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Identification of the peptide split from trypsinogen during autocatalytic activation
442
SHORT COMMUNICATIONS, PRELIMINARY NOTES
VOL. 1 1
(1953)
IDENTIFICATION OF THE PEPTIDE SPLIT FROM TRYPSINOGEN DURING AUTOCATALYTIC ACTIVATION* by E A R L W. D A V I E AND H A N S N E U R A T H
Department o/Biochemistry, University o/ Washington, Seattle, Washington (U.S.:I .)
R e c e n t s t u d i e s of t h e t e r m i n a l g r o u p s of t r y p s i n o g e n a n d D F P - t r y p s i n l , 2 h a v e led to t h e s u g g e s t i o n t h a t d u r i n g a u t o c a t a l y t i c a c t i v a t i o n of t h e z y m o g e n one or more p e p t i d e s are split off. T h i s s u g g e s t i o n was b a s e d on t h e r e s u l t t h a t t h e N - t e r m i n a l g r o u p s of t r y p s i n o g e n a n d D F P - t r y p s i n were different, w h e r e a s no C - t e r m i n a l g r o u p s reactive t o w a r d c a r b o x y p e p t i d a s e could be d e t e c t e d w h e n t h e s e p r o t e i n s were in t h e n a t i v e form. More r e c e n t s t u d i e s h a v e shown, however, t h a t after d e n a t u r a t i o n b y acid 1, D F P - t r y p s i n yields o. 3 e q u i v a l e n t s of lysine, a n d t r y p s i n o g e n o,o 5 e q u i v a l e n t s , in a d d i t i o n to lesser a m o u n t s of o t h e r a m i n o acids 1 w h i c h were identical for b o t h p r o t e i n s u b s t r a t e s . T h e a c t i o n of c a r b o x y p e p t i d a s e t h u s s u g g e s t s t h a t t h e c a r b o x y l e n d of t h e p o l y p e p t i d e c h a i n m a y n o t c h a n g e d u r i n g a c t i v a t i o n of t r y p s i n o g e n a n d t h a t t h e loss of a N - t e r m i n a l p e p t i d e gives rise to a t r y p s i n molecule m o r e susceptible to acid d e n a t u r a t i o n . W e h a v e s u c c e e d e d in i d e n t i f y i n g t h e single p e p t i d e f o r m e d d u r i n g t h e a c t i v a t i o n process, as follows: I n a t y p i c a l e x p e r i m e n t , 12o m g of twice crystallized t r y p s i n o g e n a was dissolved a t o ° in 9 ml of o.I M b o r a t e buffer, p H 8.o, c o n t a i n i n g o.o 5 M CaC12, a n d a c t i v a t e d b y t h e a d d i t i o n of o. 7 m g of t r y p s i n . A l i q u o t s were r e m o v e d a t i n t e r v a l s up to 8 h o u r s a n d a n y free a m i n o acids or p e p t i d e s were a d s o r b e d on D o w e x 5 ° resin (H+ form, 4 or 12 % cross-linked). T h e r e a c t i o n p r o d u c t s were e l u t e d f r o m t h e resin a n d s u b j e c t e d to p a p e r c h r o m a t o g r a p h y (butanol-acetic a c i d - w a t e r 4 a n d nlethanol-water-pyridineS). Control e x p e r i m e n t s d e m o n s t r a t e d t h a t u n d e r t h e s e c o n d i t i o n s a d s o r p t i o n on t h e resin was q u a n t i t a t i v e . S i m u l t a n e o u s l y , t h e r a t e of a c t i v a t i o n w a s followed b y t h e esterase m e t h o d u s i n g benzoyl-L-arginine e t h y l ester as s u b s t r a t e 6. T h e zero t i m e control s h o w e d t h e c o m p l e t e a b s e n c e of n i n h y d r i n - p o s i t i v e p r o d u c t s . A f t e r 5 % a c t i v a t i o n , a single s p o t w a s o b t a i n e d h a v i n g a n R F v a l u e of o.io (butanol-acetic acid-water), t h e i n t e n s i t y of t h i s s p o t increasing in p r o p o r t i o n to t h e e x t e n t of t r y p t i c a c t i v a t i o n . E l u t i o n of t h e spot, followed b y h y d r o l y s i s in a sealed t u b e in 6 N HC1 a t i io ° for 24 a n d 48 h o u r s i n d i c a t e d t h e presence of lysine, valine a n d a s p a r t i c acid in mole ratios of 1 : 1 : 5 or 6, a n d t r a c e s of g l u t a m i c acid a n d alanine. T h e C - t e r m i n a l position of lysine in t h e p e p t i d e is s u g g e s t e d b y t h e specificity r e q u i r e m e n t s of t r y p s i n , a n d t h e N - t e r m i n a l position of valine b y t h e r e p o r t t h a t this a m i n o acid occupies a like position in t r y p s i n o g e n , in c o n t r a s t to isoleucine for D F P - t r y p s i n 2. Hence, t h e t e n t a t i v e s t r u c t u r e of val-(asp) 5 or ~-lys, w i t h a m i n i m u m m o l e c u l a r w e i g h t of a b o u t IOOO, m a y be a s s i g n e d to t h i s peptide. T h e acidic p r o p e r t i e s of t h e p e p t i d e are in accord w i t h t h e relative positions of t h e isoionic p o i n t s of t r y p s i n o g e n a n d t r y p s i n , i.e., 9-3 a n d i o . i , respectively ~. Control e x p e r i m e n t s t e s t i n g t h e a u t o l y s i s of 1 % t r y p s i n u n d e r t h e s a m e e x p e r i m e n t a l c o n d i t i o n s revealed t h e presence of a f a i n t s p o t w i t h an R F v a l u e e q u a l to o.47 (butanolacetic acid-water) which, however, failed to increase in i n t e n s i t y w i t h t h e age of t h e solution. REFERENCES 1 E. g . DAVIE AND H. NEURATH, J. Am. Chem. Soc., 74 (1952) 63o5. ~"M. ROVERY, C. FABRE AND P. DESNUELLE, Biochim. Biophys. Acta, 9 (1952) 7 °2. 3 F. TIETZE, .]. Biol. Chem., in press. 4 j . A. GLADNER AND H. NEURATH, Biochim. Biophys. Acta, 9 (1952) 335, a n d n l a n u s c r i p t in p r e p a ration. 5 R. R. REOFIELD, Biochim. Biophys. Acla, IO (1953) 344. G. W. SCllWERT AND M. A. EISENBERG, J. Biol. Chem., 179 (1949) 665. 7 N. M. GREEN, u n p u b l i s h e d results. lleceived J u n e ist, 1953 T h i s w o r k was p e r f o r m e d u n d e r c o u t r a c t No. Nonr-477-o 4 b e t w e e n t h e U n i v e r s i t y of W a s h i n g t o n a n d t h e Office of N a v a l R e s e a r c h , D e p a r t m e n t of t h e N a v y , a n d w a s s u p p o r t e d also b y f u n d s m a d e available b y t h e people of t h e S t a t e of W a s h i n g t o n , I n i t i a t i v e I7I.
SHORT COMMUNICATIONS, PRELIMINARY NOTES
VOL. 1 1
(1953)
IDENTIFICATION OF THE PEPTIDE SPLIT FROM TRYPSINOGEN DURING AUTOCATALYTIC ACTIVATION* by E A R L W. D A V I E AND H A N S N E U R A T H
Department o/Biochemistry, University o/ Washington, Seattle, Washington (U.S.:I .)
R e c e n t s t u d i e s of t h e t e r m i n a l g r o u p s of t r y p s i n o g e n a n d D F P - t r y p s i n l , 2 h a v e led to t h e s u g g e s t i o n t h a t d u r i n g a u t o c a t a l y t i c a c t i v a t i o n of t h e z y m o g e n one or more p e p t i d e s are split off. T h i s s u g g e s t i o n was b a s e d on t h e r e s u l t t h a t t h e N - t e r m i n a l g r o u p s of t r y p s i n o g e n a n d D F P - t r y p s i n were different, w h e r e a s no C - t e r m i n a l g r o u p s reactive t o w a r d c a r b o x y p e p t i d a s e could be d e t e c t e d w h e n t h e s e p r o t e i n s were in t h e n a t i v e form. More r e c e n t s t u d i e s h a v e shown, however, t h a t after d e n a t u r a t i o n b y acid 1, D F P - t r y p s i n yields o. 3 e q u i v a l e n t s of lysine, a n d t r y p s i n o g e n o,o 5 e q u i v a l e n t s , in a d d i t i o n to lesser a m o u n t s of o t h e r a m i n o acids 1 w h i c h were identical for b o t h p r o t e i n s u b s t r a t e s . T h e a c t i o n of c a r b o x y p e p t i d a s e t h u s s u g g e s t s t h a t t h e c a r b o x y l e n d of t h e p o l y p e p t i d e c h a i n m a y n o t c h a n g e d u r i n g a c t i v a t i o n of t r y p s i n o g e n a n d t h a t t h e loss of a N - t e r m i n a l p e p t i d e gives rise to a t r y p s i n molecule m o r e susceptible to acid d e n a t u r a t i o n . W e h a v e s u c c e e d e d in i d e n t i f y i n g t h e single p e p t i d e f o r m e d d u r i n g t h e a c t i v a t i o n process, as follows: I n a t y p i c a l e x p e r i m e n t , 12o m g of twice crystallized t r y p s i n o g e n a was dissolved a t o ° in 9 ml of o.I M b o r a t e buffer, p H 8.o, c o n t a i n i n g o.o 5 M CaC12, a n d a c t i v a t e d b y t h e a d d i t i o n of o. 7 m g of t r y p s i n . A l i q u o t s were r e m o v e d a t i n t e r v a l s up to 8 h o u r s a n d a n y free a m i n o acids or p e p t i d e s were a d s o r b e d on D o w e x 5 ° resin (H+ form, 4 or 12 % cross-linked). T h e r e a c t i o n p r o d u c t s were e l u t e d f r o m t h e resin a n d s u b j e c t e d to p a p e r c h r o m a t o g r a p h y (butanol-acetic a c i d - w a t e r 4 a n d nlethanol-water-pyridineS). Control e x p e r i m e n t s d e m o n s t r a t e d t h a t u n d e r t h e s e c o n d i t i o n s a d s o r p t i o n on t h e resin was q u a n t i t a t i v e . S i m u l t a n e o u s l y , t h e r a t e of a c t i v a t i o n w a s followed b y t h e esterase m e t h o d u s i n g benzoyl-L-arginine e t h y l ester as s u b s t r a t e 6. T h e zero t i m e control s h o w e d t h e c o m p l e t e a b s e n c e of n i n h y d r i n - p o s i t i v e p r o d u c t s . A f t e r 5 % a c t i v a t i o n , a single s p o t w a s o b t a i n e d h a v i n g a n R F v a l u e of o.io (butanol-acetic acid-water), t h e i n t e n s i t y of t h i s s p o t increasing in p r o p o r t i o n to t h e e x t e n t of t r y p t i c a c t i v a t i o n . E l u t i o n of t h e spot, followed b y h y d r o l y s i s in a sealed t u b e in 6 N HC1 a t i io ° for 24 a n d 48 h o u r s i n d i c a t e d t h e presence of lysine, valine a n d a s p a r t i c acid in mole ratios of 1 : 1 : 5 or 6, a n d t r a c e s of g l u t a m i c acid a n d alanine. T h e C - t e r m i n a l position of lysine in t h e p e p t i d e is s u g g e s t e d b y t h e specificity r e q u i r e m e n t s of t r y p s i n , a n d t h e N - t e r m i n a l position of valine b y t h e r e p o r t t h a t this a m i n o acid occupies a like position in t r y p s i n o g e n , in c o n t r a s t to isoleucine for D F P - t r y p s i n 2. Hence, t h e t e n t a t i v e s t r u c t u r e of val-(asp) 5 or ~-lys, w i t h a m i n i m u m m o l e c u l a r w e i g h t of a b o u t IOOO, m a y be a s s i g n e d to t h i s peptide. T h e acidic p r o p e r t i e s of t h e p e p t i d e are in accord w i t h t h e relative positions of t h e isoionic p o i n t s of t r y p s i n o g e n a n d t r y p s i n , i.e., 9-3 a n d i o . i , respectively ~. Control e x p e r i m e n t s t e s t i n g t h e a u t o l y s i s of 1 % t r y p s i n u n d e r t h e s a m e e x p e r i m e n t a l c o n d i t i o n s revealed t h e presence of a f a i n t s p o t w i t h an R F v a l u e e q u a l to o.47 (butanolacetic acid-water) which, however, failed to increase in i n t e n s i t y w i t h t h e age of t h e solution. REFERENCES 1 E. g . DAVIE AND H. NEURATH, J. Am. Chem. Soc., 74 (1952) 63o5. ~"M. ROVERY, C. FABRE AND P. DESNUELLE, Biochim. Biophys. Acta, 9 (1952) 7 °2. 3 F. TIETZE, .]. Biol. Chem., in press. 4 j . A. GLADNER AND H. NEURATH, Biochim. Biophys. Acta, 9 (1952) 335, a n d n l a n u s c r i p t in p r e p a ration. 5 R. R. REOFIELD, Biochim. Biophys. Acla, IO (1953) 344. G. W. SCllWERT AND M. A. EISENBERG, J. Biol. Chem., 179 (1949) 665. 7 N. M. GREEN, u n p u b l i s h e d results. lleceived J u n e ist, 1953 T h i s w o r k was p e r f o r m e d u n d e r c o u t r a c t No. Nonr-477-o 4 b e t w e e n t h e U n i v e r s i t y of W a s h i n g t o n a n d t h e Office of N a v a l R e s e a r c h , D e p a r t m e n t of t h e N a v y , a n d w a s s u p p o r t e d also b y f u n d s m a d e available b y t h e people of t h e S t a t e of W a s h i n g t o n , I n i t i a t i v e I7I.