KINETIC ANALYSIS OF THE FENTON REACTION BY ESR-SPIN TRAPPING. Ynkio Mizuta*, Toshiki Masumizu*, Masahiro Kohno*, Akitane Morit and Lester Packer~

Transcription

1 Vol. 43, No. 5, December 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages KINETIC ANALYSIS OF THE FENTON REACTION BY ESR-SPIN TRAPPING Ynkio Mizuta*, Toshiki Masumizu*, Masahiro Kohno*, Akitane Morit and Lester Packer~ * ESR Application Laboratory, Analytical Instruments Division, JEOL LTD., 1-2 Musashino 3-Chome, Akishima, Tokyo 196 Japan. Department of Molecular and Cell Biology, University of California, Berkeley, California, USA. Received August 13, 1997 SUMMARY A quantitative analysis of hydroxyl radical (-OH) generated in the Fe 2+hydrogen peroxide reaction system was explored by a spin-trapping method us!rig 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) combined with electron spin resonance spectroscopy. Based on the numerical analysis of Fentonrelated reactions, the reduction of DMPO-OH adduct from t:1 stoichiometry prominent at high concentrations of Fe 2+ was consistent with a reaction model in which a molar amount of hydrogen peroxide was reduced by two molar amounts of Fe 2+. Furthermore, time-dependent decrease in DMPO-OH tqhuantity, apparent at much higher concentration of Fe 2+, was proved due to e reaction not with Fe 2+ but with Fe 3+. Keywords" ESR, spin trapping, Fenton reaction, stoichiometry, hydroxyl radical, kinetic analysis INTRODUCTION The hydroxyl radical has been considered as major species for oxygen toxicity. The most important process for generating hydroxyl radical in biological systems has been speculated to be so-called Fenton reaction as shown below. FeZ+ + H202 k, > Fe3+ + OH- +.OH ( 1 ) This reaction scheme has been investigated and confirmed by ESR [1,2] and UV photo-absorption [3] spectroscopy so far. The former has revealed the generation of Fe3+ and -OH directly. The rate constant kl of 78 M-ls -1 has been determined by the latter method. However, the quantity of hydroxyl radical has been reported to be less than the quantity of reactants. In practice, the efficiency for the generation of.oh decreases with increasing Fe 2+ concentration. For explaining the quantitative relationship between reactants and products, another reaction scheme, such as an oxoiron(iv) ion (FeO 2+) generation pathway, has also been proposed [4-6]. Fe2+ + H202 k2 > FeO2+ + H20 (2) /97/ /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved.

2 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL The ESR spin-trapping technique [7] was envisioned as a technique which could specifically identify and quantitate superoxide and hydroxyl radicals [8-10]. The resultant nitroxide radical is relatively stable and the rate constant for the reaction of the radical with the spin-trapping reagent is high enough to allow for the efficient detection of short-lived free radicals. This technique has therefore been a powerful tool for studying generation and reduction of free radicals. By use of the ESR spin-trapping technique, Yamazaki and Piette reported that the efficiency for the generation of.oh increases with decreasing Fe 2+ concentration up to t gm when the concentration of hydrogen peroxide was maintained constant at 90 gm [11]. They reported the stoichiometry decreased markedly as the Fe 2+ concentration was increased. In this study, we investigated.oh generation and/or reduction mechanism quantitatively, based on the ESR spin-trapping method using DMPO. Owing to the development of enhanced sensitivity of the ESR spectrometer used in this study, we succeeded in detecting the quantity of DMPO-OH wherein Fe 2+ concentration was much less than that already reported. In this condition, we demonstrated strict 1:1 stoichiometry between Fe 2+ and hydrogen peroxide. Furthermore, the essential reaction mechanism between Fe 2+, H202, and their derivatives could be proposed based on numerical simulation for the kinetic profile of DMPO-OH concentrations. MATERIALS AND METHODS Materials: A nitrone spin trap [5,5-dimethyl-l-pyrroline-N-oxide (DMPO)] was obtained from Dojin Chemical and used without further purification. The primary standard used for spin concentration [4-hydroxy- 2,2,6,6-tetramethyl piperidine-l-oxyl (TEMPOL)] was obtained from Sigma Chemical Co. As a source of hydroxyl radical generation, iron(ii) sulfate was obtained from Wako Pure Chemical Ins. Ltd. Hydrogen peroxide (H202) was from Mitsubishi Gas Chemical Co., Inc. Instrumentation: ESR spectra were recorded on a JEOL JES-TE200 spectrometer using an aqueous quartz flat cell (Inner size 60 mm xl0 mm x0.31 ram) with an effective sample volume of 160 ~tl. Time-dependent ESR signals were recorded from the nine just after mixing reagents using JEOL JES-SM2 stopped-flow system. Reaction Conditions: All measurements were carried out in pure water as solvent treated to remove the chelate effect by sodium phosphate buffer solution (PBS) at room temperature. Reactions were started with the flow apparatus by mixing aqueous solution containing 8.8 mm DMPO and hydrogen peroxide with an aqueous solution of Fe e+. The final concentration of aqueous H202 and DMPO solutions were 500 ~tm and 440 ~tm, respectively. The final concentration of Fe 2+ salt solution was prepared to range between ~tm to 50 ~tm. The spin concentration of DMPO-OH was determined by the double integration of its ESR spectrum compared with that of TEMPOL whose concentration was already calibrated. Once the relation between ESR signal intensity of DMPO-OH and its spin concentration were determined, the time-dependent ESR intensity were transformed to the time-dependent variation of DMPO-OH concentration. 1108

3 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Simulation Procedures: In order to analyze the time-dependent variation of DMPO-OH concentration, the results were numerically simulated by use of chemical mechanisms in which Fe 2+, H202, and their derivatives participate. In addition to reactions (1) and (2), chemical reactions such as eqs.(3)-(5) have been included as suggested by Rush et al [11,13]. 2Fe z+ + HzO 2 ~ 2 F e OH- (3) FeZ+ +Fe3+ +H202 k4 >2Fe3+ +.OH+OH- (4) Fe > +Fe 3+ +H202 k5 >FeO 2+ +Fe 3+ +H20 (5) Hydroxyl radical has been reported to behave as an electrophilic reagent in the reduction of Fe 2+ with rate constant of 3x108 M-ls -1. Fe > +-OH--Za-~ Fe 3+ + OH- (6) Once hydroxyl radical is generated, the recombination reaction occurs with rate constant of 5x109 M-ls -1. -OH +.OH--~+ H202 (7) Moreover, hydroxyl radical reacts with hydrogen peroxide to make hydroperoxyl radical with rate constant of 4x107 M'ls -1..OH + H202 kg > H20 +.OO H (8) These chemical reactions (6)-(8) and their rate constants are cited from Ref.(3). The Fe 3+ generated by above reactions has been reported to react with H202 to produce hydroperoxyl radical [14]. Fe 3+ +H202 ~ F e 2+ +.OOH + H + (9) The hydroxyl radical formed and consumed in these reactions (1)-(9) finally reacts with DMPO to make DMPO-OH, which is the only product that we can measure directly by the ESR-spin trapping method. Hydroxyl radical is trapped by DMPO with a rate constant klo = 3.4 x 109 M-ls -1 to form DMPO-OH [15]. DMPO+-OH k~0 >DMPO_OH (10) In addition to above reactions, another mechanism is necessary in which excess amounts of Fe 2+ changes DMPO-OH to the diamagnetic species. For example, Fe 2+ has been proposed to reduce DMPO-OH [11]. Fe 2+ + DMPO - OH k, > Fe3+ + DMPO - OH- (11 ) On the basis of these reaction schemes, the kinetic profile of the DMPO-OH quantity can be calculated by solving the following equation. [DMPO- OH] = kl0 fo[dmpo ] [.OH] dt (12) Equation (12) was directly solved by numerical integration. The rate constants kl, k6, k7, ks, and kl0 reported so far are listed above. These values were always used as the initial parameters in calculating eq.(12). In the simulation procedure, the rate constants of the above reactions were varied to reproduce experimental data. The parameters which have not yet reported so far, were proposed in this work from the result of the optimum fit against experimentally obtained time course of the DMPO concentration. Since the reaction rate of hydroxyl radical is supposed to be much faster compared with that of other reactions, the steady-state assumption was applied for the concentration of hydroxyl radical. 1109

4 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL RESULTS AND DISCUSSION The ESR signal due to DMPO-OH is shown in Fig. l(a). The main feature of the DMPO-OH adduct signal is composed of 4 lines with a 1:2:2:1 intensity ratio, reflecting that the nitrogen hyperfine coupling constant (hfcc) is equivalent to that of [~-hydrogen atom on DMPO-OH adduct [12]. A pair of phase-reversed signals is due to MnO/MgO used as the reference of sensitivity. By using a stopped-flow system, the time-dependence of the second signal of DMPO-OH was recorded as shown in Fig. l(b). The efficiency for the generation of DMPO-OH plotted against the concentration of Fe 2+ is shown in Figure 2. It was found that a 1:1 stoichiometry between Fe 2+ and DMPO-OH was strictly valid when [Fe 2+] was less than 0.5 ~tm, but the ratio [DMPO-OH]/[Fe 2+] markedly decreased as the Fe 2+ concentration was increased. In order to quantitate this phenomenon, the kinetic profile of DMPO-OH was examined under the same experimental conditions. The time course of the quantitative ratio of [DMPO-OH] against [Fe 2+] is shown in Fig. 3. From these results, it was found that 1:1 stoichiometry was strictly valid when [Fe 2+] was less than 0.5 ~tm. When [Fe 2+] ranged from 0.5 ~tm to 2.5 ~tm, the DMPO-OH generation efficiency decreased from 1:1 stoichiometry and approached a steady state. To explain this result, the reaction path other than equation (1), such as equation (2), seems necessary. When [Fe 2+] exceeds 2.5 ~tm, time-dependent consumption of DMPO-OH was apparent. The decay rate of DMPO-OH was found approximately proportional to the concentration of [Fe2+]. In order to explain this result, some other reaction mechanism seems warranted in which DMPO-OH is changed to some diamagnetic molecule. At first, we investigated a case where 1:1 stoichiometry was strictly valid. In Fig. 4, the experimental result appeared noisy because DMPO-OH concentration, ca. 0.1 ~tm, was nearly at the lower limit of detection. However, the final concentration of DMPO-OH was determined to be the same as the initial concentration of Fe 2+ as shown in Fig. 2. Therefore, reaction (1) should be the dominant mechanism in this case. Simulation procedures were performed with varying kl and the contribution from other kinetic processes was ignored. The best fit was obtained at k1=230 M- 1s-1. It was apparent that the final concentration of DMPO-OH was identical so long as only reaction (1) was considered. As shown in Fig. 4, kl was only reflected in the initial stage of DMPO-OH generation. When Fe 2+ concentration increased to 2.5 ~tm, the final concentration of DMPO-OH was about 40 % of Fe 2+ concentration. In order to interpret this result, another reaction path other than (1) should be necessary. In this 1110

5 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Q c~ 0 "-E < 9 -~ ~ 9 S L it 0 ~ ""4 r,o e ~.~ v g T,-- "7, (Hun'qa~) J~l!suelul 1111

6 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL 4.0 4,,"i /! i... ; '( o, r i,' t i ~/ 0.0,~ ,0 [Fe(++)] /~M Fig. 2 The efficiency for the generation of DMPO-OH plotted against the concentration of Fe SuM 6O "1" , SUM... 5.OuM 0, I, I, I, I time/s Fig. 3 The kinetic profile of the quantitative ratio of DMPO-OH plotted with varying Fe 2+ concentration. 50uM 1112

7 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL looo r 800 t'~ I- 60o SO ~i 150 >~ 230 KI / M-Is-I I/~ l, l I ~ l i time/$ Fig. 4 Comparison between experimental ESR results (noisy spectrum) and simulated results. [Fe 2+] is gm. The rate constant kl was determined to be 230 M-ls -1. case, reduction of the production efficiency for DMPO-OH was interpreted by including other reaction scheme than (1) as the perturbation effect. Fig. 5 shows the comparison between experimental and simulated results for an Fe 2+ concentration of 2.5 ~tm. The simulated result is shown in Fig. 5(a), in which reaction (2) was included in addition to reaction (1). As shown in this figure, the final concentration of DMPO-OH was reduced with increasing k2. However, the time-course profile of DMPO-OH concentration could not be reproduced. On the other hand, the experimental results was closely reproduced in the case where reaction (3) was included in addition to reaction (i). Two rate constants, k1=84 M-Is -! and k3=5.5x107 M-2s -1, were determined in this curve-fitting procedure. The rate constants k4, k5, k9, and kll were set to zero to obtain the results in Fig. 5. Though the oxoiron(iv) ion, FeO 2+, has been reported to be produced in the Fenton reaction as the intermediate species [4-6], our numerical simulation excludes the possibility for existence of FeO 2+. This result seems reasonable because the deviation from 1:1 stoichiometry was much more sensitive than the variation of Fe 2+ concentration. If reaction (2) was effective, stoichiometry between Fe 2+ and DMPO-OH should be retained 1113

8 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 5000 K I = 84M-Is -l K 2 / M-Is ( :3.6 },., ~ f 70 r \ 1 O0 \ Experimental I time/s K I - 84M-Is -j K 3 / 107M-2s -t , ,0 ' \ Experimental 0, I, I, I i I time/s Fig. 5 Comparison between experimental ESR and simulated results. [Fe 2+] is 2.5 gm. (a) Reactions (1) and (2) were assumed, (b) Reactions (1) and (3) were assumed, as the Fe 2+ consumption mechanism. 1114

9 Vol. 43, No. 5, 1997 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL because reaction(i) and reaction (2) compete for consumption of Fe 2+. This explanation does not reproduce the experimental results. On the other hand, the Fe 2+ consumption rate between reaction (1) and reaction (3) sensitively depends on the Fe 2+ concentration and this explanation closely agrees with the experimental results. For the result wherein the Fe 2+ concentration was in excess of 5 btm, the DMPO-OH consumption mechanism needs to be included to reproduce experimental results. Fig. 6 shows the simulated result in which reaction scheme (11) was included in addition to reactions (1) and (3). The rate constants k4, k5, and k9 were set to zero to obtain the results in Fig. 6. It was apparent that the rate constant kl 1 only contributed to the final steady concentration of DMPO-OH. Although Fe 2+ has been reported to contribute for the reduction of nitroxide radicals such as DMPO-OH [11], our simulated results are in disagreement with this view. Our result, however, seems reasonable because the Fe 2+ quantity for generating hydroxyl radical in reaction (1) should be reduced to some extent if excess Fe 2+ was used for the consumption of DMPO-OH. K, = 52Mqs -! K 3 = 9.4 x 106M-2s Kll / 10 3 M-is q E :3 J~ 2000,... ;>.,,.,i..a (n r 6) r looo t "~"" " ~"~"" - "- ental time/s Fig. 6 Comparison between experimental ESR and simulated results. [Fe 2+] is 50 gm. In addition to the reaction schemes (1) and (3), reaction (11) was included as the consumption mechanism os DMPO,OH

10 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Therefore, we have assumed some chemical reactions including Fe 2+ and/or Fe 3+ as the mechanism for DMPO-OH reduction. As a result of cumbersome trials of simulation procedure with varying reaction models, the most satisfying result was obtained by only including 1:1 reaction between Fe 3+ and DMPO-OH, as shown in Fig. 7. In obtaining this result, the rate constants k4, k5, k9, and kll were set to zero. Since we didn't assign any products related to this reaction, we could not yet suggest any chemical reaction scheme. However, the amount of DMPO-OH should not be controlled by the initial concentration of Fe 2+ if the Fe2+/Fe 3+ redox system was present. So we anticipated that Fe 3+ should not be reduced to Fe 2+. In other words, the reaction rate for Fe 3+ reduction, such as eq.(9), seems very slow. For further understanding of the reason why DMPO-OH was decreased by an excess amount of Fe2+, as shown in Figs. 6 and 7, we examined the reactivity between TEMPOL as nitroxide and Fe2+/Fe 3+ in aqueous solution. TEMPOL was used here instead of DMPO-OH because Fe2+/Fe3+ redox reaction must exists in using DMPO-OH, generated by Fenton reaction system. As shown in Fig. 8, we have determined the K= = 52Mqs -' J K 3 ='9.4x I06M-2s-I 0.0 K / M-is -L r J~ 2000.~ r-.-, 1000 t- Pe!O~ontal time/s Fig. 7 Comparison between experimental ESR and simulated results. [Fe 2+] is 50 gm. In addition to the reaction schemes (1) and (3), 1:1 reaction between Fe 3+ and DMPO-OH was included as the consumption mechanism of DMPO-OH. 1[16

11 Vol. 43. No BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL 600 e2+ (a) F ~-~ 500 c -~ 400 },,,, v u) 300 c ,,,,,, time Is Fig. 8 The kinetic profile of TEMPOL ESR signal. TEMPOL was mixed (a) with Fe 2+, (b) with Fe 3+ in aqueous solution. [TEMPOL] is 1 gm. [Fe 2+] is 50 gm and [Fe 3+] is 50 gm. decrease in concentration of TEMPOL by an excess amount of Fe 3+. However, the same amount of Fe 2+ didn't change the concentration of TEMPOL. This result indicates that Fe 3+ is the main species for further reduction of DMPO-OH. As noted above, we have succeeded in explaining the time dependence of DMPO-OH quantity over wide range of Fe2+ concentration. The rate constants kl and k3, obtained by these simulation procedure, were plotted against Fe 2+ concentration in Fig. 9. The kl of 78 M-ls -1 was reported so far as the result of photo-absorption experiment [3]. Our result reproduced it at relatively higher Fe2+ concentrations. However, it was found that kl increasingly changes with decreasing concentration of Fe 2+. As shown in figure 9, it was found that kl and k3 gradually decreased with increasing Fe 2+ concentration. Since we didn't use phosphate buffer to remove its chelative effect on Fe 2+, this seems due to a gradual ph change in changing Fe 2+ concentration. In practice, the ph of the Fe 2+ sulfate aqueous solution of 50 ~tm was 6.6. However, the ph of this solution of was changed to 4.6 when all of the Fe 2+ was changed to Fe 3+ through reaction (1) and reaction (3) by the addition of hydrogen peroxide. Since ph of the solution is proportional to the logarithmic concentration of the solution, the horizontal axis in Fig. 9 linearly corresponds to the ph change. However, further investigations are needed to explain ph effect in relation to the variation of rate constants. 1117

12 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL (a) "" i 1 I 10 J 100 1(],,,,.... -,,,,,,,,, t,,,,, [Fe(++)] /r~m 10 (b) '7, '~ 10 ~ V 10, t a i, i i l l J t,, i,,a_j [Fe(++)]/I~M Fig. 9 The variation of ESR determined rate constants, (a) kl and (b) k3, plotted against Fe 2+ concentration. In conclusion, 1:1 stoichiometry between Fe 2+ and DMPO-OH could be demonstrated when the Fenton reaction was investigated under conditions where Fe 2+ was much lower in concentration, ca. 0,1 to 0.5 ~tm, than the concentration of hydrogen peroxide. This result can be explained only by so-called Fenton reaction (1) as the mechanism. The efficiency for generation of DMPO-OH was gradually decreased when the Fe

13 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL concentration was increased to 2.5 gm. From the results of numerical simulation for the time course of DMPO-OH formation, it was found that reaction, in which a molar amount of hydrogen peroxide was reduced by two molar amounts of Fe 2+, mainly contributed to the reduction of the DMPO-OH yield. When [Fe 2+] exceeds 2.5 gm, DMPO-OH was found consumed time-dependently. This result was well interpreted by the reaction of DMPO-OH not with Fe 2+ but with Fe 3+. The rate constants for a series of relevant reaction was determined as the result of numerical simulation. REFERENCES 1. Buettner, G. R. (1987) Spin trapping: ESR parameters of spin adducts. Free Radical Biol. Med. 3, Mizuta, Y.; Mitsuta, K; Kohno, K. (1989) Quantitative analysis of active oxygen generated in Fe-Peroxide reaction system using ESR spin trapping method, in Medical, Biochemical and Chemical Aspects of Free Radicals. (Niki, E, Kondo, M. and Yoshikawa, T. Eds.), pp , Elsevier Science, Amsterdam. 3. Saito, I; Matsugo, S. (1988) in Protein, in Nucleic Acid and Enzyme., (Nakano, M., Asada, K. and Oyanagi, Y. Eds.), pp , Kyoritsu- Shuppan, Tokyo. 4. Walling, C. (1975) Fenton's reagent revisited Acc. Chem. Res. 8, Rush, J. D.; Koppenol, W. H. (1986) Oxidizing intermediates in the reaction of ferrous EDTA with hydrogen peroxide. J. Biol. Chem. 261, Rahhal, S.; Richter, H. W. (1988) Reduction of Hydrogen Peroxide by the Ferrous Iron Chelate of Diethytene-N,N,N',N",N"-pentaacetate J. Am. Chem. Soc. 110, Janzen, E. G. (1971) Spin trapping. Acc. Chem. Res. 4, Janzen, E. G. (1980) in Free Radicals in Biology (Pryor, W. A. Ed.), vol. IV, pp , Academic Press, New York.. 9. Harbour, J. R.; Chow, V.; Bolton, J. R. (1974) An electron spin resonance study of the spin adducts of OH and HO2 radicals with nitrones in the ultraviolet photolysis of Aqueous hydrogen Peroxide solutions Can. J. Chem. 52, Harbour, J. R.; Bolton, J. R. (1975) Superoxide formation in spinach chloroplasts: Electron spin resonance detection by spin trapping Biochem. Biophys. Res. Commun. 64, i 1. Yamazaki, I.; Piette, L. H. (19.90) ESR spin-trapping studies on the reaction of Fe 2+ ions with H202 -reactive species in oxygen toxicity in biology. J. Biol. Chem. 265, Sargent, F. P.; Gardy, E. M. (1976) Spin trapping of radicals formed during radiolysis of aqueous solutions. Direct electron spin resonance observations Can. J. Chem. 54,

14 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL 13. Rush, J. D.; Koppenol, W. H. (1987) The Reaction Between Ferrous Polyaminocarboxylate Complexes and Hydrogen Peroxide : An Investigation of the Reaction Intermediates by Stopped Flow Spectrophotometry. J. Inorg. Biochem. 29, Walling, C.; Goosen, A. (1973) Mechanism of the Ferric Ion Catalyzed Decomposition of Hydrogen Peroxide. Effect of Organic Substrates J. Am. Chem. Soc. 9S, Finkelstein, E.; Rosen, G. M.; Rauckman, E. J. (1980) Spin Trapping. Kinetics of the Reaction of Superoxide and Hydroxyl Radicals with Nitrones. J. Am. Chem. Soc. 102,