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On the supposed catalytic oxidation of thiol groups by catalase
VOL. 2 8 ( I 0 5 8 )
PRELIMINARY NO'H';S
225
On the supposed catalytic oxidation of thiol groups by catalase In a p r e l i m i n a r y c o m m u n i c a t i o n published in 1952, BOER1 AND B O N N I C I I S E N 1 reported t h a t the oxidation of thiol c o m p o u n d s such as cysteine, g l u t a t h i o n e and thioglycollic acid by molecular oxygen can be catalysed by blood or liver catalase and t h a t these thiols inhibit the catalatic decomposition of h y d r o g e n peroxide. The reaction p o s t u l a t e d by these w o r k e r s is of special interest because it resembles s o m e w h a t the oxidatiolt of d i o x y f u m a r i c acid catalysed by peroxidase. As the above c o m m u n i c a t i o n 1 was n o t followed by the promised detailed r e p o r t " w i t h d a t a on spectroscopic changes of catalase in the presence of thiol g r o u p s " , the reaction between catalase and cysteine was reinvestigated b o t h manolnetrically and spectroscopically. The results of this s t u d y t o g e t h e r with some considerations based u p o n previous w o r k can be s u m m a r i s e d as follows: I. Although both liver- and blood-catalase p r e p a r a t i o n s increase the rate of a u t o - o x i d a t i o n of cysteine, the c o n c e n t r a t i o n of catalase r.equired for this reaction is a b o u t J o ~ times higher t h a n t h a t required for the catalatic decomposition of H202 at an a p p r o x i m a t e l y similar rate. 2. Boiling catalase p r e p a r a t i o n s decreases the rate of the reaction as well as the total oxygen u p t a k e in so far as the d e n a t u r e d p r o t e i n t e n d s to a d s o r b cysteine and p r o t e c t it from oxidation. 3. S o d i u m azide, which is a powerful inhibitor of catalase, does not affect the oxidation of cysteine in this reaction. 4- S o d i u m p y r o p h o s p h a t e a n d aa' dipyridyl, which combine with free iron and other nletals, m a r k e d l y decrease the rate of oxidation of cysteine in the presence of catalase. E t h y l e n e d i a m i n e tetraacetic acid h a d little effect at high (io 2 M) cysteine concentrations, b u t w a s inhibitory at lower (io 8 M) concentrations. N N ' d i h y d r o x y e t h y l g l y c i n e (a commercial iron chelator, Versene Fe-3 specific) m a r k e d l y s t i m u l a t e d the oxidation of lO 3 31 cysteine; this s t i m u l a t i o n occurred b o t h w i t h catalase a n d a control using free iron (FeSO4). 5. Catalase, in t h e presence of cysteine undergoing auto-oxidation, changes its characteristic a b s o r p t i o n s p e c t r u m (625, 54 o, 500 m/~) to t h a t of the catalase-peroxide c o m p o u n d l I with b a n d s at 568 and 535 m/z. The effect of cysteine ill this respect resembles t h a t of ascorbic acid ~. The spectroscopic investigation of this s y s t e m m a y be complicated b y the occurrence of free H i S which, in t h e presence of peroxide, intensifies the a b s o r p t i o n b a n d of catalase in the red and shifts it to 635 m/z. 6. B o t h cysteine a n d ascorbic acid are ineffective as h y d r o g e n donors in the coupled oxidation reaction catalysed by catalase in the presence of H202 generated by a p r i m a r y oxidation s y s t e m such as n o t a t i n , glucose and o x y g e n a. 7. G l u t a t h i o n e b e h a v e s in a similar w a y to cysteine b u t o x i d i z e s a t a m u c h lower rate especially w h e n it is p r e s e n t in a low concentration. 8. M a m m a l i a n e r y t h r o c y t e s c o n t a i n high c o n c e n t r a t i o n s both of catalase and glutathione. The oxidation of g l u t a t h i o n e in isolated corpuscles is nevertheless v e r y slow 4, a n d the glntathione i n arterial blood is m a i n l y in the reduced form 5. 9. All k n o w n c a t a l y s t s of cysteine a u t o - o x i d a t i o n (such as ionic copper or iron, and haematin) u n d e r g o reversible reduction and oxidation during this process. Catalase is reduced neither lly cysteine nor by the more p o w e r f u l reducing agent s o d i u m dithionite (Na2S204). These results militate against the view p u t f o r w a r d by BOERI AND BONNICHSEN 1 and suggest t h a t the increase in the rate of the a u t o - o x i d a t i o n of thiol c o m p o u n d s in the presence of catalase is b r o u g h t a b o u t not by catalase itself b u t by n o n - h a e m a t i n iron a n d possibly some degradation p r o d u c t s of h a e m a t i n p r e s e n t in catalase p r e p a r a t i o n s . F u r t h e r m o r e , the inactivity of catalase-peroxide c o m p o u n d I I in the catalatic destruction of h y d r o g e n peroxide 2 and the f o r m a t i o n of this c o m p o u n d during the a u t o - o x i d a t i o n of cysteine explain the i n h i b i t o r y effect of the latter u p o n the a c t i v i t y of catalasel, 6. One of us (P. N.) is indebted to the Medical Research Council for a personal g r a n t enabling h i m to take p a r t in this investigation.
Molteno Institute, University o/Cambridge (England) 1 E. B. 3 I). 4 N. a H. 6 G.
D. KEILIN P. NICHOLLS
BOERI AND R. B O N N I C H S E N , Acta Chem. Scan&, 6 (1952) 968. CHANCE, 13iochem. J., 46 (195 o) 387 . KEILIN ANn E. F. HARTREE, Biochem. J,, 60 (19551 3lo. ['. MELDRUM, Biochem. J., 26 (1932) 817. MARTIN, R. RODNIGHT, H. MclLwAIN AND M. A. TRESlZE, Nalure, 179 (I9571 419. CFRIOTTI, g. SPANDRIO AND iL5. BERTI, 14iochim. t~iophys. Acta, 23 (19571 302. Received F e b r u a r y 5th, 195~
PRELIMINARY NO'H';S
225
On the supposed catalytic oxidation of thiol groups by catalase In a p r e l i m i n a r y c o m m u n i c a t i o n published in 1952, BOER1 AND B O N N I C I I S E N 1 reported t h a t the oxidation of thiol c o m p o u n d s such as cysteine, g l u t a t h i o n e and thioglycollic acid by molecular oxygen can be catalysed by blood or liver catalase and t h a t these thiols inhibit the catalatic decomposition of h y d r o g e n peroxide. The reaction p o s t u l a t e d by these w o r k e r s is of special interest because it resembles s o m e w h a t the oxidatiolt of d i o x y f u m a r i c acid catalysed by peroxidase. As the above c o m m u n i c a t i o n 1 was n o t followed by the promised detailed r e p o r t " w i t h d a t a on spectroscopic changes of catalase in the presence of thiol g r o u p s " , the reaction between catalase and cysteine was reinvestigated b o t h manolnetrically and spectroscopically. The results of this s t u d y t o g e t h e r with some considerations based u p o n previous w o r k can be s u m m a r i s e d as follows: I. Although both liver- and blood-catalase p r e p a r a t i o n s increase the rate of a u t o - o x i d a t i o n of cysteine, the c o n c e n t r a t i o n of catalase r.equired for this reaction is a b o u t J o ~ times higher t h a n t h a t required for the catalatic decomposition of H202 at an a p p r o x i m a t e l y similar rate. 2. Boiling catalase p r e p a r a t i o n s decreases the rate of the reaction as well as the total oxygen u p t a k e in so far as the d e n a t u r e d p r o t e i n t e n d s to a d s o r b cysteine and p r o t e c t it from oxidation. 3. S o d i u m azide, which is a powerful inhibitor of catalase, does not affect the oxidation of cysteine in this reaction. 4- S o d i u m p y r o p h o s p h a t e a n d aa' dipyridyl, which combine with free iron and other nletals, m a r k e d l y decrease the rate of oxidation of cysteine in the presence of catalase. E t h y l e n e d i a m i n e tetraacetic acid h a d little effect at high (io 2 M) cysteine concentrations, b u t w a s inhibitory at lower (io 8 M) concentrations. N N ' d i h y d r o x y e t h y l g l y c i n e (a commercial iron chelator, Versene Fe-3 specific) m a r k e d l y s t i m u l a t e d the oxidation of lO 3 31 cysteine; this s t i m u l a t i o n occurred b o t h w i t h catalase a n d a control using free iron (FeSO4). 5. Catalase, in t h e presence of cysteine undergoing auto-oxidation, changes its characteristic a b s o r p t i o n s p e c t r u m (625, 54 o, 500 m/~) to t h a t of the catalase-peroxide c o m p o u n d l I with b a n d s at 568 and 535 m/z. The effect of cysteine ill this respect resembles t h a t of ascorbic acid ~. The spectroscopic investigation of this s y s t e m m a y be complicated b y the occurrence of free H i S which, in t h e presence of peroxide, intensifies the a b s o r p t i o n b a n d of catalase in the red and shifts it to 635 m/z. 6. B o t h cysteine a n d ascorbic acid are ineffective as h y d r o g e n donors in the coupled oxidation reaction catalysed by catalase in the presence of H202 generated by a p r i m a r y oxidation s y s t e m such as n o t a t i n , glucose and o x y g e n a. 7. G l u t a t h i o n e b e h a v e s in a similar w a y to cysteine b u t o x i d i z e s a t a m u c h lower rate especially w h e n it is p r e s e n t in a low concentration. 8. M a m m a l i a n e r y t h r o c y t e s c o n t a i n high c o n c e n t r a t i o n s both of catalase and glutathione. The oxidation of g l u t a t h i o n e in isolated corpuscles is nevertheless v e r y slow 4, a n d the glntathione i n arterial blood is m a i n l y in the reduced form 5. 9. All k n o w n c a t a l y s t s of cysteine a u t o - o x i d a t i o n (such as ionic copper or iron, and haematin) u n d e r g o reversible reduction and oxidation during this process. Catalase is reduced neither lly cysteine nor by the more p o w e r f u l reducing agent s o d i u m dithionite (Na2S204). These results militate against the view p u t f o r w a r d by BOERI AND BONNICHSEN 1 and suggest t h a t the increase in the rate of the a u t o - o x i d a t i o n of thiol c o m p o u n d s in the presence of catalase is b r o u g h t a b o u t not by catalase itself b u t by n o n - h a e m a t i n iron a n d possibly some degradation p r o d u c t s of h a e m a t i n p r e s e n t in catalase p r e p a r a t i o n s . F u r t h e r m o r e , the inactivity of catalase-peroxide c o m p o u n d I I in the catalatic destruction of h y d r o g e n peroxide 2 and the f o r m a t i o n of this c o m p o u n d during the a u t o - o x i d a t i o n of cysteine explain the i n h i b i t o r y effect of the latter u p o n the a c t i v i t y of catalasel, 6. One of us (P. N.) is indebted to the Medical Research Council for a personal g r a n t enabling h i m to take p a r t in this investigation.
Molteno Institute, University o/Cambridge (England) 1 E. B. 3 I). 4 N. a H. 6 G.
D. KEILIN P. NICHOLLS
BOERI AND R. B O N N I C H S E N , Acta Chem. Scan&, 6 (1952) 968. CHANCE, 13iochem. J., 46 (195 o) 387 . KEILIN ANn E. F. HARTREE, Biochem. J,, 60 (19551 3lo. ['. MELDRUM, Biochem. J., 26 (1932) 817. MARTIN, R. RODNIGHT, H. MclLwAIN AND M. A. TRESlZE, Nalure, 179 (I9571 419. CFRIOTTI, g. SPANDRIO AND iL5. BERTI, 14iochim. t~iophys. Acta, 23 (19571 302. Received F e b r u a r y 5th, 195~