279
Biochimica et Biophysica Acta, 583 (1979) 279--286 © Elsevier/North-Holland Biomedical Press
BBA 28824
G E N E R A T I O N OF SUPEROXIDE RADICALS IN ALKALINE SOLUTIONS OF H Y D R O G E N P E R O X I D E AND THE E F F E C T OF SUPEROXIDE DISMUTASE ON THIS SYSTEM
M.A. SYMONYAN and R.M. NALBANDYAN Institute of Biochemistry, Academy of Sciences of the Armenian SSR, Yerevan, 375044 (U.S.S.R.) (Received April 26th, 1978) (Revised manuscript received August 22nd, 1978)
Key words: Superoxide radical generation; Hydrogen peroxide; Superoxide dismutase
Summary Superoxide radicals in high concentrations were generated from alkaline H202 w i t h o u t using catalysts or irradiation. The dependence of the intensity and parameters of the superoxide radical EPR spectrum on pH, temperature, viscosity and H202 concentration were studied. The observed changes are explained on the base of matrix effects. The addition of superoxide dismutase to alkaline H202 led initially to a drop in the EPR spectrum intensity, followed by an increase in the concentration of superoxide radicals.
Introduction Extensive studies concerning the role and properties of superoxide radicals in biological systems began in 1069, when several important data were obtained in this field almost simultaneously. Firstly, it was shown that superoxide radicals were formed in certain enzymic redox reactions [1]. Secondly, a new class of enzyme, superoxide dismutase, was discovered for which these radicals are normal substrates [2]. Thirdly, it was demonstrated that superoxide dismutase exhibited an inhibitory effect on the terminal enzymic redox reactions proceeding b y the one-electron mechanism [3--5]. Most of the methods used for the determination of superoxide dismutase activity were indirect, because they were based on the inhibitory effect of the enzyme on the reactions, accompanied by the formation of superoxide radicals as intermediates. Some investigations were carried out in which the direct reaction of superoxide dismutase with superoxide radicals was involved. The sub-
280 strate was generated by means of pulse-radiolysis, or polarographically [6,7]. However, for further elucidation of the features of the dismutation process, it is necessary to develop simpler methods for the generation of superoxide radicals in high concentrations. The radicals are known to be stable in some aprotic solvents and they may be generated therein at high concentrations. However, some of these solvents cause modification of protein structure [8,9]. Thereffore it is desirable to obtain high concentrations of the radicals in aqueous media. It has long been known that superoxide radicals are formed from hydrogen peroxide solutions in the presence of some oxidants (periodide, permanganate) or catalysts [10--12] (also, recently, Ref. 13). We show here that superoxide radicals are formed in alkaline solutions of concentrated H202, in the absence of added oxidants and catalysts. The radicals were utilized by cuprozinc superoxide dismutase, although depending on pH, viscosity and temperature, EPR signal parameters of the radicals underwent some changes. Materials and Methods Superoxide dismutase was obtained (from bovine erythrocytes) in an electrophoretically homogeneous state according to methods described in our previous paper [ 14]. Superoxide radicals were generated from alkaline H202 in a 20-ml tube, which was supplied with electrodes (for pH measurements) and a thermometer and contained 10--30% H202 (the concentration of which was established with permanganate). To generate superoxide radicals, a small piece of solid KOH or several drops of concentrated KOH solution were added. After the addition of alkali, the change of pH and temperature was traced and 0.4-ml aliquots of the mixture were frozen in liquid N2 for recording EPR spectra on a Varian E-4 instrument (modulation amplitude 6.3 G, time constant 0.3 s, scanning rate 250 G/min). Temperature-dependence was studied with the Varian E-248 variable temperature accessory. The effect of alkaline H202 on the properties of superoxide dismutase was followed after incubating 0.4-ml aliquots of superoxide dismutase with 0.1-ml aliquots of superoxide radical solution at room temperature. The effect of the enzyme on superoxide radical concentration was followed immediately after the addition of 0.1-ml aliquots of diluted superoxide dismutase to 0.4-ml aliquots of superoxide radical solutions. In control experiments, 0.1-ml aliquots were replaced by 0.1 ml 0.01 M phosphate or carbonate buffers (pH 9.5 and 10.5, respectively). All chemicals used were reagent grade. Iron and copper constants in H20: were found to be lower than the sensitivity of spectrophotometric determination methods, using the chelating agents o-phenanthroline and buthocuproin disulphonate [15,16]. Nevertheless, in special experiments, these chelators as well as salicylaldoxime, diethyldithiocarbamate, EDTA or EGTA (ethyleneglycol-bis(fl-aminoethyl ether) N,N'-tetraacetic acid) was added to the reaction mixture containing H202 to prevent the possible effect of trace metal contaminants on the phenomenon observed. Concentrations of chelators were in the order of 1 • 10-3---1 • 10 -2 M.
281 Results When the pH of H:O2 solutions was adjusted to 8.5 or higher, an EPR signal of axial s y m m e t r y was observed. The onset of the EPR signal was accompanied by an exothermic process and further increase of pH. Typical kinetic curves of these processes are shown in Fig. 1. The comparison of kinetic curves obtained for different initial pH values and concentrations of H20: led us to conclude that the EPR spectrum intensity depends on the initial pH value and concentration of H:O2 in the mixture. Thus, if the initial pH value of 30% H202 were adjusted to 10.0 (the final pH being 12.6) the maximum intensity of the EPR spectrum was three orders of magnitude as much as that the initial pH 9 of the same concentration of H202. The final pH in the last case was 10.5. It is important that the addition of the all chelators, besides EDTA, does n o t change the observed kinetic curves. Only EDTA (1 • 10 -2 M) was capable of stopping further increase of pH, temperature and generation of superoxide radicals. This effect of EDTA should n o t be connected with its chelating ability. This property of EDTA was earlier considered by Misra and Fridovich [16]. We found also that the addition of 1 • 10 -2 M Cu 2÷ or Mn 2÷ brings about a decrease of the concentration of superoxide radicals. Although the appearance of EPR signals in alkaline H202 was accompanied by heating of the medium, the free radical species were found to be very stable at alkaline pH values and room temperature. Thus, the radicals formed at initial pH 10.0 have a half-life of 2 h at 22°C. The EPR spectrum observed disappears completely and irreversibly only after heating the mixture at 80°C for 5 min. No change in the shape of the EPR spectrum was observed in the course of this thermal treatment. It was found, however, that the shape and parameters of the EPR signal depend on pH values of alkaline H202 solution. Although in all the cases studied the EPR spectra had axial shapes with two g factors, the value of g,I and the width of the low-field c o m p o n e n t of the EPR spectrum (the component at gl,) were remarkably altered with change in pH. Shapes of EPR signals observed at different initial pH values of H202 solutions are compared in Fig. 2. As can be seen, the increase of the initial pH of H202 solutions was accompanied by an increase of the EPR signal intensity as well as by increase in gll value and width. Further, it was shown that the stability of radicals generated from peroxide depends also on pH. Thus, radicals formed at lower pH
f2
~8
,nlt~al 0
l~Iv
"O'O
0
i
I~'"-C~'---0
i
i
J
,
Fig. 1. The change o f p H (~), temperature (0) and superoxide radical concentration (o) of 30% H 2 0 2 made alkaline w i t h N a O H to p H 10.0.
282
2.1f~
.-"- If20 4¢,6/..O..'°'O[
, ,W ,¢ 2.t~
],,H¢£
,"¢
t60 ]
~rW.e"'
3tO0
~2~0
~00
2.0{; ',
,
,
tOpH
,
Jo
72 t~
Fig. 2. T h e c h a n g e o f t h e s h a p e ( A ) a n d p a r a m e t e r s (B) o f t h e E P R s p e c t r u m o f s u p e r o x i d e r a d i c a l s g e n e r a t e d f r o m 3 0 % H 2 0 2 a t p H v a l u e s 8 . 2 (1), 9 . 5 (2), 1 1 . 5 (3), 1 3 . 3 (4). R e c e i v e r g a i n s w e r e : 2 • 1 0 2 . 6 . 3 101 , 1 . 2 5 - 101 , 5 • 1 0 0 , r e s p e c t i v e l y . A p H d e p e n d e n c e o f gl] value; o, p H d e p e n d e n c e o f t h e line w i d t h f o r t h e l o w - f i e l d c o m p o n e n t o f t h e E P R s p e c t r u m . All E P R s p e c t r a w e r e r e c o r d e d a t 7 7 K. Fig. 3. T h e e f f e c t o f v i s c o s i t y o n t h e s h a p e (A) a n d s t a b i l i t y (B) o f s u p e r o x i d e r a d i c a l s g e n e r a t e d . 1, r a d i cals f o r m e d f r o m 3 0 % H 2 0 2 ( p H 1 2 . 5 ) ; 2, 3 0 % p o l y ( e t h y l e n e g l y c o l ) w a s a d d e d t o I. R e c e i v e r g a i n s w e r e : 1 . 2 5 - 101 a n d 1 . 2 5 • 1 0 0 f o r 1 a n d 2, r e s p e c t i v e l y . T h e d r o p o f t h e c o n c e n t r a t i o n o f r a d i c a l s 1 a n d 2 a t 2 0 ° C is s h o w n in B ( 1 ' a n d 2').
(lower g, value) have at room temperature a shorter half-life than those formed at higher pH values. The addition of viscous solvents such as glycerol or poly(ethyleneglycol) to the H202 solutions made pH 12.0 brings a b o u t modification of the EPR signal shape. The observed signal has parameters and shape more typical for pH 9--10, although no change of pH was observed after the addition of viscous solvents. The modification of the signal was accompanied by a several-fold increase in its intensity. Surprisingly, radicals formed in the presence of viscous solvents were remarkably less stable than that obtained at pH 12.0 in the absence of the solvents. These data are presented in Fig. 3. Thus, the radicals formed in alkaline H202 at pH 12.0 in the presence of viscous solvents have EPR spectral shape and parameters which are more typical for radicals generated at pH 10.0 and, in addition, their stability resembled more that of the radicals generated at pH 10.0. Study of the effect of microwave power on the shape of EPR signals showed that the radicals formed at pH 10 and 12 do not change their shapes (at either pH value), at least in the region of 0.2--150 mW. On the other hand, the shape as well as the intensity of the EPR spectrum was found to depend on temperature. Fig. 4 shows the results obtained. As the measure of changes, the parameter Q was accepted, the mean of which is illustrated in Fig. 4C. As it can be seen, the value Q depends linearly on l/T, the change observed being completely reversible in the region --196°C to --60°C. However, the EPR signal dis-
283
I
i
$000
/
I
3~00
B. V ~ fo- ~
f/T, 10" ~
0
~OCmin) 60
Fig. 4. T e m p e r a t u r e d e p e n d e n c e o f t h e s h a p e a n d i n t e n s i t y o f t h e E P R s p e c t r u m o f s u p e r o x i d e r a d i c a l s f o r m e d 3 0 % H 2 0 2 ( p H 1 3 . 0 ) . E P R s p e c t r a ( A ) w e r e r e c o r d e d u n d e r t h e f o l l o w i n g c o n d i t i o n s : 1, r e c e i v e r g a i n , 4 - 101 , t = - - 1 6 0 ° C ; 2, r e c e i v e r g a i n , 5 - 1 0 2 , t = - - 1 0 0 ° C ; 3, r e c e i v e r g a i n , 1 . 1 0 3 , t = - - 8 0 ° C ; 4, r e c e i v e r g a i n 4 • 1 0 3 , t = - - 6 0 ° C . T h e c h a n g e o f t h e s h a p e ( p a r a m e t e r Q) is p r e s e n t e d in C. F~lled t r i a n g l e s a n d circles r e p r e s e n t d a t a o b t a i n e d f o r t h e c h a n g e o f t e m p e r a t u r e f r o m l o w e r t o h i g h e r , a n d o p e n t r i a n g l e s a n d circles are d a t a f o r t h e c h a n g e o f t e m p e r a t u r e in t h e reverse d i r e c t i o n . Fig. 5. T h e e f f e c t o f d i f f e r e n t c o n c e n t r a t i o n s o f s u p e r o x l d e d i s m u t a s e (2 • 1 0 -9 M (o), 2 • 1 0 -8 M (A)) o n the concentration of superoxide radicals. The radicals were generated form 30% H202 (pH 12.6) and had t h e E P R s p e c t r u m s h o w n in Figs. 2 - - 5 . F i l l e d circles s h o w n t h e t i m e c o u r s e in t h e a b s e n c e o f s u p e r o x i d e dismutase.
appears completely above --60°C and neither was any signal observed in the solution of alkaline H202 at r o o m temperature. When the temperature of the frozen solution fell below --60°C, the EPR spectrum was again observed. In the region --196°C to --60°C, the intensity of the signal obeys Curie's law. It is important note that radicals generated at pH 10.0 show similar temperature dependence. The addition of catalytic amounts of superoxide dismutase to alkaline solutions (pH 10.0 or 12.0) leads to the disappearance of radicals. We were unable to follow this process kinetically; however it was noted that the same concentration of the enzyme is capable of utilizing various limited concentrations of the radicals, depending on pH values (Table I). The results obtained show that the catalytic p o w e r of superoxide dismutase is maximum when H~O2 is taken to pH 11.5--12.5. After the rapid disappearance of free radicals following superoxide dismutase addition the formation of radicals was again observed, the rate of which was significantly slower than the breakdown of radicals in the course of the first stage (Fig. 5). It was found that the EPR signal shape of radicals formed in the second stage was identical to that observed before the addition of superoxide dismutase. The generation o f radicals in the course of the second step is also a catalytic process because the rate depends linearly on superoxide dismutase concentration. If superoxide dismutase and alkaline H~O2 are mixed in comparable molar
284 TABLE
I
THE CATALYTIC
POWER
OF SUPEROXIDE
DISMUTASE
IN ALKALINE
H202
SOLUTIONS
T o d e t e r m i n e t h e p o w e r o f t h e e n z y m e a t d i f f e r e n t p H v a l u e s , m i n i m a l q u a n t i t i e s o f the: e n z y m e w e r e mixed with alkaline 30% H 2 05 and concentrations of superoxide radicals before and immediately after t h e a d d i t i o n o f t h e e n z y m e w e r e c o m p a r e d . F a l l i n c o n c e n t r a t i o n (in o r d e r s o f m a g n i t u d e ) w a s c a l c u l a t e d per mole of the enzyme. pH
8.5
10.5
11.5
12.3
] 3.0
13.9
Catalytic power
2.0
3.5
4.5
5.6
4.1
2.5
amounts, modification of the EPR spectrum of the enzyme is observed. The shape of the spectrum is different from that obtained in the presence of H:O2 or alkali alone. Discussion The data obtained indicate that superoxide radicals may be easily formed from alkaline concentrated H202 solution without irradiation, catalysts or added oxidants. The exact mechanism of the generation of superoxide radicals in this system is as y e t unknown. Several more-or-less possible mechanisms of the process may be suggested, however their consideration lies b e y o n d this study. The data presented indicate that trace metal probably does not participate in the formation of superoxide radicals. The sharp increase of superoxide radical concentration at pH values above 11.0 agrees with pK = 11.6 for H~O2. Thus, deprotonation of peroxide is essential for the generation of superoxide radicals. The shape of the EPR spectrum of the radicals generated and their utilization by superoxide dismutase leave little d o u b t a b o u t the nature of these radicals. However, the question of differences of EPR spectral parameters of superoxide radicals formed b y different methods in various systems is still open. Shifts in g-factor values of the EPR spectrum of superoxide radicals generated in several more complex systems were explained b y Knowles et al. [ 1 ] as a matrix effect. We found that, although the EPR spectra of the radicals have in the alkaline scale an axial symmetry with two g factors, the gLI value and the width of the EPR spectrum were very sensitive to pH, temperature and viscosity. The microenvironment of the superoxide radicals has also a certain influence on their stability. Thus, as could be predicted from cell effect, the stability of radicals formed in more viscous media was decreased. In accordance with earlier data [18--20] to the effect that superoxide radicals are stabilized in aprotic organic and aqueous alkaline media, we noted that the concentration of the radicals formed in alkaline hydrogen peroxide was higher at more alkaline pH. However, the catalytic p o w e r of superoxide dismutase added to the hydrogen peroxide-alkali system was maximal in the range pH 11--12.5. The decrease of the power at higher pH values is probably connected with the denaturation of the enzyme at strongly basic pH [21]. On the other hand, at pH 9--11 the drop of the catalytic power may be connected with higher concentration of hydrogen peroxide in the system, which is known to inactivate superoxide dismutase [22,23]. As was established, the addition of superoxide dismutase to alkaline
285
H202 led to the increase of superoxide radical concentration in the second step (Fig. 5). It was shown that the copper environment in superoxide dismutase is sharply modified in the presence of alkaline H202. We think that the system considered is suitable for the study of b o t h direct and reverse reactions catalyzed b y superoxide dismutase. The catalysis of superoxide dismutase of the generation of superoxide radicals from H202 and oxygen, i.e., the reversal of the superoxide dismutase process, was demonstrated by Hodgson and Fridovich [24] using the indirect m e t h o d of detection of superoxide radical through its reaction with tetranitromethane. The presence of two stages in the effect of superoxide dismutase on the concentration of superoxide radicals formed in alkaline H202 solutions indicates that there are equilibria in the system in which superoxide radicals are involved. The main free radical species formed in H202 solutions are admitted to be h y d r o x y l and superoxide. H y d r o x y l radicals are formed e.g., according to the Haber-Weiss reaction [ 25,26] : H20~ + 02- ~- O H - + "OH + O2
(I)
Although there is some d o u b t in importance of this reaction in weakly alkaline media [27], nevertheless, .OH is generated in some way from H202 in the presence of superoxide radicals [28,29]. Also, the formation of superoxide and h y d r o x y l radicals under the effect of superoxide dismutase is suggested [30]: OH- + 02 -~ 02- + "OH
(2)
On the other hand, superoxide radicals possibly are generated in the reaction of hydroxyl radicals with deprotonated H202: HO2 + "OH ~ H20 + 02-
(3)
It is evident that the effect of superoxide dismutase on equilibria [1--3] and the other possible schemes with the participation of hydroxyl and superoxide radicals is very complicated. Moreover, recently Bielski and Allen [31] demonstrated that the simultaneous nonenzymic dismutation in aqueous alkaline media should occur with the participation of this protonated form of superoxide radicals, HO2, although the pK for this radical is of 4.75. Thus, superoxide radicals are in equilibrium with their protonated form and the latter is capable of dismuting at alkaline pH with a high rate constant. This finding raises the question of whether enzymic dismutation of superoxide radicals involves their preliminary protonation. In other words, it would be of interest to determine the significance of the two protons required for the formation of H2O 2 in the course of superoxide dismutase catalyzed dismutation of superoxide radicals. References 1 Knowles, P.F., Gibson, J.F., Pick, F.M. and Bray, R.C. (1969) Biochem. J. 111, 53---58 2 McCord, J. and Fridovich, I. (1969) J. Biol. Chem. 244, 6045--6055 3 Ballou, D., Palmer, G. and Massey, V. (1969) Biochem. Biophys. Res. Commun. 36,898--904 4 0 r m e - J o h n s o n , W.H. and Beinert, H. (1969) Biochem. Biophys. Res. Commun. 36, 905--911 5 Massey, V., Strikland, S., Mayhew, S.G., Howell, L.G., Engel, P.C., Matthews, R.G., Sehuman, M. a n d Sullivan, P.A. (1969) Biochem. Biophys. Res. Commun. 36, 891--897
286 6 Fielden. E.M.. Roberts, P.B.. Bray. R.C., Lowe. D.J.. Maunter. G.N., RotiIio. G. snd Calabrese. L. (1974) Biochem. J. 139.49-60 (and references therein) 7 Rigo. A., VigIino, P. and RotiUo, G. (1976) Biochem. Biophys. Res. Commun. 63.1013-1016 8 Symonyan. M.A. and Nalbandyan, R.M. (1976) Biochim. Biophys. Acta 446.432-444 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Symonyan, M.A. and Nalbandyan, R.M. (1976) Studia Biophysics 54.99-103 Lunsford. J. and Javne, J.P. (1966) J. Chem. Phys. 44.1487-1492 Atkins, P.W. and Symons, M.C.P. (1967) The Structure of Inorganic Radicals, Elsevier. Amsterdam Schumb, W.C.. Satterfield, C.N. and Wentwoth. R.L. (1966) Hydrogen peroxide. Reinold, New York One, J., Matsumura, T.. Kit&ma. N. and Shun&hi Fukuzumi (1977) J. Phys. Chem. 81.1307-1311 Symonyan. M.A. and Nalbandyan. R.M. (1976) Biochimija (Russ) 40.726-732 Cameron. B.F. (1965) Anal. Biochem. 11,164-169 Poillon, W.N. and Dawson, C.R. (1963) Biochim. Biophys. Acta 77, 27-36 Misra. H.P. and Fridovich, I. (1972) J. Biol. Chem. 247.3170-3175 Maricle, D.L. and Hodgson. W.G. (1965) Anal. Chem. 37.1562-1565 Fee, J.A. and Hildenberg, P.C. (1974) FEBS Lett. 39.79-82 Czapski. G. and Dorfman, L.M. (1964) J. Phys. Chem. 68.1169-1177 Symonuan. M.A. and Nalbandyan. R.M. (1976) Biophysics (Russ) 20.783-787 Symonyan, M.A. and Nalbandyan, R.M. (1972) FEBS Lett. 28.22-24 Bray. R.C.. Cockle, S.A.. Fielden, E.M., Roberts, P.B.. Rotilio. G. and Calabrese, L. (1974) Biochem. J. 139,43-48 Hodgson, E.K. and Fridovich, I. (1973) Biochem. Biophys. Res. Commun. 54,270-272 Haber, F. and Weiss, J. (1934) Proc. R. Sot. London. Ser. A. 247,332-351 WalIing, C. (1975) Act. Chem. Res. 8.126-128 McClune, G.J. and Fee. J.A. (1976) FEBS Lett. 67.294-298 HalIiweII. B. (1976) FEBS Lett. 72.8-10 Rigo. A., Stevanato, R.. Finazzi-Agrb. A., Rotilio. G. (1977) FEBS Lett. 80. 13-132 Symonyan, M.A. and Nalbanduan, R.M. (1976) Biochem. Biophys. Res. Common. 71,1131-1138 BieIski, B.H.J. and Allen. A.O. (1977) J. Phys. Chem. 81,1048-1064
Biochimica et Biophysica Acta, 583 (1979) 279--286 © Elsevier/North-Holland Biomedical Press
BBA 28824
G E N E R A T I O N OF SUPEROXIDE RADICALS IN ALKALINE SOLUTIONS OF H Y D R O G E N P E R O X I D E AND THE E F F E C T OF SUPEROXIDE DISMUTASE ON THIS SYSTEM
M.A. SYMONYAN and R.M. NALBANDYAN Institute of Biochemistry, Academy of Sciences of the Armenian SSR, Yerevan, 375044 (U.S.S.R.) (Received April 26th, 1978) (Revised manuscript received August 22nd, 1978)
Key words: Superoxide radical generation; Hydrogen peroxide; Superoxide dismutase
Summary Superoxide radicals in high concentrations were generated from alkaline H202 w i t h o u t using catalysts or irradiation. The dependence of the intensity and parameters of the superoxide radical EPR spectrum on pH, temperature, viscosity and H202 concentration were studied. The observed changes are explained on the base of matrix effects. The addition of superoxide dismutase to alkaline H202 led initially to a drop in the EPR spectrum intensity, followed by an increase in the concentration of superoxide radicals.
Introduction Extensive studies concerning the role and properties of superoxide radicals in biological systems began in 1069, when several important data were obtained in this field almost simultaneously. Firstly, it was shown that superoxide radicals were formed in certain enzymic redox reactions [1]. Secondly, a new class of enzyme, superoxide dismutase, was discovered for which these radicals are normal substrates [2]. Thirdly, it was demonstrated that superoxide dismutase exhibited an inhibitory effect on the terminal enzymic redox reactions proceeding b y the one-electron mechanism [3--5]. Most of the methods used for the determination of superoxide dismutase activity were indirect, because they were based on the inhibitory effect of the enzyme on the reactions, accompanied by the formation of superoxide radicals as intermediates. Some investigations were carried out in which the direct reaction of superoxide dismutase with superoxide radicals was involved. The sub-
280 strate was generated by means of pulse-radiolysis, or polarographically [6,7]. However, for further elucidation of the features of the dismutation process, it is necessary to develop simpler methods for the generation of superoxide radicals in high concentrations. The radicals are known to be stable in some aprotic solvents and they may be generated therein at high concentrations. However, some of these solvents cause modification of protein structure [8,9]. Thereffore it is desirable to obtain high concentrations of the radicals in aqueous media. It has long been known that superoxide radicals are formed from hydrogen peroxide solutions in the presence of some oxidants (periodide, permanganate) or catalysts [10--12] (also, recently, Ref. 13). We show here that superoxide radicals are formed in alkaline solutions of concentrated H202, in the absence of added oxidants and catalysts. The radicals were utilized by cuprozinc superoxide dismutase, although depending on pH, viscosity and temperature, EPR signal parameters of the radicals underwent some changes. Materials and Methods Superoxide dismutase was obtained (from bovine erythrocytes) in an electrophoretically homogeneous state according to methods described in our previous paper [ 14]. Superoxide radicals were generated from alkaline H202 in a 20-ml tube, which was supplied with electrodes (for pH measurements) and a thermometer and contained 10--30% H202 (the concentration of which was established with permanganate). To generate superoxide radicals, a small piece of solid KOH or several drops of concentrated KOH solution were added. After the addition of alkali, the change of pH and temperature was traced and 0.4-ml aliquots of the mixture were frozen in liquid N2 for recording EPR spectra on a Varian E-4 instrument (modulation amplitude 6.3 G, time constant 0.3 s, scanning rate 250 G/min). Temperature-dependence was studied with the Varian E-248 variable temperature accessory. The effect of alkaline H202 on the properties of superoxide dismutase was followed after incubating 0.4-ml aliquots of superoxide dismutase with 0.1-ml aliquots of superoxide radical solution at room temperature. The effect of the enzyme on superoxide radical concentration was followed immediately after the addition of 0.1-ml aliquots of diluted superoxide dismutase to 0.4-ml aliquots of superoxide radical solutions. In control experiments, 0.1-ml aliquots were replaced by 0.1 ml 0.01 M phosphate or carbonate buffers (pH 9.5 and 10.5, respectively). All chemicals used were reagent grade. Iron and copper constants in H20: were found to be lower than the sensitivity of spectrophotometric determination methods, using the chelating agents o-phenanthroline and buthocuproin disulphonate [15,16]. Nevertheless, in special experiments, these chelators as well as salicylaldoxime, diethyldithiocarbamate, EDTA or EGTA (ethyleneglycol-bis(fl-aminoethyl ether) N,N'-tetraacetic acid) was added to the reaction mixture containing H202 to prevent the possible effect of trace metal contaminants on the phenomenon observed. Concentrations of chelators were in the order of 1 • 10-3---1 • 10 -2 M.
281 Results When the pH of H:O2 solutions was adjusted to 8.5 or higher, an EPR signal of axial s y m m e t r y was observed. The onset of the EPR signal was accompanied by an exothermic process and further increase of pH. Typical kinetic curves of these processes are shown in Fig. 1. The comparison of kinetic curves obtained for different initial pH values and concentrations of H20: led us to conclude that the EPR spectrum intensity depends on the initial pH value and concentration of H:O2 in the mixture. Thus, if the initial pH value of 30% H202 were adjusted to 10.0 (the final pH being 12.6) the maximum intensity of the EPR spectrum was three orders of magnitude as much as that the initial pH 9 of the same concentration of H202. The final pH in the last case was 10.5. It is important that the addition of the all chelators, besides EDTA, does n o t change the observed kinetic curves. Only EDTA (1 • 10 -2 M) was capable of stopping further increase of pH, temperature and generation of superoxide radicals. This effect of EDTA should n o t be connected with its chelating ability. This property of EDTA was earlier considered by Misra and Fridovich [16]. We found also that the addition of 1 • 10 -2 M Cu 2÷ or Mn 2÷ brings about a decrease of the concentration of superoxide radicals. Although the appearance of EPR signals in alkaline H202 was accompanied by heating of the medium, the free radical species were found to be very stable at alkaline pH values and room temperature. Thus, the radicals formed at initial pH 10.0 have a half-life of 2 h at 22°C. The EPR spectrum observed disappears completely and irreversibly only after heating the mixture at 80°C for 5 min. No change in the shape of the EPR spectrum was observed in the course of this thermal treatment. It was found, however, that the shape and parameters of the EPR signal depend on pH values of alkaline H202 solution. Although in all the cases studied the EPR spectra had axial shapes with two g factors, the value of g,I and the width of the low-field c o m p o n e n t of the EPR spectrum (the component at gl,) were remarkably altered with change in pH. Shapes of EPR signals observed at different initial pH values of H202 solutions are compared in Fig. 2. As can be seen, the increase of the initial pH of H202 solutions was accompanied by an increase of the EPR signal intensity as well as by increase in gll value and width. Further, it was shown that the stability of radicals generated from peroxide depends also on pH. Thus, radicals formed at lower pH
f2
~8
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l~Iv
"O'O
0
i
I~'"-C~'---0
i
i
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,
Fig. 1. The change o f p H (~), temperature (0) and superoxide radical concentration (o) of 30% H 2 0 2 made alkaline w i t h N a O H to p H 10.0.
282
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.-"- If20 4¢,6/..O..'°'O[
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~rW.e"'
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,
,
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,
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Fig. 2. T h e c h a n g e o f t h e s h a p e ( A ) a n d p a r a m e t e r s (B) o f t h e E P R s p e c t r u m o f s u p e r o x i d e r a d i c a l s g e n e r a t e d f r o m 3 0 % H 2 0 2 a t p H v a l u e s 8 . 2 (1), 9 . 5 (2), 1 1 . 5 (3), 1 3 . 3 (4). R e c e i v e r g a i n s w e r e : 2 • 1 0 2 . 6 . 3 101 , 1 . 2 5 - 101 , 5 • 1 0 0 , r e s p e c t i v e l y . A p H d e p e n d e n c e o f gl] value; o, p H d e p e n d e n c e o f t h e line w i d t h f o r t h e l o w - f i e l d c o m p o n e n t o f t h e E P R s p e c t r u m . All E P R s p e c t r a w e r e r e c o r d e d a t 7 7 K. Fig. 3. T h e e f f e c t o f v i s c o s i t y o n t h e s h a p e (A) a n d s t a b i l i t y (B) o f s u p e r o x i d e r a d i c a l s g e n e r a t e d . 1, r a d i cals f o r m e d f r o m 3 0 % H 2 0 2 ( p H 1 2 . 5 ) ; 2, 3 0 % p o l y ( e t h y l e n e g l y c o l ) w a s a d d e d t o I. R e c e i v e r g a i n s w e r e : 1 . 2 5 - 101 a n d 1 . 2 5 • 1 0 0 f o r 1 a n d 2, r e s p e c t i v e l y . T h e d r o p o f t h e c o n c e n t r a t i o n o f r a d i c a l s 1 a n d 2 a t 2 0 ° C is s h o w n in B ( 1 ' a n d 2').
(lower g, value) have at room temperature a shorter half-life than those formed at higher pH values. The addition of viscous solvents such as glycerol or poly(ethyleneglycol) to the H202 solutions made pH 12.0 brings a b o u t modification of the EPR signal shape. The observed signal has parameters and shape more typical for pH 9--10, although no change of pH was observed after the addition of viscous solvents. The modification of the signal was accompanied by a several-fold increase in its intensity. Surprisingly, radicals formed in the presence of viscous solvents were remarkably less stable than that obtained at pH 12.0 in the absence of the solvents. These data are presented in Fig. 3. Thus, the radicals formed in alkaline H202 at pH 12.0 in the presence of viscous solvents have EPR spectral shape and parameters which are more typical for radicals generated at pH 10.0 and, in addition, their stability resembled more that of the radicals generated at pH 10.0. Study of the effect of microwave power on the shape of EPR signals showed that the radicals formed at pH 10 and 12 do not change their shapes (at either pH value), at least in the region of 0.2--150 mW. On the other hand, the shape as well as the intensity of the EPR spectrum was found to depend on temperature. Fig. 4 shows the results obtained. As the measure of changes, the parameter Q was accepted, the mean of which is illustrated in Fig. 4C. As it can be seen, the value Q depends linearly on l/T, the change observed being completely reversible in the region --196°C to --60°C. However, the EPR signal dis-
283
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i
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/
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3~00
B. V ~ fo- ~
f/T, 10" ~
0
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Fig. 4. T e m p e r a t u r e d e p e n d e n c e o f t h e s h a p e a n d i n t e n s i t y o f t h e E P R s p e c t r u m o f s u p e r o x i d e r a d i c a l s f o r m e d 3 0 % H 2 0 2 ( p H 1 3 . 0 ) . E P R s p e c t r a ( A ) w e r e r e c o r d e d u n d e r t h e f o l l o w i n g c o n d i t i o n s : 1, r e c e i v e r g a i n , 4 - 101 , t = - - 1 6 0 ° C ; 2, r e c e i v e r g a i n , 5 - 1 0 2 , t = - - 1 0 0 ° C ; 3, r e c e i v e r g a i n , 1 . 1 0 3 , t = - - 8 0 ° C ; 4, r e c e i v e r g a i n 4 • 1 0 3 , t = - - 6 0 ° C . T h e c h a n g e o f t h e s h a p e ( p a r a m e t e r Q) is p r e s e n t e d in C. F~lled t r i a n g l e s a n d circles r e p r e s e n t d a t a o b t a i n e d f o r t h e c h a n g e o f t e m p e r a t u r e f r o m l o w e r t o h i g h e r , a n d o p e n t r i a n g l e s a n d circles are d a t a f o r t h e c h a n g e o f t e m p e r a t u r e in t h e reverse d i r e c t i o n . Fig. 5. T h e e f f e c t o f d i f f e r e n t c o n c e n t r a t i o n s o f s u p e r o x l d e d i s m u t a s e (2 • 1 0 -9 M (o), 2 • 1 0 -8 M (A)) o n the concentration of superoxide radicals. The radicals were generated form 30% H202 (pH 12.6) and had t h e E P R s p e c t r u m s h o w n in Figs. 2 - - 5 . F i l l e d circles s h o w n t h e t i m e c o u r s e in t h e a b s e n c e o f s u p e r o x i d e dismutase.
appears completely above --60°C and neither was any signal observed in the solution of alkaline H202 at r o o m temperature. When the temperature of the frozen solution fell below --60°C, the EPR spectrum was again observed. In the region --196°C to --60°C, the intensity of the signal obeys Curie's law. It is important note that radicals generated at pH 10.0 show similar temperature dependence. The addition of catalytic amounts of superoxide dismutase to alkaline solutions (pH 10.0 or 12.0) leads to the disappearance of radicals. We were unable to follow this process kinetically; however it was noted that the same concentration of the enzyme is capable of utilizing various limited concentrations of the radicals, depending on pH values (Table I). The results obtained show that the catalytic p o w e r of superoxide dismutase is maximum when H~O2 is taken to pH 11.5--12.5. After the rapid disappearance of free radicals following superoxide dismutase addition the formation of radicals was again observed, the rate of which was significantly slower than the breakdown of radicals in the course of the first stage (Fig. 5). It was found that the EPR signal shape of radicals formed in the second stage was identical to that observed before the addition of superoxide dismutase. The generation o f radicals in the course of the second step is also a catalytic process because the rate depends linearly on superoxide dismutase concentration. If superoxide dismutase and alkaline H~O2 are mixed in comparable molar
284 TABLE
I
THE CATALYTIC
POWER
OF SUPEROXIDE
DISMUTASE
IN ALKALINE
H202
SOLUTIONS
T o d e t e r m i n e t h e p o w e r o f t h e e n z y m e a t d i f f e r e n t p H v a l u e s , m i n i m a l q u a n t i t i e s o f the: e n z y m e w e r e mixed with alkaline 30% H 2 05 and concentrations of superoxide radicals before and immediately after t h e a d d i t i o n o f t h e e n z y m e w e r e c o m p a r e d . F a l l i n c o n c e n t r a t i o n (in o r d e r s o f m a g n i t u d e ) w a s c a l c u l a t e d per mole of the enzyme. pH
8.5
10.5
11.5
12.3
] 3.0
13.9
Catalytic power
2.0
3.5
4.5
5.6
4.1
2.5
amounts, modification of the EPR spectrum of the enzyme is observed. The shape of the spectrum is different from that obtained in the presence of H:O2 or alkali alone. Discussion The data obtained indicate that superoxide radicals may be easily formed from alkaline concentrated H202 solution without irradiation, catalysts or added oxidants. The exact mechanism of the generation of superoxide radicals in this system is as y e t unknown. Several more-or-less possible mechanisms of the process may be suggested, however their consideration lies b e y o n d this study. The data presented indicate that trace metal probably does not participate in the formation of superoxide radicals. The sharp increase of superoxide radical concentration at pH values above 11.0 agrees with pK = 11.6 for H~O2. Thus, deprotonation of peroxide is essential for the generation of superoxide radicals. The shape of the EPR spectrum of the radicals generated and their utilization by superoxide dismutase leave little d o u b t a b o u t the nature of these radicals. However, the question of differences of EPR spectral parameters of superoxide radicals formed b y different methods in various systems is still open. Shifts in g-factor values of the EPR spectrum of superoxide radicals generated in several more complex systems were explained b y Knowles et al. [ 1 ] as a matrix effect. We found that, although the EPR spectra of the radicals have in the alkaline scale an axial symmetry with two g factors, the gLI value and the width of the EPR spectrum were very sensitive to pH, temperature and viscosity. The microenvironment of the superoxide radicals has also a certain influence on their stability. Thus, as could be predicted from cell effect, the stability of radicals formed in more viscous media was decreased. In accordance with earlier data [18--20] to the effect that superoxide radicals are stabilized in aprotic organic and aqueous alkaline media, we noted that the concentration of the radicals formed in alkaline hydrogen peroxide was higher at more alkaline pH. However, the catalytic p o w e r of superoxide dismutase added to the hydrogen peroxide-alkali system was maximal in the range pH 11--12.5. The decrease of the power at higher pH values is probably connected with the denaturation of the enzyme at strongly basic pH [21]. On the other hand, at pH 9--11 the drop of the catalytic power may be connected with higher concentration of hydrogen peroxide in the system, which is known to inactivate superoxide dismutase [22,23]. As was established, the addition of superoxide dismutase to alkaline
285
H202 led to the increase of superoxide radical concentration in the second step (Fig. 5). It was shown that the copper environment in superoxide dismutase is sharply modified in the presence of alkaline H202. We think that the system considered is suitable for the study of b o t h direct and reverse reactions catalyzed b y superoxide dismutase. The catalysis of superoxide dismutase of the generation of superoxide radicals from H202 and oxygen, i.e., the reversal of the superoxide dismutase process, was demonstrated by Hodgson and Fridovich [24] using the indirect m e t h o d of detection of superoxide radical through its reaction with tetranitromethane. The presence of two stages in the effect of superoxide dismutase on the concentration of superoxide radicals formed in alkaline H202 solutions indicates that there are equilibria in the system in which superoxide radicals are involved. The main free radical species formed in H202 solutions are admitted to be h y d r o x y l and superoxide. H y d r o x y l radicals are formed e.g., according to the Haber-Weiss reaction [ 25,26] : H20~ + 02- ~- O H - + "OH + O2
(I)
Although there is some d o u b t in importance of this reaction in weakly alkaline media [27], nevertheless, .OH is generated in some way from H202 in the presence of superoxide radicals [28,29]. Also, the formation of superoxide and h y d r o x y l radicals under the effect of superoxide dismutase is suggested [30]: OH- + 02 -~ 02- + "OH
(2)
On the other hand, superoxide radicals possibly are generated in the reaction of hydroxyl radicals with deprotonated H202: HO2 + "OH ~ H20 + 02-
(3)
It is evident that the effect of superoxide dismutase on equilibria [1--3] and the other possible schemes with the participation of hydroxyl and superoxide radicals is very complicated. Moreover, recently Bielski and Allen [31] demonstrated that the simultaneous nonenzymic dismutation in aqueous alkaline media should occur with the participation of this protonated form of superoxide radicals, HO2, although the pK for this radical is of 4.75. Thus, superoxide radicals are in equilibrium with their protonated form and the latter is capable of dismuting at alkaline pH with a high rate constant. This finding raises the question of whether enzymic dismutation of superoxide radicals involves their preliminary protonation. In other words, it would be of interest to determine the significance of the two protons required for the formation of H2O 2 in the course of superoxide dismutase catalyzed dismutation of superoxide radicals. References 1 Knowles, P.F., Gibson, J.F., Pick, F.M. and Bray, R.C. (1969) Biochem. J. 111, 53---58 2 McCord, J. and Fridovich, I. (1969) J. Biol. Chem. 244, 6045--6055 3 Ballou, D., Palmer, G. and Massey, V. (1969) Biochem. Biophys. Res. Commun. 36,898--904 4 0 r m e - J o h n s o n , W.H. and Beinert, H. (1969) Biochem. Biophys. Res. Commun. 36, 905--911 5 Massey, V., Strikland, S., Mayhew, S.G., Howell, L.G., Engel, P.C., Matthews, R.G., Sehuman, M. a n d Sullivan, P.A. (1969) Biochem. Biophys. Res. Commun. 36, 891--897
286 6 Fielden. E.M.. Roberts, P.B.. Bray. R.C., Lowe. D.J.. Maunter. G.N., RotiIio. G. snd Calabrese. L. (1974) Biochem. J. 139.49-60 (and references therein) 7 Rigo. A., VigIino, P. and RotiUo, G. (1976) Biochem. Biophys. Res. Commun. 63.1013-1016 8 Symonyan. M.A. and Nalbandyan, R.M. (1976) Biochim. Biophys. Acta 446.432-444 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Symonyan, M.A. and Nalbandyan, R.M. (1976) Studia Biophysics 54.99-103 Lunsford. J. and Javne, J.P. (1966) J. Chem. Phys. 44.1487-1492 Atkins, P.W. and Symons, M.C.P. (1967) The Structure of Inorganic Radicals, Elsevier. Amsterdam Schumb, W.C.. Satterfield, C.N. and Wentwoth. R.L. (1966) Hydrogen peroxide. Reinold, New York One, J., Matsumura, T.. Kit&ma. N. and Shun&hi Fukuzumi (1977) J. Phys. Chem. 81.1307-1311 Symonyan. M.A. and Nalbandyan. R.M. (1976) Biochimija (Russ) 40.726-732 Cameron. B.F. (1965) Anal. Biochem. 11,164-169 Poillon, W.N. and Dawson, C.R. (1963) Biochim. Biophys. Acta 77, 27-36 Misra. H.P. and Fridovich, I. (1972) J. Biol. Chem. 247.3170-3175 Maricle, D.L. and Hodgson. W.G. (1965) Anal. Chem. 37.1562-1565 Fee, J.A. and Hildenberg, P.C. (1974) FEBS Lett. 39.79-82 Czapski. G. and Dorfman, L.M. (1964) J. Phys. Chem. 68.1169-1177 Symonuan. M.A. and Nalbandyan. R.M. (1976) Biophysics (Russ) 20.783-787 Symonyan, M.A. and Nalbandyan, R.M. (1972) FEBS Lett. 28.22-24 Bray. R.C.. Cockle, S.A.. Fielden, E.M., Roberts, P.B.. Rotilio. G. and Calabrese, L. (1974) Biochem. J. 139,43-48 Hodgson, E.K. and Fridovich, I. (1973) Biochem. Biophys. Res. Commun. 54,270-272 Haber, F. and Weiss, J. (1934) Proc. R. Sot. London. Ser. A. 247,332-351 WalIing, C. (1975) Act. Chem. Res. 8.126-128 McClune, G.J. and Fee. J.A. (1976) FEBS Lett. 67.294-298 HalIiweII. B. (1976) FEBS Lett. 72.8-10 Rigo. A., Stevanato, R.. Finazzi-Agrb. A., Rotilio. G. (1977) FEBS Lett. 80. 13-132 Symonyan, M.A. and Nalbanduan, R.M. (1976) Biochem. Biophys. Res. Common. 71,1131-1138 BieIski, B.H.J. and Allen. A.O. (1977) J. Phys. Chem. 81,1048-1064
