M6moires originaux
BIOCHIMIE, 1'975, 57, 265-270.
Kinetic evidence of horseradish peroxidase oxidation by compound I. Marius SANTIMONE. Laboratoire de Phgsiologie Cellulaire Vdg~lale associ~ au C.N.R.S., Universitd d'Aix-Marseille II, Centre de L u m i n g , 70, Route L{'on L a c h a m p , 13288 Marseille Cddex 2, France. (11-10-197~). Summary. - - The kinetics of c o m p o u n d II f o r m a t i o n , o b t a i n e d upon mixing a highly purified h o r s e r a d i s h peroxidase and h y d r o g e n peroxide, was s p e c t r o p h o t o m e t r i c a l l y studied at t h r e e w a v e l e n g h t s in the absence of an added reducing agent. Our e x p e r i m e n t s confirm George's finding t h a t more t h a n one mole of compound II is formed per mole of h y d r o g e n peroxide added. The new m e c h a n i s m t h a t we propose, c o n t r a r y to the mechan i s m of George, is only valid w h e n c o m p o u n d II is o b t a i n e d in the absenee of a n added donor. Moreover, it is not i n c o n s i s t e n t w i t h the elassieal Chance m e c h a n i s m of oxidation of a n added d o n o r b y the system peroxidase - - hydrogen peroxide. Aeeording to t h i s new m e c h a n i s m , in the absence of a n added donor, compound II format.ion involves two p a t h w a y s . The first p a t h w a y is the m o n o m o l e c u l a r reduction of c o m p o u n d I by the endogenous donor, and the second p a t h w a y is t h e f o r m a t i o n of two moles of c o m p o u n d II t h r o u g h the oxidoreduetion reaction between one mole of peroxidase and one mole of compound I.
JNT1RODUCTION. Horseradish peroxidase catalyzes the oxidation of a w i d e v a r i e t y of e l e c t r o n - d o n o r s b y h y d r o g e n p e r o x i d e . T h e g e n e r a l l y a c c e p t e d r o l e of t h e enzyme can be represented by the following mechan i s m ~ v h i c h o w e s a g r e a t d e a l to t h e c o n t r i b u t i o n of C h a n c e [1-31 a n d G e o r g e [4-.5]. E + Co I + Co II +
S AH AH
- - • .... • - - ~
Co 1 Co II + E + A
A
(1) (2) (3)
w h e r e E r e p r e s e n t s t h e n a t i v e e n z y m e , S is t h e h y d r o g e n p e r o x i d e , Co I a n d Co II a r e tile o x i d i z e d f o r m s of t h e e n z y m e , c a l l e d c o m p o u n d I a n d c o m p o u n d II, r e s p e c t i v e l y , a n d AH is a r e d u c e d one-electron donor. Thus this classical reaction s c h e m e , w h i c h is c u r r e . n t l y a d m i t t e d w h e n AH is a n a d d e d d o n o r , c o r r e s p o n d s to t h e f o r m a t i o n of o n e m o l e of c o m p o u n d II f r o m o n e m o l e of h y d r o gen peroxide. This observation, which agrees w i t h t h o s e of p r e v i o u s w o r k e r s (e.g. A l t s c h u ] , A b r a m s a n d H o g n e s s [6]) a n d m o r e r e c e n t l y w i t h t h o s e of Y o n e t a n i [7], d o e s n o t a g r e e w i t h G e o r g e ' s f i n d i n g [511 t h a t t h e a d d i t i o n of o n e m o l e of H 2 0 e to H R P r e s u l t s i n t h e f o r m a t i o n of m o r e t h a n o n e m o l e of c o m p o u n d II. A l t h o u g h h e h a d previously presented e q u a t i o n s 1 a n d 2 [8], George proposed another mechanism which was c o n f o r m to h i s t i t r a t i o n r e s u l t s .
E + S -> Co I (1) Co I + A H , - - ~ Co II + AH (4) E + AH - - - > Co I I (5) w h e r e AH 2 r e p r e s e n t s e i t h e r r e d u c i n g g r o u p s p r e sent in the enzyme preparation or the deliberately a d d e d r e d u c i n g a g e n t , AH is a n o x i d i z i n g r a d i c a l a n d t h e o t h e r a b b r e v i a t i o n s a r e as d e f i n e d f o r e q u a t i o n s 1, 2 a n d 3. In this paper, we present a kinetic study which c o n f i r m s t i , t r a t i o n s t u d y of George. W e s h o w t h a t G e o r g e ' s o b s e r v a t i o n , t h a t m o r e t h a n o n e m o l e of compound II is o b t a i n e d f r o m o n m o l e of h y d r o g e n p e r o x i d e , is o n l y v a l i d w h e n n o a d d e d d o n o r is p r e s e n t i n t h e m e d i m n . I n t h i s case, c o m p o u n d II is p r o d u c e d b y t w o r e a c t i o n s w h i c h o c c u r s i m u l t a n e o u s l y . T h e first p a t h w a y ( r e a c t i o n 6) is t h e o x i d o r e d u c t i o n r e a c t i o n E + Co I - - > 2 Co [1 (6) b e t w e e n f e r r i p e r o x i d a s e a n d c o m p o u n d I. S i n c e t h e f o r m a l v a l e n c y s t a t e s of i r o n i n p e r o x i d a s e , c o m p o u n d 1 a n d c o m p o u n d II a r e r e s p e c t i v e l y (3-]-), ( 5 + ) a n d ( 4 + ) , t h i s r e a c t i o n c a n b e w r i t t e n as fol'lows : Fe~a ~- ~ + ]~'e(~-" ~ ~ 2FeI~+~ (7) T h e s e c o n d p a t h w a y ( r e a c t i o n 8) is t h e i n t r a - m o lecular reduction Co I . BH - • Co I I . B (8) of c o m p o u n d I to c o m p o u n d II b y t h e 20
266
M. S a n t i m o n e .
endogenous donor BH, p r e s e n t i n a.l~l peroxidases, a n d w h i c h is associated w i t h the enzyme p r o t e i n [5, 9]. One can observe that reaction 6 supposes that, i n the absence of added donor, one mole of c o m p o u n d I can react w i t h peroxidase i n excess giving two moles of c o m p o u n d II, while r e a c t i o n 8 supposes that only one mole of c o m p o u n d II is o b t a i n e d from one mole of c o m p o u n d I. Therefore, by the two pathways, one tootle of h y d r o g e n p,eroxide gives n m r e t h a n one ulole of c o m p o u n d II. MATERIALS AND METHODS. The h o r s e r a d i s h peroxidase isoenzyme used in the p r e s e n t w o r k designated P2 [10] is very similar to isoenzyure C of HR~P p,reviously described by S h a n n o n et al. [11]. It was isolated and purified from the c o m m e r c i a l e n z y m e p r e p a r a t i o n o b t a i n e d from Fluka. The purifica~ion method [10] was derived from that of Mazza et al. [12]. The hemop r o t e i n thus o b t a i n e d was homogeneous on anatytical centrifugatio,n and on electrophoresis. Its R. Z. (Reinheitszahl) was close to 3. H y d r o g e n peroxide (30 p. cent H~Oo p r o d u c e d by Merck) w h i c h was a p p r o p r i a t e l y di.luted immediately before use, was accurately calibrated against a s t a n d a r d solution of potassium p e r m a n ganate in an acidic nredium [13]. The kinetics of c o m p o u n d I and II a p p e a r a n c e a n d d i s a p p e a r a n c e were r e c o r d e d w i t h a Spectralux 1800 s p e c t r o p h o t o m e t e r equipped w i t h a Sefram record,er. This appa,ratus was modified so as to be ca:pable o.f r a p i d l y i n j e c t i n g a r e a c t a n t into the m e d i u m and r e c o r d i n g the a b s o r b a n c e change at a fixed wawelength. Mixing was effected in less than 3 secondes by a m e c h a n i c a l s t i r r e r p r e v i o u s l y described It0]. A kinetic analysis of the curves r e c o r d e d was then possible b e y o n d this time. RESULTS. It is well established [5, 7] that HRP is r a p i d l y a n d i r r e v e r s i b l y converted to c o m p o t m d I by the a d d i t i o n of an equal q u a n t i t y of h y d r o g e n peroxide. W h e n the peroxidas.e isoenzyme P2 is used, this reaction can be followed at 410 rim, the isosbestic p o i n t b e t w e e n p,eroxidase P , a n d comp o u n d II. This wavelenght is close to the corresp o n d i n g value of 410.5 n m p r e v i o u s l y r e p o r t e d by Chance [2] a n d Yonetani [7]. ,Curve 1 of figure 1A shows a r a p i d decrease in the absorb anee w h i c h corresponds to the stoic h i o m e t r i c f o r m a t i o n of c o m p o u n d I p r o d u c e d BIOCHIMIE, 1975, 57, n ° 3.
w h e n peroxidase a n d h y d r o g e n peroxide are put together i n equal ccmcentrations. Then, a slow increase in the a b s o r b a n c e is observed, w h i c h c o r r e s p o n d s .to the r e d u c t i o n of c o m p o u n d I. Since no added d o n o r is presen~ in the m e d i u m , this c o m p o u n d I r e d u c t i o n is not possible through C~aance's m e c h a n i s m . As we shall show, the hypothesis that the enzyme p r e p a r a t i o n a n d / o r the h y d r o g e n p e r o x i d e sotlution may c o n t a i n a reducing i m p u r i t y must be excluded. I n d e e d , if it is assumed that p is the c o n c e n t r a tion of both peroxidase and h y d r o g e n peroxide, a n d i E, the c o n c e n t r a t i o n of IH, an h y p o t h e t i c a l i m p u r i t y p r e s e n t in the enzyme p r e p a r a t i o n , then c o m p o u n d I is r a p i d l y formed at p c o n c e n t r a t i o n w h e n peroxidase and h y d r o g e n p e r o x i d e are mixed. C o m p o u n d I is, afterwards, r e d u c e d to c o m p o u n d II through the r e a c t i o n Co I + IH - - - > Co II + I (9) The velocity of this reaction can be m e a s u r e d by the slope of curve 1 of figure 1A w h i c h shows a relatively slow increase i n the absorbance. This velocity is vp = - - k (p - - x) (ij~ - - x) w h e r e k is the rate constant of reaction 9, a n d x the c o n c e n t r a t i o n of c o m p o u n d I w h i c h has been reduced. W h e n the h y d r o g e n p e r o x i d e c o n c e n t r a tion used is two times lower, the c o n c e n t r a t i o n of c o m p o u n d I formed is -p~ and the velocity is 2 vp/2 = - - k ( p-- - - x) (i~ - - x) (10) 2 Equations 9 a n d 10 show that the velocity must be r e d u c e d w h e n the hydroge,n peroxide c o n c e n t r a tion is decreased. On the contrary, c o m p a r i s o n of curves 1 a n d 2 of figure 1 A shows an increase in the i n i t i a l velocity w h e n the h y d r o g e n peroxide c o n c e n t r a t i o n is decreased. Therefore, the hypothesis that this p h e n o m e n o m is caused by a reducing i m p u r i t y present in the enzyme p r e p a r a t i o n must be rejected. Assurni:ng that the r e d u c i n g matter is p r e s e n t i n the h y d r o g e n p e r o x i d e solution, w h e n equal concentrations of peroxidase and h y d r o g e n p e r o x i d e are used, the velocity is v s = - - k ( p - - x ) (i s - x) (11) w h e r e i s is the r e d u c i n g i m p u r i t y c o n c e n t r a t i o n in lhe h y d r o g e n p e r o x i d e solution. W h e n the hydrogen Feroxide c o n c e n t r a t i o n used is two times lower, the velocity becomes Vs/2 = -
k (-~P - - x) ( i s _ _ _ x) 2 2
(12)
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FIG. 1. - - Kinetics of the reduction of peroxidase c o m p o u n d I to c o m p o u n d II. A. - - Absorbance changes recorded at ~10 n m ( i s o s b e s t i c p o i n t of p e r o x i d a s e P 2 - - c o m p o u n d II). I n j e c t i o n of h y d r o g e n p e r o x i d e ( A r r o w a) in t h e m e d i u m c o n t a i n i n g p e r o x i d a s e g i v e s n e g a t i v e A A w h i c h c o r r e s p o n d to t h e t r a n s f o r m a t i o n of f e r r i p e r o x i d a s e to c o m p o u n d I. T h e p o s i t i v e A A t h e n r e c o r d e d c o r r e s p o n d to its d i s a p p e a r a n c e . T h e r e s p e c t i v e a m o u n t s of perox.idase a n d h y d r o g e n p e r o x i d e u s e d are 6.87 × %0-3 ,vmole a n d 6.87 × 10-.3 ;vmole (Curv.e 1), 6.87 X 1,0-3 Ivmole a n d 3.43 × 10-3 ~xmole (Curve 2), 3.43 × 10-3 t~mole a n d 3.43 × 10:-3 ~xmole (Curve 33. B. - - Absorbance changes recorded at /~27 n m ( i s o s b e s t i c p o i n t p e r o x i d a s e P_~ - - c o m p o u n d I). T h e p o s i t i v e A A o b s e r v e d c o r r e s p o n d to t h e c o m p o u n d II f o r m a t i o n . E x p e r i m e n t a l c o n d i t i o n s o f c u r v e s 1, 2 a n d 3 a r e a s in figure i A. C. - - Absorbance changes recorded at 397 n m ( i s o s b e s t i c p o i n t c o m p o u n d I - - c o m p o u n d II). T h e n e g a t i v e A A o b s e r v e d c o r r e s p o n d to t h e t r a n s f o r m a t i o n of f e r r i p e r o x i d a s e to c o m p o u n d I. T h e a b s o r b a n c e v a r i a t i o n s t h e n o b s e r v e d a r e v e r y s l i g h t d u r i n g t h e first five m i n u t e s of t h e r e a c t i o n . T h i s s h o w s t h a t t h e r e d u c t i o n o f c o m p o u n d II to f e r r i p e r o x i d a s e is n e g l i g i b l e . T h e a m o u n t s of p e r o x i d a s e a n d h y d r o g e n p e r o x i d e u s e d a r e r e s p e c t i v e l y 6.87 × 10-3 ,~tmole a n d 3.43 × 10.-3 ~ m o l e . T h e final v o l u m e s of t h e r e a c t i o n med,ia a r c 3 m l . T h e p H is 6.8 (0.01 M p h o s p h a t e b u f f e r ) . T h e t e m p e r a t u r e i s 25°C. BIOCHIMIE, 1975, 57, n ° 3.
268
M. S a n t i m o n e .
E q u a t i o n 1X a n d 12 show that the velocity must be r e d u c e d wh,en the h y d r o g e n p e r o x i d e c o n c e n t r a t i o n is decreased. C o m p a r i s o n of curves 1 a n d 2 of figure 1A p e r m i t s to conclude, as above, thai the presence of a r e d u c i n g i m p u r i t y i n the h y d r o g e n peroxide so~u.tion c a n n o t explain the increase of the velocity w h e n the h y d r o g e n peroxide c o n c e n t r a t i o n is decreased. Th.e kinetics of c o m p o u n d II formation can be r e c o r d e d (Figure 1 B) at 427 nm, the isosbestic p o i n t o.f peroxidase P o a n d c o m p o u n d I (This w a v e l e n g h t is close to the value of 426 n m reported b y Chance [2]). C o m p a r i s o n of curves t a n d 2 of figure 1 B p.ermi~s to conclude, as above, that the presence of a r e d u c i n g i m p u r i t y in lh.e peroxidase solution and/oT in the h y d r o g e n p e r o x i d e solution oan.not e x p l a i n the increase of velocity w h e n the h y d r o g e n peroxide c o n c e n t r a t i o n is decreased. The only p,lausible hypothesis to explain these u n e x p e c t e d results is that, w h e n equal peroxidase and h y d r o g e n p e r o x i d e co,ncentrations are used (Curves 1 and 3 of figures 1 A a n d 1 B) the reducing agent of c o m p o u n d I is exclusive'ly the endogenous donor, p r e s e n t i n p'eroxidase, (Reaction 8), whereas w h e n the peroxidase c o n c e n t r a t i o n used is h i g h e r than thai of h y d r o g e n peroxide (Curves 2 of figures 1 A a n d 1 B), there are two r e d u c i n g agents o,f eompoun, d I w h i c h are the endogenous d o n o r (Reaction 8) a n d the peroxidase i n excess (Reaction 8). The hypothesis that c o m p o u n d I is r e d u c e d exclusively by the e.n.dogenous d o n o r w h e n peroxidase is i n excess must be also rejected. I n d e e d , i n assuming that this r e a c t i o n is i r r e v e r s i b l e (Equation 8) or reversibl, e (,Equation 13) Col. BH .( the half-life is Tirrev.
~ ColI. B log. 2 -
-
k]
i n the first case and
log, Trey.
(13)
k1-- k1 2 k1
1:-1 + k_l w h e r e k 1 and k_1 are respectively the f o r w a r d a n d the reverse rate constants of reaction 13. Tile expvessio~ns of ~irrev. a n d T...... are in,d e p e n d a n t of the c o m p o u n d I c o n c e n i r a t i o n . However, compar i s o n of curves 1 and 2 of figure 1 A clearly shows that the half-life of c o m p o u n d I is d i m i n i s h e d w h e n its c o n c e n t r a t i o n is decreased, i.e. w h e n the h y d r o g e n p e r o x i d e c o n c e n t r a t i o n used is decreased. This result is in agreement with Yonetani's BIOCHIMIE, 1975, 57, n ° 3.
observation E7] that the half-life of c o m p o u n d I d e p e n d s on the conce,ntration of h y d r o g e n peroxide. Therefore, these results seem to confirm the idea that c o m p o u n d I is not only r e d u c e d b y the endogenous d o n o r but also by free peroxidase w h e n the enzyme is in excess. This hypothesis can be easily d e m o n s t r a t e d b y the following kinetic analysis.
B
Co'rr
$ - CoI -\
\ 2 CoK
FIG. 2. Proposed scheme to explain kinetic data. E, S, Co I, Co II, BH and B are the ferriperoxidase, the hydrogen peroxide, the compound I, the compound II, the reduced and oxidized endogenous donor of peroxidase. -
-
The proposed m e c h a n i s m (figure 2) predicts that w h e n peroxid,ase is i n excess, the ratio, i n f u n c t i o n of time, [formed c o m p o u n d I I ] / [reduced c o m p o u n d I] is 1 for r e a c t i o n 8 a n d 2 for reaction 6, so that in any case, this ratio must be greater t h a n 1 a n d less t h a n 2. The results s h o w n in curve 1 of figure 3 con.firm this hypothesis. The c o n c e n l r a t i o n of c o m p o u n d I r e d u c e d by the endogenous d o n o r alone can be d e t e r m i n e d w i t h curve 3 of figure I:A ~vhere eqtml peroxidase a n d h y d r o g e n p e r o x i d e c o n c e n t r a t i o n s are used. Curve 2 of figure 1A shows the r e d u c t i o n of comp o u n d I both by the endogenous d o n o r a n d by peroxidase, this one b e i n g i n excess. We can therefore obt'ain the c o n c e n t r a t i o n of c o m p o u n d I r e d u c e d by the peroxidase in excess (Reaction 6) by substrac~ting from this value the one o b t a i n e d i n curve 3 of figure 1A w h i c h c o r r e s p o n d s to the c o n c e n t r a t i o n of c o m p o u n d I r e d u c e d by the endogenous donor. I n the same way, the c o n c e n t r a t i o n of comp o u n d II exclusively formed b y r e d u c t i o n of comp o u n d I by peroxidase i n excess (Reaction 6) can be o b t a i n e d by s u b s t r a e t i n g from the total a m o u n t of formed c o m p o u n d IX (curve 2 of figure 1 B) the
Oxidation of horseradish peroxidase by c o m p o u n d I. a m o u n t of c o m p o u n d II f o r m e d by r e d u c t i o n of c o m p o u n d I by the endogenous d o n o r (curve 3 of figure 1 B).
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t (rain) Fro. 3 . - - Values of the ratio [formed Co lI]/[reduced Co I]. ( o ) Values of the ratio [formed CoII]/[redueed Co I] iu the experimental conditions of curve 2 of figures 1 A and 1 B, where peroxidase is in excess. (A) Values of the ratio [formed CoII]/[reduced Co I] corresponding to the reduction of compound I to compound II exclusively by peroxidase. The amount of compound I reduced, by the endogenous donor (obtained by curve 3 of figure 1 A) has been substraeted from the total amount of reduced compound I (obtained by curve 2 of figure 1 A). In the same way, the amount of compound II formed by reduction of compound I by the endogenous donor (obtained by curve 3 of figure 1 B) has been substraeted from the total amount of formed compound II (obtained by curve 2 of figure 1 B). ( o ) Values of the ratio [formed CoII]/[redueed Co I] in the experimental conditions of curve 3 of figures 1 A and 1 B, where equal concentrations of peroxidase and hydrogen peroxide are used. The calculations of concentrations were made using the following values for the differences of the relevant milliumlar absorption coefficients : 410
nm
A~ ('o I -- E 27 nm Ae Co II -- Co I
= - - ~'mM-1 era-l, = 42 ln~I-i enl-1.
R e a c t i o n 6 p r e d i c t s that, in f u n c t i o n of time, the ratio [fo,rmed c o m p o u n d I I ] / [ r e d u c e d comp o u n d I] = 2. These results can be also verified in curve 2 of figure 3. When equal amounts of h y d r o g e n p e r o x i d e and p e r o x i d a s e are used, c o m p o u n d II f o r m a t i o n proceeds by r e d u c t i o n of c o m p o u n d II by the endogenous d o n o r Mone (Reaction 8). In these conditions, the ratio [ f o r m e d c o m p o u n d I I ] / [ r e d u c e d c o m p o u n d I] in f u n c t i o n of time is equal to 1 for each value (curve 3 of figure 3).
BIOCHIMIE, 1975, 57, n ° 3.
269
These p r e d i c t i o n s of the p r o p o s e d m e c h a n i s n l (Figure 2) are v al i d only w h e n the r e d u c l i o n of c o m p o u n d ]I to p e r o x i d a s e is not significant. F i g u r e 1 C sh o w s that this is the case d u r i n g the first five m i n u t e s of l h e r e a c t i o n . The r e l a t i v e l y slow c o m p o u n d II r e d u c t i o n to p e r o x i d a s e observed is in a g r e e m e n t w i t h the idea [3, 7~ that the c o n v e r s i o n of c o m p o u n d II to p e r o x i d a s e is cons i d e r a b l y s l o w e r than that of c o m p o u n d I to comp o u n d II. DISCUSSION. The results p r e s e n t e d in this p a p e r s h o w that, at least in the case of h i g h l y p u r i f i e d h o r s e r a d i s h peroxidase P2, c o m p o u n d II is o b t a i n e d no~ only by r e d u c t i o n of c o m p o u n d I by an e l e c t r o n d o n o r but also by o x i d a t i o n of p e r o x i d a s e by c o m p o u n d I in a o n e - o x i d i z i n g e q u i v a l e n t reaction. It must be o u t l i n e d that a high degree of p u r i f i c a t i o n is essential before this p h e n o m e n o m can be observed. Indeed, it is likely that the p r e s e n c e of sonde cont a m i n a t i n g r e d u c i n g substance in the e n z y m a t i c p r e p a r a t i o n w o u l d accelerate the r e d u c t i o n of the t w o c o m p o u n d s t h r o u g h the m e c h a n i s m p r o p o s e d by Chance [3]. The p r e s e n c e of a r e d u c i n g impurity in the m e d i u m is p r o b a b l y the reason w h y Yonetani [7] did not confirm ttde results of George [5] w h o found that m o r e than one mole of c o m p o u n d II p e r mole of h y d r o g e n p e r o x i d e a d d e d w e r e f o r m e d f r o m HRP. Indeed, in the e x p e r i n l e n t a l results of Yonetani, the t r a n s i t i o n of c o m p o u n d II to HRP is r e l a t i v e l y rapid, w h i c h is not tide case w h e n no r e d u c i n g i m p u r i t y is present in the m e d i u m (Figure 1 C). The n e w kinetic m o d e l that w e p r o p o s e (figure 2) is in ag r eem en t w i t h George's e x p e r i m e n t a l results [5]. In particular, r e a c t i o n 6, w h i c h is only possible w h e n p e r o x i d a s e is in excess, explains George's observation that the n u m b e r o$ mole of c o m p o u n d II f o r m e d f r o m one mole of h y d r o g e n p e r o x i d e app r o a c h e d two, only at h i g h [ p e r o x i d a s e ] / [ h y d r o gen p e r o x i d e ] ratio va*lues. It is i m p o r t a n t to outline the fact that the speeds of the t w o p a t h w a y s of the p r o p o s e d m o d e l (figure 2) are p r o b a b l y s l o w e r than the speed of c o m p o u n d I r e d u c t i o n to c o m p o u n d II by an a d d e d d o n o r t h r o u g h Chance's m e c h a n i s m (Reaction 3) since w h e n an a d d e d d o n o r (or a r e d u c i n g i m p u r i t y ) is p r esen t in the m e d i u m , the ratio [ f o r m e d c o m p o u n d I I ] / [ r e d u c e d c o m p o u n d I] is equal to 1 [7]. In this case, h y d r o g e n p e r o x i d e is utilized t h r o u g h Chance's m e c h a n i s m and cannot be utilized t h r o u g h these t w o p a t h w a y s . It should be noted that the p r o p o s e d m e c h a n i s m of figure 2 is p r o b a b l y over-simplified. F o r ins-
M. S a n t i m o n e .
270
t a n c e , r e a c t i o n 6 cou~ld b e r e a l i z e d b y m o r e t h a n o n e step. F o r t h e t i m e b e i n g , w e c a n n o t f o r m u l a t e a precise mechanism for this new reaction. Howev e r , w e t h i n k t h a t t h e e n d o g e n o u s d o n o r [5, 9], p r e s e n t i n p e r o x i d a s e P2, c a n b e o x i d i z e d i n t h e intermoleculav reaction E ~ E + Co I - - ~ E B + CoII (9) w h i c h is p e r f o r m e d w i t h o n e m o l e c u l e of c o m p o u n d I. The,n t h e prosLthetie g r o u p o f t h e p e r oxidase molecule bearing the oxidized endogenous donor, can be oxidized by this donor in the intram o l e c u l a r r e a c t i o n 10. F o n o w i n g t h i s , t h e p e r o x i d a s e P2 is c o n v e r t e d to c o m p o u n d I I Eg >- Co II~H (10) T h i s i n t e r p r e t a t i o n i,s s u g g e s t e d b y t h e p a r t i c i p a t i o n of t h i s e n d o g e n o u s do,nor to t h e f e r r o c y t o chrome c oxidation by HRP in the kinetic model p r o p o s e d b y Nichol'Is [9].
Acknowledgements. We w i s h to express our g r a t i t u d e to Professor J. Rieard, in whose l a b o r a t o r y t h i s work was earried out. We are grateful "~o Professor Dr. G. Noat for helpful discussions a n d criticism. The help of Dr. M. M. S m i t h a n d Dr. P. P e n o n in t h e E n g l i s h version of t h i s m a n u s cript was greatly appreciated. The s.kilful technical a s s i s t a n c e of Mrs. Woud, s¢ra is also g r a t e f u l l y acknowledged. R~SUM~. La ein~tique de ~a f o r m a t i o n du eompos6 II, o b t e n u en f a i s a n t r~agir u n e peroxydase de r a i f o r t h a u t e m e n t puvifide et l'eau oxyg6n6e, a dtd dtudi6e spectrophoto-
BIOCH1M1E, 1975, 57, n ° 3.
m 6 t r i q u e m e n t /t trois l o n g u e u r s d'onde en l'absence d'agent r~ducteur ajout6 d a n s le milieu r6actionnel. Nos expdriences confirment certes de George qui obten a i t plus d ' u n e mole de compos6 II p a r mole d'eau oxyg6nde. Le n o u v e a u m~canisme que nous proposons, c o n t r a i r e m e n t au m6canisme de George, est s e u l e m e n t valable lorsque le composd II est o h t e n u en l'absence de donneur. De plus, il n ' e s t pas i n c o m p a t i b l e avec le m6canisme propos6 p a r Chance p o u r l ' o x y d a t i o n des d o n n e u r s d'61ectrons p a r le syst6me peroxydase-eau oxyg6n6e. S u i v a n t ce n o u v e a u m6canismc, en Fabsence de donneur, l'a f o r m a t i o n du compos6 It s~effectuerait s u i v a n t deux voles. La p r e m i 6 r e voie correspond h ta r~duction du compos6 I p a r le d o n n e u r endog6ne, et la seeonde correspond h }a f o r m a t i o n de deux moles de compos~ II s u i v a n t la r~action d'oxydor6duction entre une mole de peroxydase et u n e mole de compos6 I. REFERENCES. 1. Chance, B. (1954) Adoan. Enzymol., 12, 153,-190. 2. Chance, B. (1'962) Arch. Biochem. Biophys., 41, 416424. 3. Chance, B. (195,2) Arch. Biochem. Biophys., 44, 404415. 4. George, P. (1953)' Biochem. J., 54, 267~277. 5. George, P. (1963) Bioehem. J., 55, ~20-230. 6. Altsehul, A. M., Abrams, R. & ttogness, T. R. (1940) J. Biol. Chem., 136, 777-794. 7. Yone~ani, T. (196'6) J. Biol. Chem., 241, 2662,-2571. 8. George, P. (19,52) Advau. Catalysis, 4, 367-428. 9. Nieholl,s, P. (1964) Arch. Bioehem. Biophys., 106, 25-48. 1.0. Santimone, M. (1'973) Th~se, Universit~ d'Aix-MarseiHe II', Centre de L~miny, France, pp. 1-190. 11. S h a n n o n , L. M., Kay, E. & Lew, J. Y. (1.9,66) J. Biol. Chem., 241, 2,166-2172. 12. Mazza, G., Charles, CI., Bouehet, M., Rieard, J. & Reynaud, J. (1,968) Biochim. Biophys. Acta, 167, 89-98. 13. Kolthoff, I. M. & Bel'eher, R. (1957) Volumetris Analysis, Vol. III, Interseien.ee P u b l i s h e r s , Inc. NewYork, pp. 75'-76.
BIOCHIMIE, 1'975, 57, 265-270.
Kinetic evidence of horseradish peroxidase oxidation by compound I. Marius SANTIMONE. Laboratoire de Phgsiologie Cellulaire Vdg~lale associ~ au C.N.R.S., Universitd d'Aix-Marseille II, Centre de L u m i n g , 70, Route L{'on L a c h a m p , 13288 Marseille Cddex 2, France. (11-10-197~). Summary. - - The kinetics of c o m p o u n d II f o r m a t i o n , o b t a i n e d upon mixing a highly purified h o r s e r a d i s h peroxidase and h y d r o g e n peroxide, was s p e c t r o p h o t o m e t r i c a l l y studied at t h r e e w a v e l e n g h t s in the absence of an added reducing agent. Our e x p e r i m e n t s confirm George's finding t h a t more t h a n one mole of compound II is formed per mole of h y d r o g e n peroxide added. The new m e c h a n i s m t h a t we propose, c o n t r a r y to the mechan i s m of George, is only valid w h e n c o m p o u n d II is o b t a i n e d in the absenee of a n added donor. Moreover, it is not i n c o n s i s t e n t w i t h the elassieal Chance m e c h a n i s m of oxidation of a n added d o n o r b y the system peroxidase - - hydrogen peroxide. Aeeording to t h i s new m e c h a n i s m , in the absence of a n added donor, compound II format.ion involves two p a t h w a y s . The first p a t h w a y is the m o n o m o l e c u l a r reduction of c o m p o u n d I by the endogenous donor, and the second p a t h w a y is t h e f o r m a t i o n of two moles of c o m p o u n d II t h r o u g h the oxidoreduetion reaction between one mole of peroxidase and one mole of compound I.
JNT1RODUCTION. Horseradish peroxidase catalyzes the oxidation of a w i d e v a r i e t y of e l e c t r o n - d o n o r s b y h y d r o g e n p e r o x i d e . T h e g e n e r a l l y a c c e p t e d r o l e of t h e enzyme can be represented by the following mechan i s m ~ v h i c h o w e s a g r e a t d e a l to t h e c o n t r i b u t i o n of C h a n c e [1-31 a n d G e o r g e [4-.5]. E + Co I + Co II +
S AH AH
- - • .... • - - ~
Co 1 Co II + E + A
A
(1) (2) (3)
w h e r e E r e p r e s e n t s t h e n a t i v e e n z y m e , S is t h e h y d r o g e n p e r o x i d e , Co I a n d Co II a r e tile o x i d i z e d f o r m s of t h e e n z y m e , c a l l e d c o m p o u n d I a n d c o m p o u n d II, r e s p e c t i v e l y , a n d AH is a r e d u c e d one-electron donor. Thus this classical reaction s c h e m e , w h i c h is c u r r e . n t l y a d m i t t e d w h e n AH is a n a d d e d d o n o r , c o r r e s p o n d s to t h e f o r m a t i o n of o n e m o l e of c o m p o u n d II f r o m o n e m o l e of h y d r o gen peroxide. This observation, which agrees w i t h t h o s e of p r e v i o u s w o r k e r s (e.g. A l t s c h u ] , A b r a m s a n d H o g n e s s [6]) a n d m o r e r e c e n t l y w i t h t h o s e of Y o n e t a n i [7], d o e s n o t a g r e e w i t h G e o r g e ' s f i n d i n g [511 t h a t t h e a d d i t i o n of o n e m o l e of H 2 0 e to H R P r e s u l t s i n t h e f o r m a t i o n of m o r e t h a n o n e m o l e of c o m p o u n d II. A l t h o u g h h e h a d previously presented e q u a t i o n s 1 a n d 2 [8], George proposed another mechanism which was c o n f o r m to h i s t i t r a t i o n r e s u l t s .
E + S -> Co I (1) Co I + A H , - - ~ Co II + AH (4) E + AH - - - > Co I I (5) w h e r e AH 2 r e p r e s e n t s e i t h e r r e d u c i n g g r o u p s p r e sent in the enzyme preparation or the deliberately a d d e d r e d u c i n g a g e n t , AH is a n o x i d i z i n g r a d i c a l a n d t h e o t h e r a b b r e v i a t i o n s a r e as d e f i n e d f o r e q u a t i o n s 1, 2 a n d 3. In this paper, we present a kinetic study which c o n f i r m s t i , t r a t i o n s t u d y of George. W e s h o w t h a t G e o r g e ' s o b s e r v a t i o n , t h a t m o r e t h a n o n e m o l e of compound II is o b t a i n e d f r o m o n m o l e of h y d r o g e n p e r o x i d e , is o n l y v a l i d w h e n n o a d d e d d o n o r is p r e s e n t i n t h e m e d i m n . I n t h i s case, c o m p o u n d II is p r o d u c e d b y t w o r e a c t i o n s w h i c h o c c u r s i m u l t a n e o u s l y . T h e first p a t h w a y ( r e a c t i o n 6) is t h e o x i d o r e d u c t i o n r e a c t i o n E + Co I - - > 2 Co [1 (6) b e t w e e n f e r r i p e r o x i d a s e a n d c o m p o u n d I. S i n c e t h e f o r m a l v a l e n c y s t a t e s of i r o n i n p e r o x i d a s e , c o m p o u n d 1 a n d c o m p o u n d II a r e r e s p e c t i v e l y (3-]-), ( 5 + ) a n d ( 4 + ) , t h i s r e a c t i o n c a n b e w r i t t e n as fol'lows : Fe~a ~- ~ + ]~'e(~-" ~ ~ 2FeI~+~ (7) T h e s e c o n d p a t h w a y ( r e a c t i o n 8) is t h e i n t r a - m o lecular reduction Co I . BH - • Co I I . B (8) of c o m p o u n d I to c o m p o u n d II b y t h e 20
266
M. S a n t i m o n e .
endogenous donor BH, p r e s e n t i n a.l~l peroxidases, a n d w h i c h is associated w i t h the enzyme p r o t e i n [5, 9]. One can observe that reaction 6 supposes that, i n the absence of added donor, one mole of c o m p o u n d I can react w i t h peroxidase i n excess giving two moles of c o m p o u n d II, while r e a c t i o n 8 supposes that only one mole of c o m p o u n d II is o b t a i n e d from one mole of c o m p o u n d I. Therefore, by the two pathways, one tootle of h y d r o g e n p,eroxide gives n m r e t h a n one ulole of c o m p o u n d II. MATERIALS AND METHODS. The h o r s e r a d i s h peroxidase isoenzyme used in the p r e s e n t w o r k designated P2 [10] is very similar to isoenzyure C of HR~P p,reviously described by S h a n n o n et al. [11]. It was isolated and purified from the c o m m e r c i a l e n z y m e p r e p a r a t i o n o b t a i n e d from Fluka. The purifica~ion method [10] was derived from that of Mazza et al. [12]. The hemop r o t e i n thus o b t a i n e d was homogeneous on anatytical centrifugatio,n and on electrophoresis. Its R. Z. (Reinheitszahl) was close to 3. H y d r o g e n peroxide (30 p. cent H~Oo p r o d u c e d by Merck) w h i c h was a p p r o p r i a t e l y di.luted immediately before use, was accurately calibrated against a s t a n d a r d solution of potassium p e r m a n ganate in an acidic nredium [13]. The kinetics of c o m p o u n d I and II a p p e a r a n c e a n d d i s a p p e a r a n c e were r e c o r d e d w i t h a Spectralux 1800 s p e c t r o p h o t o m e t e r equipped w i t h a Sefram record,er. This appa,ratus was modified so as to be ca:pable o.f r a p i d l y i n j e c t i n g a r e a c t a n t into the m e d i u m and r e c o r d i n g the a b s o r b a n c e change at a fixed wawelength. Mixing was effected in less than 3 secondes by a m e c h a n i c a l s t i r r e r p r e v i o u s l y described It0]. A kinetic analysis of the curves r e c o r d e d was then possible b e y o n d this time. RESULTS. It is well established [5, 7] that HRP is r a p i d l y a n d i r r e v e r s i b l y converted to c o m p o t m d I by the a d d i t i o n of an equal q u a n t i t y of h y d r o g e n peroxide. W h e n the peroxidas.e isoenzyme P2 is used, this reaction can be followed at 410 rim, the isosbestic p o i n t b e t w e e n p,eroxidase P , a n d comp o u n d II. This wavelenght is close to the corresp o n d i n g value of 410.5 n m p r e v i o u s l y r e p o r t e d by Chance [2] a n d Yonetani [7]. ,Curve 1 of figure 1A shows a r a p i d decrease in the absorb anee w h i c h corresponds to the stoic h i o m e t r i c f o r m a t i o n of c o m p o u n d I p r o d u c e d BIOCHIMIE, 1975, 57, n ° 3.
w h e n peroxidase a n d h y d r o g e n peroxide are put together i n equal ccmcentrations. Then, a slow increase in the a b s o r b a n c e is observed, w h i c h c o r r e s p o n d s .to the r e d u c t i o n of c o m p o u n d I. Since no added d o n o r is presen~ in the m e d i u m , this c o m p o u n d I r e d u c t i o n is not possible through C~aance's m e c h a n i s m . As we shall show, the hypothesis that the enzyme p r e p a r a t i o n a n d / o r the h y d r o g e n p e r o x i d e sotlution may c o n t a i n a reducing i m p u r i t y must be excluded. I n d e e d , if it is assumed that p is the c o n c e n t r a tion of both peroxidase and h y d r o g e n peroxide, a n d i E, the c o n c e n t r a t i o n of IH, an h y p o t h e t i c a l i m p u r i t y p r e s e n t in the enzyme p r e p a r a t i o n , then c o m p o u n d I is r a p i d l y formed at p c o n c e n t r a t i o n w h e n peroxidase and h y d r o g e n p e r o x i d e are mixed. C o m p o u n d I is, afterwards, r e d u c e d to c o m p o u n d II through the r e a c t i o n Co I + IH - - - > Co II + I (9) The velocity of this reaction can be m e a s u r e d by the slope of curve 1 of figure 1A w h i c h shows a relatively slow increase i n the absorbance. This velocity is vp = - - k (p - - x) (ij~ - - x) w h e r e k is the rate constant of reaction 9, a n d x the c o n c e n t r a t i o n of c o m p o u n d I w h i c h has been reduced. W h e n the h y d r o g e n p e r o x i d e c o n c e n t r a tion used is two times lower, the c o n c e n t r a t i o n of c o m p o u n d I formed is -p~ and the velocity is 2 vp/2 = - - k ( p-- - - x) (i~ - - x) (10) 2 Equations 9 a n d 10 show that the velocity must be r e d u c e d w h e n the hydroge,n peroxide c o n c e n t r a tion is decreased. On the contrary, c o m p a r i s o n of curves 1 a n d 2 of figure 1 A shows an increase in the i n i t i a l velocity w h e n the h y d r o g e n peroxide c o n c e n t r a t i o n is decreased. Therefore, the hypothesis that this p h e n o m e n o m is caused by a reducing i m p u r i t y present in the enzyme p r e p a r a t i o n must be rejected. Assurni:ng that the r e d u c i n g matter is p r e s e n t i n the h y d r o g e n p e r o x i d e solution, w h e n equal concentrations of peroxidase and h y d r o g e n p e r o x i d e are used, the velocity is v s = - - k ( p - - x ) (i s - x) (11) w h e r e i s is the r e d u c i n g i m p u r i t y c o n c e n t r a t i o n in lhe h y d r o g e n p e r o x i d e solution. W h e n the hydrogen Feroxide c o n c e n t r a t i o n used is two times lower, the velocity becomes Vs/2 = -
k (-~P - - x) ( i s _ _ _ x) 2 2
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FIG. 1. - - Kinetics of the reduction of peroxidase c o m p o u n d I to c o m p o u n d II. A. - - Absorbance changes recorded at ~10 n m ( i s o s b e s t i c p o i n t of p e r o x i d a s e P 2 - - c o m p o u n d II). I n j e c t i o n of h y d r o g e n p e r o x i d e ( A r r o w a) in t h e m e d i u m c o n t a i n i n g p e r o x i d a s e g i v e s n e g a t i v e A A w h i c h c o r r e s p o n d to t h e t r a n s f o r m a t i o n of f e r r i p e r o x i d a s e to c o m p o u n d I. T h e p o s i t i v e A A t h e n r e c o r d e d c o r r e s p o n d to its d i s a p p e a r a n c e . T h e r e s p e c t i v e a m o u n t s of perox.idase a n d h y d r o g e n p e r o x i d e u s e d are 6.87 × %0-3 ,vmole a n d 6.87 × 10-.3 ;vmole (Curv.e 1), 6.87 X 1,0-3 Ivmole a n d 3.43 × 10-3 ~xmole (Curve 2), 3.43 × 10-3 t~mole a n d 3.43 × 10:-3 ~xmole (Curve 33. B. - - Absorbance changes recorded at /~27 n m ( i s o s b e s t i c p o i n t p e r o x i d a s e P_~ - - c o m p o u n d I). T h e p o s i t i v e A A o b s e r v e d c o r r e s p o n d to t h e c o m p o u n d II f o r m a t i o n . E x p e r i m e n t a l c o n d i t i o n s o f c u r v e s 1, 2 a n d 3 a r e a s in figure i A. C. - - Absorbance changes recorded at 397 n m ( i s o s b e s t i c p o i n t c o m p o u n d I - - c o m p o u n d II). T h e n e g a t i v e A A o b s e r v e d c o r r e s p o n d to t h e t r a n s f o r m a t i o n of f e r r i p e r o x i d a s e to c o m p o u n d I. T h e a b s o r b a n c e v a r i a t i o n s t h e n o b s e r v e d a r e v e r y s l i g h t d u r i n g t h e first five m i n u t e s of t h e r e a c t i o n . T h i s s h o w s t h a t t h e r e d u c t i o n o f c o m p o u n d II to f e r r i p e r o x i d a s e is n e g l i g i b l e . T h e a m o u n t s of p e r o x i d a s e a n d h y d r o g e n p e r o x i d e u s e d a r e r e s p e c t i v e l y 6.87 × 10-3 ,~tmole a n d 3.43 × 10.-3 ~ m o l e . T h e final v o l u m e s of t h e r e a c t i o n med,ia a r c 3 m l . T h e p H is 6.8 (0.01 M p h o s p h a t e b u f f e r ) . T h e t e m p e r a t u r e i s 25°C. BIOCHIMIE, 1975, 57, n ° 3.
268
M. S a n t i m o n e .
E q u a t i o n 1X a n d 12 show that the velocity must be r e d u c e d wh,en the h y d r o g e n p e r o x i d e c o n c e n t r a t i o n is decreased. C o m p a r i s o n of curves 1 a n d 2 of figure 1A p e r m i t s to conclude, as above, thai the presence of a r e d u c i n g i m p u r i t y i n the h y d r o g e n peroxide so~u.tion c a n n o t explain the increase of the velocity w h e n the h y d r o g e n peroxide c o n c e n t r a t i o n is decreased. Th.e kinetics of c o m p o u n d II formation can be r e c o r d e d (Figure 1 B) at 427 nm, the isosbestic p o i n t o.f peroxidase P o a n d c o m p o u n d I (This w a v e l e n g h t is close to the value of 426 n m reported b y Chance [2]). C o m p a r i s o n of curves t a n d 2 of figure 1 B p.ermi~s to conclude, as above, that the presence of a r e d u c i n g i m p u r i t y in lh.e peroxidase solution and/oT in the h y d r o g e n p e r o x i d e solution oan.not e x p l a i n the increase of velocity w h e n the h y d r o g e n peroxide c o n c e n t r a t i o n is decreased. The only p,lausible hypothesis to explain these u n e x p e c t e d results is that, w h e n equal peroxidase and h y d r o g e n p e r o x i d e co,ncentrations are used (Curves 1 and 3 of figures 1 A a n d 1 B) the reducing agent of c o m p o u n d I is exclusive'ly the endogenous donor, p r e s e n t i n p'eroxidase, (Reaction 8), whereas w h e n the peroxidase c o n c e n t r a t i o n used is h i g h e r than thai of h y d r o g e n peroxide (Curves 2 of figures 1 A a n d 1 B), there are two r e d u c i n g agents o,f eompoun, d I w h i c h are the endogenous d o n o r (Reaction 8) a n d the peroxidase i n excess (Reaction 8). The hypothesis that c o m p o u n d I is r e d u c e d exclusively by the e.n.dogenous d o n o r w h e n peroxidase is i n excess must be also rejected. I n d e e d , i n assuming that this r e a c t i o n is i r r e v e r s i b l e (Equation 8) or reversibl, e (,Equation 13) Col. BH .( the half-life is Tirrev.
~ ColI. B log. 2 -
-
k]
i n the first case and
log, Trey.
(13)
k1-- k1 2 k1
1:-1 + k_l w h e r e k 1 and k_1 are respectively the f o r w a r d a n d the reverse rate constants of reaction 13. Tile expvessio~ns of ~irrev. a n d T...... are in,d e p e n d a n t of the c o m p o u n d I c o n c e n i r a t i o n . However, compar i s o n of curves 1 and 2 of figure 1 A clearly shows that the half-life of c o m p o u n d I is d i m i n i s h e d w h e n its c o n c e n t r a t i o n is decreased, i.e. w h e n the h y d r o g e n p e r o x i d e c o n c e n t r a t i o n used is decreased. This result is in agreement with Yonetani's BIOCHIMIE, 1975, 57, n ° 3.
observation E7] that the half-life of c o m p o u n d I d e p e n d s on the conce,ntration of h y d r o g e n peroxide. Therefore, these results seem to confirm the idea that c o m p o u n d I is not only r e d u c e d b y the endogenous d o n o r but also by free peroxidase w h e n the enzyme is in excess. This hypothesis can be easily d e m o n s t r a t e d b y the following kinetic analysis.
B
Co'rr
$ - CoI -\
\ 2 CoK
FIG. 2. Proposed scheme to explain kinetic data. E, S, Co I, Co II, BH and B are the ferriperoxidase, the hydrogen peroxide, the compound I, the compound II, the reduced and oxidized endogenous donor of peroxidase. -
-
The proposed m e c h a n i s m (figure 2) predicts that w h e n peroxid,ase is i n excess, the ratio, i n f u n c t i o n of time, [formed c o m p o u n d I I ] / [reduced c o m p o u n d I] is 1 for r e a c t i o n 8 a n d 2 for reaction 6, so that in any case, this ratio must be greater t h a n 1 a n d less t h a n 2. The results s h o w n in curve 1 of figure 3 con.firm this hypothesis. The c o n c e n l r a t i o n of c o m p o u n d I r e d u c e d by the endogenous d o n o r alone can be d e t e r m i n e d w i t h curve 3 of figure I:A ~vhere eqtml peroxidase a n d h y d r o g e n p e r o x i d e c o n c e n t r a t i o n s are used. Curve 2 of figure 1A shows the r e d u c t i o n of comp o u n d I both by the endogenous d o n o r a n d by peroxidase, this one b e i n g i n excess. We can therefore obt'ain the c o n c e n t r a t i o n of c o m p o u n d I r e d u c e d by the peroxidase in excess (Reaction 6) by substrac~ting from this value the one o b t a i n e d i n curve 3 of figure 1A w h i c h c o r r e s p o n d s to the c o n c e n t r a t i o n of c o m p o u n d I r e d u c e d by the endogenous donor. I n the same way, the c o n c e n t r a t i o n of comp o u n d II exclusively formed b y r e d u c t i o n of comp o u n d I by peroxidase i n excess (Reaction 6) can be o b t a i n e d by s u b s t r a e t i n g from the total a m o u n t of formed c o m p o u n d IX (curve 2 of figure 1 B) the
Oxidation of horseradish peroxidase by c o m p o u n d I. a m o u n t of c o m p o u n d II f o r m e d by r e d u c t i o n of c o m p o u n d I by the endogenous d o n o r (curve 3 of figure 1 B).
r~ 0
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t (rain) Fro. 3 . - - Values of the ratio [formed Co lI]/[reduced Co I]. ( o ) Values of the ratio [formed CoII]/[redueed Co I] iu the experimental conditions of curve 2 of figures 1 A and 1 B, where peroxidase is in excess. (A) Values of the ratio [formed CoII]/[reduced Co I] corresponding to the reduction of compound I to compound II exclusively by peroxidase. The amount of compound I reduced, by the endogenous donor (obtained by curve 3 of figure 1 A) has been substraeted from the total amount of reduced compound I (obtained by curve 2 of figure 1 A). In the same way, the amount of compound II formed by reduction of compound I by the endogenous donor (obtained by curve 3 of figure 1 B) has been substraeted from the total amount of formed compound II (obtained by curve 2 of figure 1 B). ( o ) Values of the ratio [formed CoII]/[redueed Co I] in the experimental conditions of curve 3 of figures 1 A and 1 B, where equal concentrations of peroxidase and hydrogen peroxide are used. The calculations of concentrations were made using the following values for the differences of the relevant milliumlar absorption coefficients : 410
nm
A~ ('o I -- E 27 nm Ae Co II -- Co I
= - - ~'mM-1 era-l, = 42 ln~I-i enl-1.
R e a c t i o n 6 p r e d i c t s that, in f u n c t i o n of time, the ratio [fo,rmed c o m p o u n d I I ] / [ r e d u c e d comp o u n d I] = 2. These results can be also verified in curve 2 of figure 3. When equal amounts of h y d r o g e n p e r o x i d e and p e r o x i d a s e are used, c o m p o u n d II f o r m a t i o n proceeds by r e d u c t i o n of c o m p o u n d II by the endogenous d o n o r Mone (Reaction 8). In these conditions, the ratio [ f o r m e d c o m p o u n d I I ] / [ r e d u c e d c o m p o u n d I] in f u n c t i o n of time is equal to 1 for each value (curve 3 of figure 3).
BIOCHIMIE, 1975, 57, n ° 3.
269
These p r e d i c t i o n s of the p r o p o s e d m e c h a n i s n l (Figure 2) are v al i d only w h e n the r e d u c l i o n of c o m p o u n d ]I to p e r o x i d a s e is not significant. F i g u r e 1 C sh o w s that this is the case d u r i n g the first five m i n u t e s of l h e r e a c t i o n . The r e l a t i v e l y slow c o m p o u n d II r e d u c t i o n to p e r o x i d a s e observed is in a g r e e m e n t w i t h the idea [3, 7~ that the c o n v e r s i o n of c o m p o u n d II to p e r o x i d a s e is cons i d e r a b l y s l o w e r than that of c o m p o u n d I to comp o u n d II. DISCUSSION. The results p r e s e n t e d in this p a p e r s h o w that, at least in the case of h i g h l y p u r i f i e d h o r s e r a d i s h peroxidase P2, c o m p o u n d II is o b t a i n e d no~ only by r e d u c t i o n of c o m p o u n d I by an e l e c t r o n d o n o r but also by o x i d a t i o n of p e r o x i d a s e by c o m p o u n d I in a o n e - o x i d i z i n g e q u i v a l e n t reaction. It must be o u t l i n e d that a high degree of p u r i f i c a t i o n is essential before this p h e n o m e n o m can be observed. Indeed, it is likely that the p r e s e n c e of sonde cont a m i n a t i n g r e d u c i n g substance in the e n z y m a t i c p r e p a r a t i o n w o u l d accelerate the r e d u c t i o n of the t w o c o m p o u n d s t h r o u g h the m e c h a n i s m p r o p o s e d by Chance [3]. The p r e s e n c e of a r e d u c i n g impurity in the m e d i u m is p r o b a b l y the reason w h y Yonetani [7] did not confirm ttde results of George [5] w h o found that m o r e than one mole of c o m p o u n d II p e r mole of h y d r o g e n p e r o x i d e a d d e d w e r e f o r m e d f r o m HRP. Indeed, in the e x p e r i n l e n t a l results of Yonetani, the t r a n s i t i o n of c o m p o u n d II to HRP is r e l a t i v e l y rapid, w h i c h is not tide case w h e n no r e d u c i n g i m p u r i t y is present in the m e d i u m (Figure 1 C). The n e w kinetic m o d e l that w e p r o p o s e (figure 2) is in ag r eem en t w i t h George's e x p e r i m e n t a l results [5]. In particular, r e a c t i o n 6, w h i c h is only possible w h e n p e r o x i d a s e is in excess, explains George's observation that the n u m b e r o$ mole of c o m p o u n d II f o r m e d f r o m one mole of h y d r o g e n p e r o x i d e app r o a c h e d two, only at h i g h [ p e r o x i d a s e ] / [ h y d r o gen p e r o x i d e ] ratio va*lues. It is i m p o r t a n t to outline the fact that the speeds of the t w o p a t h w a y s of the p r o p o s e d m o d e l (figure 2) are p r o b a b l y s l o w e r than the speed of c o m p o u n d I r e d u c t i o n to c o m p o u n d II by an a d d e d d o n o r t h r o u g h Chance's m e c h a n i s m (Reaction 3) since w h e n an a d d e d d o n o r (or a r e d u c i n g i m p u r i t y ) is p r esen t in the m e d i u m , the ratio [ f o r m e d c o m p o u n d I I ] / [ r e d u c e d c o m p o u n d I] is equal to 1 [7]. In this case, h y d r o g e n p e r o x i d e is utilized t h r o u g h Chance's m e c h a n i s m and cannot be utilized t h r o u g h these t w o p a t h w a y s . It should be noted that the p r o p o s e d m e c h a n i s m of figure 2 is p r o b a b l y over-simplified. F o r ins-
M. S a n t i m o n e .
270
t a n c e , r e a c t i o n 6 cou~ld b e r e a l i z e d b y m o r e t h a n o n e step. F o r t h e t i m e b e i n g , w e c a n n o t f o r m u l a t e a precise mechanism for this new reaction. Howev e r , w e t h i n k t h a t t h e e n d o g e n o u s d o n o r [5, 9], p r e s e n t i n p e r o x i d a s e P2, c a n b e o x i d i z e d i n t h e intermoleculav reaction E ~ E + Co I - - ~ E B + CoII (9) w h i c h is p e r f o r m e d w i t h o n e m o l e c u l e of c o m p o u n d I. The,n t h e prosLthetie g r o u p o f t h e p e r oxidase molecule bearing the oxidized endogenous donor, can be oxidized by this donor in the intram o l e c u l a r r e a c t i o n 10. F o n o w i n g t h i s , t h e p e r o x i d a s e P2 is c o n v e r t e d to c o m p o u n d I I Eg >- Co II~H (10) T h i s i n t e r p r e t a t i o n i,s s u g g e s t e d b y t h e p a r t i c i p a t i o n of t h i s e n d o g e n o u s do,nor to t h e f e r r o c y t o chrome c oxidation by HRP in the kinetic model p r o p o s e d b y Nichol'Is [9].
Acknowledgements. We w i s h to express our g r a t i t u d e to Professor J. Rieard, in whose l a b o r a t o r y t h i s work was earried out. We are grateful "~o Professor Dr. G. Noat for helpful discussions a n d criticism. The help of Dr. M. M. S m i t h a n d Dr. P. P e n o n in t h e E n g l i s h version of t h i s m a n u s cript was greatly appreciated. The s.kilful technical a s s i s t a n c e of Mrs. Woud, s¢ra is also g r a t e f u l l y acknowledged. R~SUM~. La ein~tique de ~a f o r m a t i o n du eompos6 II, o b t e n u en f a i s a n t r~agir u n e peroxydase de r a i f o r t h a u t e m e n t puvifide et l'eau oxyg6n6e, a dtd dtudi6e spectrophoto-
BIOCH1M1E, 1975, 57, n ° 3.
m 6 t r i q u e m e n t /t trois l o n g u e u r s d'onde en l'absence d'agent r~ducteur ajout6 d a n s le milieu r6actionnel. Nos expdriences confirment certes de George qui obten a i t plus d ' u n e mole de compos6 II p a r mole d'eau oxyg6nde. Le n o u v e a u m~canisme que nous proposons, c o n t r a i r e m e n t au m6canisme de George, est s e u l e m e n t valable lorsque le composd II est o h t e n u en l'absence de donneur. De plus, il n ' e s t pas i n c o m p a t i b l e avec le m6canisme propos6 p a r Chance p o u r l ' o x y d a t i o n des d o n n e u r s d'61ectrons p a r le syst6me peroxydase-eau oxyg6n6e. S u i v a n t ce n o u v e a u m6canismc, en Fabsence de donneur, l'a f o r m a t i o n du compos6 It s~effectuerait s u i v a n t deux voles. La p r e m i 6 r e voie correspond h ta r~duction du compos6 I p a r le d o n n e u r endog6ne, et la seeonde correspond h }a f o r m a t i o n de deux moles de compos~ II s u i v a n t la r~action d'oxydor6duction entre une mole de peroxydase et u n e mole de compos6 I. REFERENCES. 1. Chance, B. (1954) Adoan. Enzymol., 12, 153,-190. 2. Chance, B. (1'962) Arch. Biochem. Biophys., 41, 416424. 3. Chance, B. (195,2) Arch. Biochem. Biophys., 44, 404415. 4. George, P. (1953)' Biochem. J., 54, 267~277. 5. George, P. (1963) Bioehem. J., 55, ~20-230. 6. Altsehul, A. M., Abrams, R. & ttogness, T. R. (1940) J. Biol. Chem., 136, 777-794. 7. Yone~ani, T. (196'6) J. Biol. Chem., 241, 2662,-2571. 8. George, P. (19,52) Advau. Catalysis, 4, 367-428. 9. Nieholl,s, P. (1964) Arch. Bioehem. Biophys., 106, 25-48. 1.0. Santimone, M. (1'973) Th~se, Universit~ d'Aix-MarseiHe II', Centre de L~miny, France, pp. 1-190. 11. S h a n n o n , L. M., Kay, E. & Lew, J. Y. (1.9,66) J. Biol. Chem., 241, 2,166-2172. 12. Mazza, G., Charles, CI., Bouehet, M., Rieard, J. & Reynaud, J. (1,968) Biochim. Biophys. Acta, 167, 89-98. 13. Kolthoff, I. M. & Bel'eher, R. (1957) Volumetris Analysis, Vol. III, Interseien.ee P u b l i s h e r s , Inc. NewYork, pp. 75'-76.
