Evidence against the Generation of Free Hydroxyl Radicals from the Interaction of Copper,Zinc-Superoxide Dismutase and Hydrogen Peroxide
1999; Elsevier BV; Volume: 274; Issue: 49 Linguagem: Inglês
10.1074/jbc.274.49.34576
ISSN1083-351X
AutoresSornampillai Sankarapandi, Jay L. Zweíer,
Tópico(s)Metal-Catalyzed Oxygenation Mechanisms
ResumoPrior spin trapping studies reported that H2O2 is metabolized by copper,zinc-superoxide dismutase (SOD) to form ⋅OH that is released from the enzyme, serving as a source of oxidative injury. Although this mechanism has been invoked in a number of diseases, controversy remains regarding whether the hydroxylation of spin traps by SOD is truly derived from free ⋅OH or ⋅OH scavenged off the Cu2+ catalytic site. To distinguish whether⋅OH is released from the enzyme, a comprehensive EPR investigation of radical production and the kinetics of spin trapping was performed in the presence of a series of structurally different⋅OH scavengers including ethanol, formate, and azide. Although each of these have similar potency in scavenging ⋅OH as the spin trap 5,5-dimethyl-1-pyrroline-N-oxide and form secondary radical adducts, each exhibited very different potency in scavenging⋅OH from SOD. Ethanol was 1400-fold less potent than would be expected for reaction with free ⋅OH. The anionic scavenger formate, which readily accesses the active site, was still 10-fold less effective than would be predicted for free ⋅OH, whereas azide was almost 2-fold more potent than would be predicted. Analysis of initial rates of adduct formation indicated that these reactions did not involve free ⋅OH. EPR studies of the copper center demonstrated that while high H2O2concentrations induce release of Cu2+, the magnitude of spin adducts produced by free Cu2+ was negligible compared with that from intact SOD. Further studies with a series of peroxidase substrates demonstrated that characteristic radicals formed by peroxidases were also efficiently generated by H2O2 and SOD. Thus, SOD and H2O2 oxidize and hydroxylate substrates and spin traps through a peroxidase reaction with bound ⋅OH not release of ⋅OH from the enzyme. Prior spin trapping studies reported that H2O2 is metabolized by copper,zinc-superoxide dismutase (SOD) to form ⋅OH that is released from the enzyme, serving as a source of oxidative injury. Although this mechanism has been invoked in a number of diseases, controversy remains regarding whether the hydroxylation of spin traps by SOD is truly derived from free ⋅OH or ⋅OH scavenged off the Cu2+ catalytic site. To distinguish whether⋅OH is released from the enzyme, a comprehensive EPR investigation of radical production and the kinetics of spin trapping was performed in the presence of a series of structurally different⋅OH scavengers including ethanol, formate, and azide. Although each of these have similar potency in scavenging ⋅OH as the spin trap 5,5-dimethyl-1-pyrroline-N-oxide and form secondary radical adducts, each exhibited very different potency in scavenging⋅OH from SOD. Ethanol was 1400-fold less potent than would be expected for reaction with free ⋅OH. The anionic scavenger formate, which readily accesses the active site, was still 10-fold less effective than would be predicted for free ⋅OH, whereas azide was almost 2-fold more potent than would be predicted. Analysis of initial rates of adduct formation indicated that these reactions did not involve free ⋅OH. EPR studies of the copper center demonstrated that while high H2O2concentrations induce release of Cu2+, the magnitude of spin adducts produced by free Cu2+ was negligible compared with that from intact SOD. Further studies with a series of peroxidase substrates demonstrated that characteristic radicals formed by peroxidases were also efficiently generated by H2O2 and SOD. Thus, SOD and H2O2 oxidize and hydroxylate substrates and spin traps through a peroxidase reaction with bound ⋅OH not release of ⋅OH from the enzyme. superoxide dismutase copper,zinc-superoxide dismutase 5,5-dimethyl-1-pyrroline-N-oxide 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide familial amyotrophic lateral sclerosis 2,2,6,6-tetramethyl-1-piperidinyl-1-oxy 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid horseradish peroxidase Superoxide dismutases catalyze the dismutation of the toxic superoxide anion, O·̄2, to O2 and H2O2 at a rate close to the diffusion limit, over the entire range of pH from 5 to 10 (1McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar, 2Bannister J.V. Bannister W.H. Rotilio G. CRC Crit. Rev. Biochem. 1987; 22: 111-180Crossref PubMed Scopus (781) Google Scholar). Because of the important role of oxygen free radicals in normal biological function and disease, there has been increasing interest in the therapeutic use of SOD1 (3Fridovich I. Science. 1978; 201: 875-880Crossref PubMed Scopus (2746) Google Scholar, 4McCord J.M. Free Radical Biol. Med. 1988; 4: 9-14Crossref PubMed Scopus (272) Google Scholar, 5Zweier J.L. Flaherty J.T. Weisfelt M.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1404-1407Crossref PubMed Scopus (1016) Google Scholar). Humans contain three distinct isozymes which include a cytosolic, homodimeric copper,zinc-enzyme (CuZnSOD or SOD1), a manganese-containing mitochondrial SOD (or SOD2) and an extracellular form of CuZnSOD (SOD3) (2Bannister J.V. Bannister W.H. Rotilio G. CRC Crit. Rev. Biochem. 1987; 22: 111-180Crossref PubMed Scopus (781) Google Scholar). CuZnSOD, but not manganese-SOD, undergoes inactivation by its own product H2O2 at pH > 9 (6Symonyan M.A. Nalbandyan R.M. FEBS Lett. 1972; 28: 22-34Crossref PubMed Scopus (47) Google Scholar, 7Bray R.C. Cockle S.H. Fielden E.M. Roberts P.B. Rotilio G. Calabrese L. Biochem. J. 1974; 139: 43-48Crossref PubMed Scopus (332) Google Scholar, 8Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5294-5299Crossref Scopus (681) Google Scholar, 9Blech D.M. Borders Jr., C.L. Arch. Biochem. Biophys. 1983; 224: 579-586Crossref PubMed Scopus (92) Google Scholar, 10Borders Jr., C.L. Fridovich I. Arch. Biochem. Biophys. 1985; 241: 472-476Crossref PubMed Scopus (55) Google Scholar, 11Salo D.C. Pacifici R.E. Lin S.W. Giulivi C. Davies K.J.A. J. Biol. Chem. 1990; 265: 11919-11927Abstract Full Text PDF PubMed Google Scholar). The inactivation is understood to be caused by the oxidation of the active site histidine, His118 (7Bray R.C. Cockle S.H. Fielden E.M. Roberts P.B. Rotilio G. Calabrese L. Biochem. J. 1974; 139: 43-48Crossref PubMed Scopus (332) Google Scholar, 8Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5294-5299Crossref Scopus (681) Google Scholar, 9Blech D.M. Borders Jr., C.L. Arch. Biochem. Biophys. 1983; 224: 579-586Crossref PubMed Scopus (92) Google Scholar, 10Borders Jr., C.L. Fridovich I. Arch. Biochem. Biophys. 1985; 241: 472-476Crossref PubMed Scopus (55) Google Scholar, 11Salo D.C. Pacifici R.E. Lin S.W. Giulivi C. Davies K.J.A. J. Biol. Chem. 1990; 265: 11919-11927Abstract Full Text PDF PubMed Google Scholar). Hodgson and Fridovich (8Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5294-5299Crossref Scopus (681) Google Scholar,12Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5299-5303Crossref PubMed Scopus (215) Google Scholar) proposed a mechanism in which H2O2 first reduces the Cu(II) and then reacts with the Cu(I) to give a Cu2+-bound ⋅OH, which can attack an adjacent histidine and destroy the integrity of the catalytic site. Exogenous reductants such as xanthine, urate, formate, and azide protect the enzyme by scavenging the Cu2+-bound ⋅OH (8Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5294-5299Crossref Scopus (681) Google Scholar, 12Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5299-5303Crossref PubMed Scopus (215) Google Scholar). This single electron oxidation of substances by Cu2+-bound⋅OH is referred to as the peroxidase function of CuZnSOD because of its similarity to the one electron oxidation by horseradish peroxidase and H2O2 (8Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5294-5299Crossref Scopus (681) Google Scholar, 12Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5299-5303Crossref PubMed Scopus (215) Google Scholar).Yim et al. (13Yim M.B. Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5006-5010Crossref PubMed Scopus (315) Google Scholar, 14Yim M.B. Chock P.B. Stadtman E.R. J. Biol. Chem. 1993; 268: 4099-4105Abstract Full Text PDF PubMed Google Scholar) observed the generation of intense EPR signals of DMPO-OH while studying the interaction between SOD and H2O2 by EPR spin trapping. They hypothesized that the ⋅OH reactivity was not restricted to the active site of CuZnSOD but that free ⋅OH was released from the active site. Fridovich (15Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2690) Google Scholar), while arguing against this free ⋅OH generation, suggested that CuZnSOD could have acted as a peroxidase toward DMPO producing DMPO-OH, which appeared as though free ⋅OH reacted with DMPO. Other reports (15Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2690) Google Scholar, 16Sato K. Akaike T. Kohno M. Ando M. Maeda H. J. Biol. Chem. 1992; 267: 25371-25377Abstract Full Text PDF PubMed Google Scholar, 17Konningsberger J.C. van Asback B.S. van Faassen E. Wiegman L.J.J.M. van Hattum J. van Berge Henegouwen G.P. Marx J.J.M. Clin. Chim. Acta. 1994; 230: 51-61Crossref PubMed Scopus (23) Google Scholar) claimed that H2O2 damaged a portion of the enzyme releasing free copper, which catalyzed free ⋅OH generation similar to the iron-mediated Fenton reaction. It was also suggested that in the presence of excess of H2O2, reversal of the second step of the superoxide dismutation pathway becomes significant resulting in the formation of O·̄2 (18Hodgson E.K. Fridovich I. Biochem. Biophys. Res. Commun. 1973; 54: 270-274Crossref PubMed Scopus (177) Google Scholar). If O·̄2 were trapped by DMPO, the superoxide adduct DMPO-OOH may decompose into DMPO-OH. Although this controversy remains unresolved, this phenomenon has been already implicated in the gain-of-function of CuZnSOD mutants associated with the familial form of amyotrophic lateral sclerosis (FALS), a progressive degenerative disorder of motor neurons leading to paralysis (19Wiedau-Pazos M. Goto J.J. Rabizadeh S. Gralla E.B. Roe J.A. Lee M.K. Valentine J.S. Bredesen D.E. Science. 1996; 271: 515-518Crossref PubMed Scopus (659) Google Scholar, 20Yim M.B. Kang J.-H Yim H.-S Kwak H.-S Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5709-5714Crossref PubMed Scopus (428) Google Scholar, 21Yim H.S. Kang J.H. Chock P.B. Stadtman E.R. J. Biol. Chem. 1997; 272: 8861-8863Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 22Goto J.J. Gralla E.B. Valentine J.S. Cabelli D.E. J. Biol. Chem. 1998; 273: 30104-30109Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). However, recent reports (23Liochev S.I. Chen L.L. Hallewell R.A. Fridovich I. Arch. Biochem. Biophys. 1998; 352: 237-239Crossref PubMed Scopus (32) Google Scholar, 24Singh R.J. Karoui H. Gunther M.R. Beckman J.S. Mason R.P. Kalyanaraman B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6675-6680Crossref PubMed Scopus (109) Google Scholar) questioned the relationship between this phenomenon and FALS.Although all of these spin trapping studies were performed only in bicarbonate buffer, they failed to acknowledge the role of bicarbonate in this phenomenon. In our previous report (25Sankarapandi S. Zweier J.L. J. Biol. Chem. 1999; 274: 1226-1232Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) we clearly established that large magnitude DMPO-OH generation occurs only in the presence of HCO3− or structurally similar anions. We also presented evidence to show that HCO3− binds to the anion-binding site of the enzyme and facilitates anchoring of the neutral H2O2 at the active site that is surrounded by highly positively charged residues (25Sankarapandi S. Zweier J.L. J. Biol. Chem. 1999; 274: 1226-1232Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Otherwise, H2O2 with a pK a value of 11.9 cannot gain access into the positive charged active channel in large quantities, and it would be impossible to explain the rapid and intense generation of DMPO-OH (25Sankarapandi S. Zweier J.L. J. Biol. Chem. 1999; 274: 1226-1232Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar).In the present study, we characterize the CuZnSOD-mediated reaction of hydrogen peroxide with spin traps to understand which of the two mechanisms, peroxidation of DMPO or free ⋅OH generation, primarily result in the signal of DMPO-OH. Our results provide several lines of evidence for a direct reaction of DMPO with a copper-bound oxidant, most likely ⋅OH, and demonstrate that the function of SOD is similar to that of a peroxidase.RESULTSCuZnSOD (2 μm) dissolved in bicarbonate buffer at pH 7.4 gave rise to a large EPR signal when treated with H2O2 (1 mm) in the presence of DMPO (50 mm) (Fig. 1 A). The quartet signal observed with a typical intensity ratio (1:2:2:1) and hyperfine couplings (a H =a N = 14.9 G) corresponds to DMPO-OH (29Zweier J.L. J. Biol. Chem. 1988; 263: 1353-1357Abstract Full Text PDF PubMed Google Scholar, 30Zweier J.L. Broderick R. Kuppusamy P. Thompsun-Gorman S. Lutty G.A. J. Biol. Chem. 1994; 269: 24156-24162Abstract Full Text PDF PubMed Google Scholar). Because higher concentrations of H2O2 (30 mm) were used in many prior reports, we also recorded the signals at these concentrations (Fig. 1 B). Several reports attributed the signals to free ⋅OH generation catalyzed by Cu2+ released from the enzyme because of the usage of high doses of H2O2 (15Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2690) Google Scholar, 16Sato K. Akaike T. Kohno M. Ando M. Maeda H. J. Biol. Chem. 1992; 267: 25371-25377Abstract Full Text PDF PubMed Google Scholar, 17Konningsberger J.C. van Asback B.S. van Faassen E. Wiegman L.J.J.M. van Hattum J. van Berge Henegouwen G.P. Marx J.J.M. Clin. Chim. Acta. 1994; 230: 51-61Crossref PubMed Scopus (23) Google Scholar). Hence, we compared the signals obtained from the enzyme with that derived from an equivalent amount of free Cu2+ or Fe2+ ions. The corresponding EPR spectra (Fig. 1, C and D) indicate that signals obtained from these ions were negligible when compared with that obtained with CuZnSOD. Thus, free Cu2+released from CuZnSOD contributes very little, if any, to the large signal of DMPO-OH. In the presence of 2% ethanol, no detectable hydroxyethyl radical adduct, DMPO-Et, was observed (spectrum not shown).The magnitude of DMPO-OH signal was dependent on the concentration of H2O2, and this EPR signal was clearly detected at [H2O2] as low as 30 μm (Fig.2). At low H2O2concentrations signal generation persisted until all the H2O2 was consumed, whereas at H2O2 concentrations higher than 1 mm, there was an initial burst in the signal followed by a steep decline with time (Fig. 2). Because this could be due to damage to the enzyme, we analyzed the structural changes in the active site at high doses of H2O2. EPR spectra of the active site Cu2+ at 77 K was observed from 0.5 mm SOD with different H2O2 concentrations (Fig.3). The reaction between the two was allowed to proceed for different times before freezing the sample in liquid nitrogen. As seen from the EPR spectra, H2O2 reduces Cu2+ to diamagnetic Cu+, resulting in the reduction in the EPR intensity. In addition, H2O2 also causes changes in the Cu2+ coordination geometry from the rhombic symmetry of the native enzyme into axial symmetry resulting in an increase in the anisotropic copper hyperfine splitting, A ∥from 135 to 180 G (31Rotilio G. Morpurgo L. Calabrese L. Mondovi B. Biochim. Biophys. Acta. 1973; 302: 229-235Crossref PubMed Scopus (44) Google Scholar, 32Rotilio G. Morpurgo L. Giovagnoli C. Calabrese L. Mondovi B. Biochemistry. 1972; 11: 2187-2192Crossref PubMed Scopus (139) Google Scholar). The extent of reduction in the intensity and increase in A ∥ depends on the amount of H2O2 and reaction time. Reduction in the intensity was partially reversible over time because of reoxidation, whereas the change in A∥ was not, suggesting that millimolar quantities of H2O2 can cause irreversible damage to the enzyme resulting in altered copper coordination. This was even more marked in experiments with lower micromolar amounts of the enzyme.Figure 2Time courses of DMPO-OH generation by CuZnSOD at different H2O2 concentrations. Spectra were recorded with CuZnSOD (1.25 μm) and varying amounts of H2O2 under conditions described in the legend to Fig. 1. The concentration of the observed DMPO-OH adduct was quantitated by computer simulation and comparison with the signal from a standard TEMPO solution (10 μm), which was measured under identical conditions.View Large Image Figure ViewerDownload (PPT)Figure 3Effect of high concentrations of H2O2 on the EPR spectra of the active site Cu2+ of SOD . Spectra observed from 0.5 mmCuZnSOD in phosphate buffer (50 mm, pH 7.0) at 77 K. Spectra were recorded with different H2O2concentrations at different time intervals of mixing the reactants before freezing in liquid nitrogen as indicated below. A, no H2O2; B, 30 mm, 2 min;C, 30 mm, 10 min; D, 30 mm, 30 min; E, 100 mm, 2 min;F, 100 mm, 10 min; G, 300 mm, 10 min; Spectra were recorded at a microwave frequency of 9.35 GHz, a microwave power of 20 mW, and a modulation amplitude, 4 G.View Large Image Figure ViewerDownload (PPT)The EPR signal of DMPO-OH can arise from direct trapping of ⋅OH or the decomposition of the adduct from O·̄2, DMPO-OOH, whose half-life is less than 1 min (30Zweier J.L. Broderick R. Kuppusamy P. Thompsun-Gorman S. Lutty G.A. J. Biol. Chem. 1994; 269: 24156-24162Abstract Full Text PDF PubMed Google Scholar). CuZnSOD, by virtue of its primary function as O·̄2 scavenger, may not leave any O·̄2 for trapping by DMPO. However, if H2O2, the main product of dismutation is available in excess amounts, it is suggested to reverse the second step in the dismutation pathway thereby forming O·̄2 (18Hodgson E.K. Fridovich I. Biochem. Biophys. Res. Commun. 1973; 54: 270-274Crossref PubMed Scopus (177) Google Scholar). To examine this possibility, we used DEPMPO, the structural analog of DMPO whose O·̄2 adduct, DEPMPO-OOH (half-life > 14 min), is 15-fold more stable than DMPO-OOH (33Roubaud V. Sankarapandi S. Kuppusamy P. Tordo P. Zweier J.L. Anal. Biochem. 1997; 247: 404-411Crossref PubMed Scopus (159) Google Scholar). Only a signal of DEPMPO-OH was observed from CuZnSOD and H2O2 in bicarbonate buffer, and addition of 2% ethanol did not generate any detectable amounts of the hydroxyethyl adduct DEPMPO-Et (Fig. 4, Aand B), similar to the results obtained with DMPO.Figure 4Identification of the radical using DEPMPO . Spectra observed from CuZnSOD (1.25 μm), H2O2 (1 mm), and DEPMPO (20 mm) in 23.5 mm NaHCO3 buffer (pH 7.4) balanced with 5% CO2 and 95% N2containing no ethanol (A) or 2% ethanol (B, 400 mm).View Large Image Figure ViewerDownload (PPT)If the signal of DMPO-OH or DEPMPO-OH arose from the trapping of free⋅OH, scavengers such as ethanol, formate, and azide would yield corresponding spin adducts derived from the scavenger molecules (30Zweier J.L. Broderick R. Kuppusamy P. Thompsun-Gorman S. Lutty G.A. J. Biol. Chem. 1994; 269: 24156-24162Abstract Full Text PDF PubMed Google Scholar,34Castelhano A.L. Perkins M.J. Griller D. Can J. Chem. 1983; 61: 298-299Crossref Google Scholar, 35Morehouse K.M. Mason R.P. J. Biol. Chem. 1988; 263: 1204-1211Abstract Full Text PDF PubMed Google Scholar, 36Kremers W. Singh A. Can J. Chem. 1980; 58: 1592-1595Crossref Google Scholar). Failure to obtain the ethanol adducts was reasoned by Yimet al. (13Yim M.B. Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5006-5010Crossref PubMed Scopus (315) Google Scholar) to be due to inability of the apolar ethanol molecules to enter into the highly positively charged channel of the SOD active site, whereas DMPO can gain access inside the channel and form the DMPO-OH adduct. On the other hand, Fridovich (15Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2690) Google Scholar) proposed this inertness of alcohol as evidence against the generation of free⋅OH and reaffirmed his previous observation that alcohols did not protect the enzyme against H2O2inactivation. To distinguish between these two proposed mechanisms, we performed a series of experiments evaluating radical generation from EtOH and other ⋅OH scavengers. Our experiments were based on the premise that ethanol is not totally nonpolar when compared with DMPO and that if inaccessibility of the active site relative to DMPO was the only deficiency of ethanol, decreasing [DMPO] and increasing [EtOH] could overcome this problem. Experiments performed with 2 mm DMPO and 5% ethanol (1000 mm) yielded a small signal of DMPO-Et (hyperfine splittings,a N = 15.8 G, a H = 22.8 G) in addition to the large 1:2:2:1 quartet signal of DMPO-OH. This DMPO-Et signal further increased when the amount of ethanol was increased up to 20% (Fig.5 A). However, the increase in [DMPO-Et]/[DMPO-OH], calculated by computer simulation and quantitation of the corresponding EPR signals, was not commensurate with the large ratio of [EtOH]/[DMPO] (Fig. 5 B). Although the formation and decay kinetics of the two adducts are reported to be similar, a ratio of 1,400 was required to reach a DMPO-Et/DMPO-OH ratio of 1 (34Castelhano A.L. Perkins M.J. Griller D. Can J. Chem. 1983; 61: 298-299Crossref Google Scholar, 35Morehouse K.M. Mason R.P. J. Biol. Chem. 1988; 263: 1204-1211Abstract Full Text PDF PubMed Google Scholar, 37Lown J.W. Chen H.H. Can J. Chem. 1981; 59: 390-395Crossref Scopus (72) Google Scholar). In contrast, with a ⋅OH generating system the ratio of these adducts should be similar to the ratio of [EtOH]/[DMPO]. Experiments performed with varying ratios and amounts of DMPO and EtOH showed similar kinetics, indicating that this nonaqueous solvent change did not disrupt the catalytic function of the enzyme (Fig. 5 C). The 10-fold decrease in the overall concentration of the adducts was due to the decrease in the availability of DMPO. These results suggest that ethanol can reach the active site but that it is much less effective in scavenging the⋅OH species formed than would be expected for its spontaneous reaction with free ⋅OH.Figure 5Effect of ethanol concentration on the generation of DMPO-Et. A, spectra observed from CuZnSOD (1.25 μm), H2O2 (1 mm), and DMPO (2 mm) in 23.5 mmNaHCO3 buffer (pH 7.4) balanced with 5% CO2and 95% N2 containing 5, 10, 15, and 20% ethanol. Spectra were recorded with parameters as described in the legend to Fig. 1.B, EPR signals from DMPO-OH and DMPO-Et were quantitated by computer simulation and comparison with the signal from a standard TEMPO solution (10 μm), which was measured under identical conditions. C, concentration of the DMPO-OH spectrum was measured as a function of time with either 50 or 2 mm DMPO.View Large Image Figure ViewerDownload (PPT)The efficiency of trapping the ⋅OH species formed by SOD was further tested with other ⋅OH scavenger molecules. Formate and azide, both anions that readily access the active site of SOD, normally react with free ⋅OH to form spin adducts with characteristic EPR spectra, DMPO-COO·̄ (a N = 15.9 G anda H = 19.3), and DMPO-N3·(a N = a H = 14.7 G anda NN3 = 3.2 G), respectively (34Castelhano A.L. Perkins M.J. Griller D. Can J. Chem. 1983; 61: 298-299Crossref Google Scholar, 35Morehouse K.M. Mason R.P. J. Biol. Chem. 1988; 263: 1204-1211Abstract Full Text PDF PubMed Google Scholar, 36Kremers W. Singh A. Can J. Chem. 1980; 58: 1592-1595Crossref Google Scholar, 37Lown J.W. Chen H.H. Can J. Chem. 1981; 59: 390-395Crossref Scopus (72) Google Scholar). Formate (20 mm) and DMPO (20 mm) gave rise to a mixture of two adducts, DMPO-OH and DMPO-COO3·, the former being predominant (Fig. 6 B). Computer simulation and quantitation of the EPR signals showed that DMPO-COO·̄ constituted only about 15% of the total signal (Fig.7 B). If the DMPO-OH signals obtained in the absence of scavengers (Figs. 6 A and7 A) were from free ⋅OH, we should have obtained 50% DMPO-COO·̄ from a mixture of equimolar concentrations of DMPO and formate, because the formation and decay kinetics of the two adducts are almost identical (34Castelhano A.L. Perkins M.J. Griller D. Can J. Chem. 1983; 61: 298-299Crossref Google Scholar, 35Morehouse K.M. Mason R.P. J. Biol. Chem. 1988; 263: 1204-1211Abstract Full Text PDF PubMed Google Scholar, 36Kremers W. Singh A. Can J. Chem. 1980; 58: 1592-1595Crossref Google Scholar). Even when 200 mm formate was used, the concentration of DMPO-COO·̄ grew only to 40% of the total spin adduct concentration (Figs. 6 C and7 C). Because the anion formate is known to readily access the catalytic site of SOD, this further indicates that most of the DMPO-OH obtained in the absence of scavengers emanate from a mechanism other than the reaction of free ⋅OH with DMPO (35Morehouse K.M. Mason R.P. J. Biol. Chem. 1988; 263: 1204-1211Abstract Full Text PDF PubMed Google Scholar, 37Lown J.W. Chen H.H. Can J. Chem. 1981; 59: 390-395Crossref Scopus (72) Google Scholar). Furthermore, the overall spin adduct concentration was enhanced by 30–40% at 20 mm formate and by 350–400% at 200 mm formate (Fig. 7, A–C), reflecting the increase in the total concentration of substrates that can react with the bound ⋅OH. Azide has been reported to completely protect the enzyme against H2O2 mediated damage (8Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5294-5299Crossref Scopus (681) Google Scholar). With equal 20 mm concentrations of azide and DMPO, which would yield equal concentrations of N3· and ⋅OH adducts if derived from free ⋅OH, in contrast, >80% of the observed adducts were azide-derived, indicating that the bound⋅OH was efficiently scavenged by azide in accordance with its known protection of the enzyme (Figs. 6 D and 7 D). It is also notable that azide caused a small reduction of the total signal, by 30–35% at 20 mm concentrations, but a large (>90%) decrease at 200 mm (Fig. 7, D andE). This is consistent with the inhibition of SOD that occurs at high azide concentrations because of its direct binding to the copper (38Fee J.A. Gaber B.P. J. Biol. Chem. 1972; 247: 60-65Abstract Full Text PDF PubMed Google Scholar, 39Djinovic-Carugo K. Polticelli F. Desideri A. Rotilio G. Wilson K.S. Bolognesi M. J. Mol. Biol. 1994; 240: 179-183Crossref PubMed Scopus (23) Google Scholar).Figure 6EPR spectra in the presence of ⋅OH scavengers formate and azide. Spectra observed from CuZnSOD (1.25 μm), DMPO (20 mm), and H2O2 (1 mm) in 23.5 mmNaHCO3 buffer (pH 7.4) balanced with 5% CO2and 95% N2 containing the following. A, no anions (control); B, formate (20 mm);C, formate (200 mm); D, azide (20 mm); E, azide (200 mm). Spectra were recorded with parameters as described in the legend to Fig. 1. The observed spectrum was scaled down by three times in the case ofC.View Large Image Figure ViewerDownload (PPT)Figure 7Effect of formate and azide on the adduct generation. EPR signals of the DMPO-OH and DMPO-R adducts were quantitated by computer simulation of the spectra observed from CuZnSOD (1.25 μm), DMPO (20 mm), and H2O2 (1 mm) in the absence and presence of formate and azide. Quantitation was performed by comparison of the simulated spectra with the signal from a standard TEMPO solution (10 μm), which was measured under identical conditions.A, no anions (control); B, formate (20 mm); C, formate (200 mm);D, azide (20 mm); E, azide (200 mm). Conditions were as described for Fig. 6.View Large Image Figure ViewerDownload (PPT)Kinetic criteria have been developed to distinguish between the reaction of free ⋅OH with DMPO and species that merely hydroxylate DMPO (34Castelhano A.L. Perkins M.J. Griller D. Can J. Chem. 1983; 61: 298-299Crossref Google Scholar, 35Morehouse K.M. Mason R.P. J. Biol. Chem. 1988; 263: 1204-1211Abstract Full Text PDF PubMed Google Scholar, 37Lown J.W. Chen H.H. Can J. Chem. 1981; 59: 390-395Crossref Scopus (72) Google Scholar). To further test whether the observed DMPO-OH formation is derived from free ⋅OH, a study of the initial rates of spin trapping was performed along the lines of these prior reports. In this kinetic approach, the initial rate of spin trapping rather than steady state adduct concentrations are measured because the latter depend not only on the rate of formation but also upon their rate of decay (34Castelhano A.L. Perkins M.J. Griller D. Can J. Chem. 1983; 61: 298-299Crossref Google Scholar, 35Morehouse K.M. Mason R.P. J. Biol. Chem. 1988; 263: 1204-1211Abstract Full Text PDF PubMed Google Scholar). The competition between DMPO and the scavengers for the ⋅OH enables differentiation of the two types of ⋅OH species and the competitive equations are presented below. ⋅OH+DMPO→k1DMPO−⋅OHEquation 1 ⋅OH+HCOO−→k2⋅COO−+H2OEquation 2 ⋅COO−+DMPO→k3DMPO−⋅COO−Equation 3 ⋅OH+EtOH→k4⋅Et+H2OEquation 4 ⋅Et+DMPO→k5DMPO−⋅EtEquation 5 The ratio k 1/k 2 for a system where the ⋅OH is derived from free ⋅OH is expected to be different from that involving a different hydroxylating species (34Castelhano A.L. Perkins M.J. Griller D. Can J. Chem. 1983; 61: 298-299Crossref Google Scholar, 35Morehouse K.M. Mason R.P. J. Biol. Chem. 1988; 263: 1204-1211Abstract Full Text PDF PubMed Google Scholar). We monitored the spin trapping kinetics for the different spin-adducts (DMPO-OH, DMPO-Et and DMPO-COO·̄) by EPR measurements from samples containing 1.25 μm SOD, 10 mm DMPO, 1 mm H2O2, and the respective scavengers in bicarbonate buffer (pH 7.4
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