIMANN, Arch. Biochem. Biophys., 33 (1951) 487 . 8 H. TAUBER AND E. L. PETIT, J. Biol. Chem., 195 (1952) 703 • Received A u g u s t 26th, IQ53 * This i n v e s t i g a t i o n w a s s u p p o r t e d in p a r t b y a g r a n t f r o m t h e D a m o n R u n y o n Memorial F u n d .
LA
STRUCTURE
DE
LA VASOPRESSINE
DE
BOEUF
par R O G E R A C H E R ET J A C Q U E L I N E
CHAUVET
Laboratoire de Chimie biologique de la Facultg des Sciences, Paris (France)
L ' 6 t u d e de la s t r u c t u r e de la v a s o p r e s s i n e de boeuf a 6t6 e n t r e p r i s e en t e n a n t conlpte du fait q u e la moldcule c o n t i e n t u n rdsidu de c h a c u n des h u i t acides a m i n 6 s s u i v a n t s : cystine, arginine, acide a s p a r t i q u e , acide g l u t a m i q u e , proline, tyrosine, p h 6 n y l a l a n i n e , glycine, corinne F o n t m o n t r 6 TURNER, PIERCE ET D u VIGNEAUD 1. C o n t i n u a n t nos i n v e s t i g a t i o n s d a n s ce d o m a i n e -°, n o u s a v o n s p u m e t t r e en 6vidence les faits s u i v a n t s : i. L o r s q u e la v a s o p r e s s i n e , purifi4e selon le proc6d6 de FROMAaEOT el al. a, est oxyd6e a u n l o y e n de l'acide p e r f o r m i q u e de fagon ~ couper le p o n t disulfure de la c y s t i n e 4, on ne p e u t d6celer q u ' u n seul p r o d u i t p a r c h r o m a t o g r a p h i e ou ionophor6se s u r p a p i e r : les d e u x r6sidus d ' a c i d e c y s t 6 i q u e ainsi form6s p a r t i c i p e n t d o n c /z u n e c h a i n e p e p t i d i q u e u n i q u e . L a v a s o p r e s s i n e o x y d 6 e est alors copul6e a v e c le dinitrofluorobenz~ne en prdsence de t r i m 6 t h y l a m i n O . Apr6s h y d r o l y s e p a r l'acide chlorh y d r i q u e , u n seul d6riv6 dinitroph6nyl6 j a u n e , l'acide d i n i t r o p h 6 n y l c y s t 6 i q u e , p e u t &tre identifi6 p a r c h r o m a t o g r a p h i e s u r papier. L a v a s o p r e s s i n e poss~de donc u n seul groupe a m i n 4 libre a p p a r t e n a n t A la cystine. D ' a u t r e p a r t , la v a s o p r e s s i n e oxyd6e, s o u m i s e & l ' a c t i o n de la t r y p s i n e (rapport e n z y m e /
31.
SHORT COMMUNICATIONS, PRELIMINARY NOTES METHOD
FOR
PREPARING
FROM
RAT
CRYSTALLINE
487 CATALASE
LIVER
by VIRA KENNEDY,
L L O Y D H. N E W M A N
AND F E L I X
FRIEDBERG
Department o[ Biochemistry, College o~ Medicine, Howard University, Washington, D.C. (U.S.A.)
T h e r e h a v e been p u b l i s h e d several m e t h o d s for t h e p r e p a r a t i o n of crystalline beef liver c a t a lasO a. No m e t h o d , however, is available for t h e p r e p a r a t i o n of t h e e n z y m e from o r g a n s of t h e r a t . Described below is a simple w a y suitable for o b t a i n i n g eatalase f r o m r a t liver. I t r e p r e s e n t s a m o d i fication of t h e p r o c e d u r e s u g g e s t e d b y TAUBER AND PETIT ~. A p p r o x i m a t e l y 2oo g r a m s of tissue are h o m o g e n i z e d in a \ V a r i n g b l e n d o r w i t h a n e q u a l a m o u n t of cold distilled water. T h e h o m o g e n a t e is cooled a n d o. 4 v o l u m e of cold a c e t o n e is a d d e d slowly w i t h stirring. T h e m i x t u r e is filtered a n d t h e filtrate o b t a i n e d is allowed to s t a n d u n t i l a red p r e c i p i t a t e a p p e a r s a r o u n d t h e walls of t h e container. T h e n t h e filtrate is c e n t r i f u g e d a n d t h e p r e c i p i t a t e s u s p e n d e d in t h e s m a l l e s t v o l u m e of o.I M p o t a s s i u m p h o s p h a t e buffer of p H 7.4. T h e r e s u l t i n g s u s p e n s i o n is c e n t r i f u g e d a n d t h e s u p e r n a t a n t placed into t h e refrigerator. Crystals f o r m on s t a n d i n g . A f t e r c e n t r i f u g a t i o n t h e y are r e s u s p e n d e d in I ml of p h o s p h a t e buffer. T h e s u p e r n a t a n t o b t a i n e d b y c e n t r i f u g a t i o n is placed into t h e refrigerator. I n it crystals will a p p e a r w h i c h d i s p l a y t h e c h a r a c teristic s h e e n of catalase. R e - e x t r a c t i o n of t h e v a r i o u s p r e c i p i t a t e s f o r m e d in t h e a b o v e p r o c e d u r e c a n be u s e d to i m p r o v e t h e yield. A b s o r p t i o n s p e c t r a of r a t liver c a t a l a s e in t h e u l t r a v i o l e t a n d visible r a n g e are given in Fig. 4.
REFERENCES 1 j . 13. SUMNER AND A. L. DOUNCE, J. Biol. Chem., 121 (1937) 417 • ,2 ~V. MOSIMANN, Arch. Biochem. Biophys., 33 (1951) 487 . 8 H. TAUBER AND E. L. PETIT, J. Biol. Chem., 195 (1952) 703 • Received A u g u s t 26th, IQ53 * This i n v e s t i g a t i o n w a s s u p p o r t e d in p a r t b y a g r a n t f r o m t h e D a m o n R u n y o n Memorial F u n d .
LA
STRUCTURE
DE
LA VASOPRESSINE
DE
BOEUF
par R O G E R A C H E R ET J A C Q U E L I N E
CHAUVET
Laboratoire de Chimie biologique de la Facultg des Sciences, Paris (France)
L ' 6 t u d e de la s t r u c t u r e de la v a s o p r e s s i n e de boeuf a 6t6 e n t r e p r i s e en t e n a n t conlpte du fait q u e la moldcule c o n t i e n t u n rdsidu de c h a c u n des h u i t acides a m i n 6 s s u i v a n t s : cystine, arginine, acide a s p a r t i q u e , acide g l u t a m i q u e , proline, tyrosine, p h 6 n y l a l a n i n e , glycine, corinne F o n t m o n t r 6 TURNER, PIERCE ET D u VIGNEAUD 1. C o n t i n u a n t nos i n v e s t i g a t i o n s d a n s ce d o m a i n e -°, n o u s a v o n s p u m e t t r e en 6vidence les faits s u i v a n t s : i. L o r s q u e la v a s o p r e s s i n e , purifi4e selon le proc6d6 de FROMAaEOT el al. a, est oxyd6e a u n l o y e n de l'acide p e r f o r m i q u e de fagon ~ couper le p o n t disulfure de la c y s t i n e 4, on ne p e u t d6celer q u ' u n seul p r o d u i t p a r c h r o m a t o g r a p h i e ou ionophor6se s u r p a p i e r : les d e u x r6sidus d ' a c i d e c y s t 6 i q u e ainsi form6s p a r t i c i p e n t d o n c /z u n e c h a i n e p e p t i d i q u e u n i q u e . L a v a s o p r e s s i n e o x y d 6 e est alors copul6e a v e c le dinitrofluorobenz~ne en prdsence de t r i m 6 t h y l a m i n O . Apr6s h y d r o l y s e p a r l'acide chlorh y d r i q u e , u n seul d6riv6 dinitroph6nyl6 j a u n e , l'acide d i n i t r o p h 6 n y l c y s t 6 i q u e , p e u t &tre identifi6 p a r c h r o m a t o g r a p h i e s u r papier. L a v a s o p r e s s i n e poss~de donc u n seul groupe a m i n 4 libre a p p a r t e n a n t A la cystine. D ' a u t r e p a r t , la v a s o p r e s s i n e oxyd6e, s o u m i s e & l ' a c t i o n de la t r y p s i n e (rapport e n z y m e /
31.