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The inactivation of ribonuclease by restricted pepsin digestion
v o L 17 (1955)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
593
The inactivation of ribonuclease by restricted pepsin digestion Recent studies on the chemical and physical properties of ribonuclease I 6 begin to p e r m i t a rational examination of the s t r u c t u r a l basis of its enzymic activity. Ribonuclease consists of a single peptide chain I of 1_,6 amino acids folded into a relatively c o m p a c t s t r u c t u r e u n d e r the influence of four disulfide cross-linkages and a secondary n e t w o r k of non-covalent bonds. The former m a v lw b r o k e n by controlled oxidation yielding a molecule w i t h o u t enzymic aetivityk The latter are easily r u p t u r e d in strong urea or guanidine chloride solutions and a p p e a r to be nonessential from the s t a n d p o i n t of catalytic activity since the urea " d e n a t u r e d " molecule, possessing a degree of disorientation nearly as great as the r a n d o m l y coiled oxidized chain, retains the catah'tic function of the native e n z y m e s . The native ribonuclease molecule has been subjected to the limited action of several proteolyt ie e n z y m e s and, on the basis of the s t r u c t u r a l studies mentioned above, the sites of attack of these proteases can be considered in relation to the enzymically active region of the molecule. Thus, the e x t r e m e C-terminal end of the chain does not a p p e a r to be catalytically inw)lved since several amino acid residues m a y be removed by c a r b o x y p e p t i d a s e w i t h o u t loss in activity v. Similarly, limited a t t a c k by the bacterial protease, subtilisin, yields a fully active derivative containing a new Nterminal end group, the fairly short chain w h k h it t e r m i n a t e s being a p p a r e n t l y attached to the body of the molecule lay disulfide bonding ~,". Earlier studies on the action of pepsin on ribonuclease suggested t h a t an initial p r o d u c t was produced diflering only \'ery slightly from native ribonuclease1°, n. Its , w and ¢~-amino nitrogen c o n t e n t were, withiu experimental error, unchanged in s')ite of complete loss of enzymic activity. This reaction has now been studied in greater detail and its kinetics have been examined by the c h r o m a t o g r a p h i c procedure of H1RS, ),IOORF AND S T E I N 1'2. The isolation and partial characterizati()n of the initial p r o d u c t suggest t h a t a very limited 8250 change has occurred within the enzymieally active ~280 portion of the molecule. Q4 In Fig. [ are s h o w n the behaviour of solutions of ribonuclease and ribonuclease digests on 0.2 IRC-5o c o l u m n s following digestion with pepsin at p H 1.8 for s h o r t periods of time. In these experi26 ments, the action of pepsin was stopped by adC 24 j u s t m e n t of the p H to 6. 5 . The figure shows the ~4 progressive p r o d u c t i o n of a new c h r o m a t o g r a p h i c 22~ 8 0.~ species at the expense of the native e n z y m e (appearing in the usual double peak form, b o t h peaks ~.6 "~ being fully active)..ks shown, the new derivatiw." is B & devoid of catalytic activity against R N A as tested 9./* 0.4 by m e a s u r e m e n t of the production of u r a n y l a c e t a t e 0.2 soluble fragmentsL C h r o m a t o g r a p h y of pepsin-treatQ2 ed ribonuclease at p H 6.o 5 r a t h e r t h a n 6.45 as employed here does not p e r m i t the elution of the inactive ,4 0.8 molecule from IRC-5 o c o l u m n s even after hmg 0.0 periods of development. By p a p e r c h r o m a t o g r a p h y 0.5 0.6 (in butanol-acetic acid-water; 4: l:5) of samples of 0.4 the digestion m i x t u r e taken after different time &4 periods, it was found t h a t a peptide was released in 0.2 0.2 progressively larger a m o u n t s . This peptide contained aspartic acid, serine, alanine and valine. The rate 50 80 20 40 of appearance of the f r a g m e n t was qualitatively corml effluent volume related with the rate of p r o d u c t i o n of the inactive Fig. I. C h r o m a t o g r a p h y (I 2) of l o mg samples protein c o m p o n e n t in Fig. I. of native and pepsin-digested ribonuclease on To characterize the new protein derivative more IRC-5o columns, 9 / 3 ° ram. Eluting buffer, fully, large samples of digest {identical with the phosphate, o.2 31, p H 6.45. Digestion condismaller sample employed for c h r o m a t o g r a m C in Fig. tions: ribonuclease o.92 % ; pepsin (Armour i were run t h r o u g h large columns u n d e r the same crystallized) ca. o.oo2°0; 3 7 ° C , p H 1.8. conditions as those described above. The flow rate was R e m a i n i n g ribonuclease activity (i): Curve adjusted to a speed which p e r m i t t e d the separation of A, IOO%; B, 6o°//o (5 min); C, 4 0 % (lo min) ; the main inactive c o m p o n e n t from the more drastiD, 15% (I0 min). Solid points, enzyme accally changed products, shown in Fig. )-C as a tivity. Open circles, protein concentration. shoulder on the principal inactive peak. The purified (The peak appearing at T8 19 effluent ml is a derivative was freed of salt b y dialysis against disninhydrin-negative artefact, uniformly seen tilled w a t e r and lyophilized, as was the remaining with this preparation of resin.) native enzyme which had emerged in earlier tubes.
594
SHORT COMMUNIC:\TIONS, PRELIMINARY NOTF,S
VOL. 1 7
(~955)
-2o I{oth t h e a c t i v e a n d i n a c t i v e c o m p o n e n t s showed t h e s a m e s e d i m e n t ~ t i o n c o n s t a n t , 5 w , (~ .8 ~.9 for ~ % solutions) a n d i n t r i n s i c v i s c o s i t y (3.3 3.9) i n d i c a t i n g t h a t t h e p h y s i c a l s ha pe a n d size ha d n o t been s i g n i f i c a n t l y a l t e r e d . P e r f o r m i c acid o x i d a t i o n of t he t w o m a t e r i a l s y i e l d e d p r o d u c t s w i t h c o m p a r a b l e s e d i m e n t a t i o n c h a r a c t e r i s t i c s (, ~v (in/lnite di l ut i on) ~.78) a l t h o u g h t h e s e d i m e n t a t i o n ' c o n s t a n t of t h e o x i d i z e d p e p s i n d e r i v a t i v e showed s o m e w h a t less c o n c e n t r a t i o n d e p e n d e n c e (S-21~ w ( 1 o,o) for o x i d i z e d n a t i v e R N a s e , ~.43; for o x i d i z e d d e r i w t t i v e , 1.()o). The r e d u c e d v i s c o s i t y (~%) of t h e o x i d i z e d d e r i v a t i v e (~o.3) w a s s l i g h t l y less t h a n t h a t of t h e o x i d i z e d n a t i v e e n z y m e (~2.31. Upon f l t e r p a p e r e l e c t r o p h o r e s i s of t h e o x i d i z e d c o m p o u n d s a t p l t 8.6 in b a r b i t a l buffer t he i n a c t i v e m a t e r i a l was s e p a r a t e d from t h e n a t i v e enzyme , b e i n g more e l e c t r o p o s i t i v e . The o x i d i z e d p e p s i n - m o d i f i e d mo lecule s h o w e d a second, n e u t r a l , c o m p o n e n t which w a s less r e a c t i v e to n i n h y d r i n . The c h a r a c t e r i z a t i o n of this f r a g m e n t which was p r e s u m a b l e l i nke d to t he body of t he i n a c t i v e m olecu le t h r o u g h disulfide l i n k a g e is now in progress. The p r e s e n t s t u d i e s c l e a r l y e x c l u d e t h e occurence of t h e " a l l - o r n o n e " or " e xpl os i x e" p r o t e o l y s i s s u g g e s t e d in earlier w o r k w i t h p e p s i n and v a r i o u s p r o t e i n s u b s t r a t e s a t p i t v a l u e s in t h e n e i g h b o r h o o d of ~.8 2.o 14, la,~
Ca~lsberg /.abora/orium, Cope~hagc~z (l)c~mart,') 1 C. 13. ANFINSEN, R. I~-EDFIELD, \V. CHOATE, J. PAGE AND \V. CANORR,J. l~iol. Chem., _'o 7 (I954) _~or. 2 C. H. \ g . H1RS, \g . H. STEIN AND S. MOORE, J. Biol. Chem., 2 I z (1954) 941 • 3 ]2. REI)FIELD AND C. B. ANFINSI~2N, lrederati(l~ Prec., 14 (1955) 208. 4 C. H. W. HIRS, Federa/io~z Prec., 13 (I9541 23o. 5 C. 1~. ANFINSEN, \V. i t. HARRINGTON, A. HVIDT, K. I.INI)ERSTROM-I.ANG, ~I. OTTESEN AND J. ~CHELLMAN, t3iochi~7. 13iophys. Acla, ~7 (1955) J4 [. 6 }~. LINDERSTROM-]~ANG, CJl('l~t. ,~oc. N),~n]5. o~1 Peptide Che~nislr)', Special P u b l i c a t i o n 2, 1955, ill press. 7 G. KALNITSKY AXD E. E. AYDERSOY, l~iochim, l~iophys. Acla, I6 0 9 5 5 ) 3 o2. S. M. KAL~IAN, K. LINDERSTROM-I,ANC;, M. OTTESEN AND F. M. RICHaRd)S, t3iochim, tJioph3's..lcta, ~6 (1955) 297. " 1~'. M. RICHARDS, Comfit. rend. lrav. lab. Carlsberg, S(r. chim., in press. 10 C. B. ANFINSEN, J. Biol. CheJ~z., 196 (19521 2Ol. 11 C. B. :\NFINSEN, in Mechanism o~ l'2n.zyme Action, e d i t e d b y \V. D. MCELRO\" AND B. GLASS, B a l t i m o r e , 1954, pp. l r 3 - t I 5. le C. It. \V. HIRS, S. MOORE AXD \V. if. STHN, J. l~iol. Che*n., 2oo (~953) 493. la E. O. P. Tl4o.~u,SON, 13iochim. l~iophys, dcta, ~5 0 9 5 4 ) 44 °. 1.1 \ . TISELIUS AND I. ERIKSSON-QUENSEL, Biochem. J., 33 (1939) ~752. la T. \VINxrCK, J. 13iol. Chem., 152 (1944) 465 . 16 .k. BELOFFAND C. B. ANFINSEX,,/. 13i0l. Clle~z., 176 (1948) 803 . iv (;. HaUGAARD AND R. M. ROBERTS, J..q~J:. Chem. ,S'oc., 64 (1942) 2004 . 18 }~. ]ANI)ERSTRflM-LANG AND 51. OTTESEN, Coll¢/)f. re~d. lrav. lab. Carlsberg, Sdr. chim., 26 (~949) 4o3 • 19 K. [ , I N I - ) E R S T R O I ~ I - | . A N G , (;old Spri~g Harbor .~3m@osia on QuaRt. t4iol., 14 (t95 o) 1~ 7. R e c e i v e d M a y i S t h , 19.55 • R o c k e f e l l e r P u b l i c Service Award, B e t h e s d a , Md. (U.S.A.).
~954
I055; p r e s e n t a d d r e s s : N a t i o n a l t t e a r t i n s t i t u t e ,
SHORT COMMUNICATIONS, PRELIMINARY NOTES
593
The inactivation of ribonuclease by restricted pepsin digestion Recent studies on the chemical and physical properties of ribonuclease I 6 begin to p e r m i t a rational examination of the s t r u c t u r a l basis of its enzymic activity. Ribonuclease consists of a single peptide chain I of 1_,6 amino acids folded into a relatively c o m p a c t s t r u c t u r e u n d e r the influence of four disulfide cross-linkages and a secondary n e t w o r k of non-covalent bonds. The former m a v lw b r o k e n by controlled oxidation yielding a molecule w i t h o u t enzymic aetivityk The latter are easily r u p t u r e d in strong urea or guanidine chloride solutions and a p p e a r to be nonessential from the s t a n d p o i n t of catalytic activity since the urea " d e n a t u r e d " molecule, possessing a degree of disorientation nearly as great as the r a n d o m l y coiled oxidized chain, retains the catah'tic function of the native e n z y m e s . The native ribonuclease molecule has been subjected to the limited action of several proteolyt ie e n z y m e s and, on the basis of the s t r u c t u r a l studies mentioned above, the sites of attack of these proteases can be considered in relation to the enzymically active region of the molecule. Thus, the e x t r e m e C-terminal end of the chain does not a p p e a r to be catalytically inw)lved since several amino acid residues m a y be removed by c a r b o x y p e p t i d a s e w i t h o u t loss in activity v. Similarly, limited a t t a c k by the bacterial protease, subtilisin, yields a fully active derivative containing a new Nterminal end group, the fairly short chain w h k h it t e r m i n a t e s being a p p a r e n t l y attached to the body of the molecule lay disulfide bonding ~,". Earlier studies on the action of pepsin on ribonuclease suggested t h a t an initial p r o d u c t was produced diflering only \'ery slightly from native ribonuclease1°, n. Its , w and ¢~-amino nitrogen c o n t e n t were, withiu experimental error, unchanged in s')ite of complete loss of enzymic activity. This reaction has now been studied in greater detail and its kinetics have been examined by the c h r o m a t o g r a p h i c procedure of H1RS, ),IOORF AND S T E I N 1'2. The isolation and partial characterizati()n of the initial p r o d u c t suggest t h a t a very limited 8250 change has occurred within the enzymieally active ~280 portion of the molecule. Q4 In Fig. [ are s h o w n the behaviour of solutions of ribonuclease and ribonuclease digests on 0.2 IRC-5o c o l u m n s following digestion with pepsin at p H 1.8 for s h o r t periods of time. In these experi26 ments, the action of pepsin was stopped by adC 24 j u s t m e n t of the p H to 6. 5 . The figure shows the ~4 progressive p r o d u c t i o n of a new c h r o m a t o g r a p h i c 22~ 8 0.~ species at the expense of the native e n z y m e (appearing in the usual double peak form, b o t h peaks ~.6 "~ being fully active)..ks shown, the new derivatiw." is B & devoid of catalytic activity against R N A as tested 9./* 0.4 by m e a s u r e m e n t of the production of u r a n y l a c e t a t e 0.2 soluble fragmentsL C h r o m a t o g r a p h y of pepsin-treatQ2 ed ribonuclease at p H 6.o 5 r a t h e r t h a n 6.45 as employed here does not p e r m i t the elution of the inactive ,4 0.8 molecule from IRC-5 o c o l u m n s even after hmg 0.0 periods of development. By p a p e r c h r o m a t o g r a p h y 0.5 0.6 (in butanol-acetic acid-water; 4: l:5) of samples of 0.4 the digestion m i x t u r e taken after different time &4 periods, it was found t h a t a peptide was released in 0.2 0.2 progressively larger a m o u n t s . This peptide contained aspartic acid, serine, alanine and valine. The rate 50 80 20 40 of appearance of the f r a g m e n t was qualitatively corml effluent volume related with the rate of p r o d u c t i o n of the inactive Fig. I. C h r o m a t o g r a p h y (I 2) of l o mg samples protein c o m p o n e n t in Fig. I. of native and pepsin-digested ribonuclease on To characterize the new protein derivative more IRC-5o columns, 9 / 3 ° ram. Eluting buffer, fully, large samples of digest {identical with the phosphate, o.2 31, p H 6.45. Digestion condismaller sample employed for c h r o m a t o g r a m C in Fig. tions: ribonuclease o.92 % ; pepsin (Armour i were run t h r o u g h large columns u n d e r the same crystallized) ca. o.oo2°0; 3 7 ° C , p H 1.8. conditions as those described above. The flow rate was R e m a i n i n g ribonuclease activity (i): Curve adjusted to a speed which p e r m i t t e d the separation of A, IOO%; B, 6o°//o (5 min); C, 4 0 % (lo min) ; the main inactive c o m p o n e n t from the more drastiD, 15% (I0 min). Solid points, enzyme accally changed products, shown in Fig. )-C as a tivity. Open circles, protein concentration. shoulder on the principal inactive peak. The purified (The peak appearing at T8 19 effluent ml is a derivative was freed of salt b y dialysis against disninhydrin-negative artefact, uniformly seen tilled w a t e r and lyophilized, as was the remaining with this preparation of resin.) native enzyme which had emerged in earlier tubes.
594
SHORT COMMUNIC:\TIONS, PRELIMINARY NOTF,S
VOL. 1 7
(~955)
-2o I{oth t h e a c t i v e a n d i n a c t i v e c o m p o n e n t s showed t h e s a m e s e d i m e n t ~ t i o n c o n s t a n t , 5 w , (~ .8 ~.9 for ~ % solutions) a n d i n t r i n s i c v i s c o s i t y (3.3 3.9) i n d i c a t i n g t h a t t h e p h y s i c a l s ha pe a n d size ha d n o t been s i g n i f i c a n t l y a l t e r e d . P e r f o r m i c acid o x i d a t i o n of t he t w o m a t e r i a l s y i e l d e d p r o d u c t s w i t h c o m p a r a b l e s e d i m e n t a t i o n c h a r a c t e r i s t i c s (, ~v (in/lnite di l ut i on) ~.78) a l t h o u g h t h e s e d i m e n t a t i o n ' c o n s t a n t of t h e o x i d i z e d p e p s i n d e r i v a t i v e showed s o m e w h a t less c o n c e n t r a t i o n d e p e n d e n c e (S-21~ w ( 1 o,o) for o x i d i z e d n a t i v e R N a s e , ~.43; for o x i d i z e d d e r i w t t i v e , 1.()o). The r e d u c e d v i s c o s i t y (~%) of t h e o x i d i z e d d e r i v a t i v e (~o.3) w a s s l i g h t l y less t h a n t h a t of t h e o x i d i z e d n a t i v e e n z y m e (~2.31. Upon f l t e r p a p e r e l e c t r o p h o r e s i s of t h e o x i d i z e d c o m p o u n d s a t p l t 8.6 in b a r b i t a l buffer t he i n a c t i v e m a t e r i a l was s e p a r a t e d from t h e n a t i v e enzyme , b e i n g more e l e c t r o p o s i t i v e . The o x i d i z e d p e p s i n - m o d i f i e d mo lecule s h o w e d a second, n e u t r a l , c o m p o n e n t which w a s less r e a c t i v e to n i n h y d r i n . The c h a r a c t e r i z a t i o n of this f r a g m e n t which was p r e s u m a b l e l i nke d to t he body of t he i n a c t i v e m olecu le t h r o u g h disulfide l i n k a g e is now in progress. The p r e s e n t s t u d i e s c l e a r l y e x c l u d e t h e occurence of t h e " a l l - o r n o n e " or " e xpl os i x e" p r o t e o l y s i s s u g g e s t e d in earlier w o r k w i t h p e p s i n and v a r i o u s p r o t e i n s u b s t r a t e s a t p i t v a l u e s in t h e n e i g h b o r h o o d of ~.8 2.o 14, la,~
Ca~lsberg /.abora/orium, Cope~hagc~z (l)c~mart,') 1 C. 13. ANFINSEN, R. I~-EDFIELD, \V. CHOATE, J. PAGE AND \V. CANORR,J. l~iol. Chem., _'o 7 (I954) _~or. 2 C. H. \ g . H1RS, \g . H. STEIN AND S. MOORE, J. Biol. Chem., 2 I z (1954) 941 • 3 ]2. REI)FIELD AND C. B. ANFINSI~2N, lrederati(l~ Prec., 14 (1955) 208. 4 C. H. W. HIRS, Federa/io~z Prec., 13 (I9541 23o. 5 C. 1~. ANFINSEN, \V. i t. HARRINGTON, A. HVIDT, K. I.INI)ERSTROM-I.ANG, ~I. OTTESEN AND J. ~CHELLMAN, t3iochi~7. 13iophys. Acla, ~7 (1955) J4 [. 6 }~. LINDERSTROM-]~ANG, CJl('l~t. ,~oc. N),~n]5. o~1 Peptide Che~nislr)', Special P u b l i c a t i o n 2, 1955, ill press. 7 G. KALNITSKY AXD E. E. AYDERSOY, l~iochim, l~iophys. Acla, I6 0 9 5 5 ) 3 o2. S. M. KAL~IAN, K. LINDERSTROM-I,ANC;, M. OTTESEN AND F. M. RICHaRd)S, t3iochim, tJioph3's..lcta, ~6 (1955) 297. " 1~'. M. RICHARDS, Comfit. rend. lrav. lab. Carlsberg, S(r. chim., in press. 10 C. B. ANFINSEN, J. Biol. CheJ~z., 196 (19521 2Ol. 11 C. B. :\NFINSEN, in Mechanism o~ l'2n.zyme Action, e d i t e d b y \V. D. MCELRO\" AND B. GLASS, B a l t i m o r e , 1954, pp. l r 3 - t I 5. le C. It. \V. HIRS, S. MOORE AXD \V. if. STHN, J. l~iol. Che*n., 2oo (~953) 493. la E. O. P. Tl4o.~u,SON, 13iochim. l~iophys, dcta, ~5 0 9 5 4 ) 44 °. 1.1 \ . TISELIUS AND I. ERIKSSON-QUENSEL, Biochem. J., 33 (1939) ~752. la T. \VINxrCK, J. 13iol. Chem., 152 (1944) 465 . 16 .k. BELOFFAND C. B. ANFINSEX,,/. 13i0l. Clle~z., 176 (1948) 803 . iv (;. HaUGAARD AND R. M. ROBERTS, J..q~J:. Chem. ,S'oc., 64 (1942) 2004 . 18 }~. ]ANI)ERSTRflM-LANG AND 51. OTTESEN, Coll¢/)f. re~d. lrav. lab. Carlsberg, Sdr. chim., 26 (~949) 4o3 • 19 K. [ , I N I - ) E R S T R O I ~ I - | . A N G , (;old Spri~g Harbor .~3m@osia on QuaRt. t4iol., 14 (t95 o) 1~ 7. R e c e i v e d M a y i S t h , 19.55 • R o c k e f e l l e r P u b l i c Service Award, B e t h e s d a , Md. (U.S.A.).
~954
I055; p r e s e n t a d d r e s s : N a t i o n a l t t e a r t i n s t i t u t e ,