The oxidation of tiron by superoxide anion. Kinetics of the reaction in aqueous solution and in chloroplasts

The rate of reaction between superoxide anion (O^¯.₂) and 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron) was measured with pulse radiolysis-generated O^¯.₂. A kinetic spectrophotometric method utilizing competition betweenp-benzoquinoneand tiron for O^¯.₂ was employed. In this system, the known rate of reduction ofp-benzoquinonewas compared with the rate of oxidation of tiron to the semiquinone. From the concentration dependence of the rate of tiron oxidation, the absolute second order rate constant for the reaction was determined to be 5 · 10⁸ M^?·s^?1. Ascorbat reduced O^¯.₂ to hydrogen peroxide with a rate constant of 10⁸ M^?1 · s^?1 as determined by the same method. The tiron semiquinone may be used as an indicator free radical for the formation of superoxide anion in biological systems because of the rapid rate of oxidation of the catechol by O^¯.₂ compared to the rate of O^¯.₂ formation in most enzymatic systems.Tiron oxidation was used to follow the formation of superoxide anion in swollen chloroplasts. The chloroplasts photochemically reduced molecular oxygen which was further reduced to hydrogen peroxide by tiron. Tiron oxidation specifically required O^¯.₂ since O₂ was consumed in the reaction and tiron did not reduce the P₇₀₀ cation radical or other components of Photosystem I under anaerobic conditions.     

The tiron free radical as a sensitive indicator of chloroplastic photoautoxidation.

Wheat chloroplasts photochemically reduced molecular oxygen, as a Hill oxidant in the Mehler reaction, to superoxide anion which then oxidized added 1,2-dihydroxybenzene-3,5-disulfonate to its semiquinone, a comparatively stable free radical at pH 7. The last mentioned reaction was rapid in aqueous solution, but the rate of formation of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone by the chloroplast system was calculated as T1 of 0.6 s. The Mehler reaction, or more specifically the univalent reduction of oxygen by Photosystem I, was rate-limiting so that the 1,2-dihydroxybenzene-3,5-disulfonate seniquinone was a useful spin probe for superoxide anion production at room temperature. The ESR signal of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was proportional to its steady state concentration and decayed in the dark with a T1/2 of 5-6 s. This oxygen-dependent signal was enhanced by mediation of chloroplastic oxygen reduction through methyl viologen. The superoxide anion scavengers ascorbate and L-epinephrine competitively obscured 1,2-dihydroxybenzene-3,5-disulfonate semiquinone formation, butadded superoxide dismutase was not as effective in this role. Partial inhibition by superoxide dismutase was achieved only by preincubation of Photosystem I enriched particles with ten times the endogenous concentration of superoxide dismutase. This and the persistence of a small amount of a 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) oxidizing species in the dark supports the concept of Tiron accessibility but not the superoxide dismutase accessibility of superoxide anion bound in its formative enzyme complex. Benzoquinone and naphthoquinone disulfonate also reacted with superoxide anion, and supported both the Hill reaction and the Mehler reaction as final oxidants of both water and superoxide anion.     

Kinetic analysis and mechanistic aspects of autoxidation of catechins

A peroxidase-based bioelectrochemical sensor of hydrogen peroxide (H(2)O(2)) and a Clark-type oxygen electrode were applied to continuous monitoring and kinetic analysis of the autoxidation of catechins. Four major catechins in green tea, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, and (-)-epigallocatechin gallate, were used as model compounds. It was found that dioxygen (O(2)) is quantitatively reduced to H(2)O(2). The initial rate of autoxidation is suppressed by superoxide dismutase and H(+), but is independent of buffer capacity. Based on these results, a mechanism of autoxidation is proposed; the initial step is the one-electron oxidation of the B ring of catechins by O(2) to generate a superoxide anion (O(2)(*-)) and a semiquinone radical, as supported in part by electron spin resonance measurements. O(2)(*-) works as a stronger one-electron oxidant than O(2) against catechins and is reduced to H(2)O(2). The semiquinone radical is more susceptible to oxidation with O(2) than fully reduced catechins. The autoxidation rate increases with pH. This behavior can be interpreted in terms of the increase in the stability of O(2)(*-) and the semiquinone radical with increasing pH, rather than the acid dissociation of phenolic groups. Cupric ion enhances autoxidation; most probably it functions as a catalyst of the initial oxidation step of catechins. The product cuprous ion can trigger a Fenton reaction to generate hydroxyl radical. On the other hand, borate ion suppresses autoxidation drastically, due to the strong complex formation with catechins. The biological significance of autoxidation and its effectors are also discussed.     

The Tiron free radical as a sensitive indicator of chloroplastic photoautoxidation

Wheat chloroplasts photochemically reduced molecular oxygen, as a Hill oxidant in the Mehler reaction, to superoxide anion which then oxidized added 1,2-dihydroxybenzene-3,5-disulfonate to its semiquinone, a comparatively stable free radical at pH 7. The last mentioned reaction was rapid in aqueous solution, but the rate of formation of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone by the chloroplast system was calculated as a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.6 s. The Mehler reaction, or more specifically the univalent reduction of oxygen by Photosystem I, was rate-limiting so that the 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was a useful spin probe for superoxide anion production at room temperature. The ESR signal of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was proportional to its steady state concentration and decayed in the dark with a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 5–6 s. This oxygen-dependent signal was enhanced by mediation of chloroplastic oxygen reduction through methyl viologen. The superoxide anion scavengers ascorbate and l-epinephrine competitively obscured 1,2-dihydroxybenzene-3,5-disulfonate semiquinone formation, but added superoxide dismutase was not as effective in this role. Partial inhibition by superoxide dismutase was achieved only by preincubation of Photosystem I enriched particles with ten times the endogenous concentration of superoxide dismutase. This and the persistence of a small amount of a 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) oxidizing species in the dark supports the concept of Tiron accessibility but not the superoxide dismutase accessibility of superoxide anion bound in its formative enzyme complex. Benzoquinone and naphthoquinone disulfonate also reacted with superoxide anion, and supported both the Hill reaction and the Mehler reaction as final oxidants of both water and superoxide anion.     

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) has been studied. The ability of DOPA to react with O2*- has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by O2*- was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by O2*- was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by O2*- using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and O2*- being equal to (3.4+/-0.6)x10(5) M(-1) s(-1).The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with O2*-.     

Pulse-radiolytic investigations of catechols and catecholamines. II. Reactions of Tiron with oxygen radical species.

Transient spectra and kinetic data of Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) are reported, obtained after pulse-radiolytic oxidation by hydroxyl radicals (.OH), superoxide anions (O-2) or a combination of both oxygen radicals. The rate constant with .OH radicals was determined at 1.0.10(9) M-1.s-1. Contrary to a previous report (Greenstock, C.L. and Miller, R.W. (1975) Biochim. Biophys. Acta 396, 11--16), the rate constant with O-2 of 1.0.10(7) M-1.s-1 is lower by one order of magnitude; also the semiquinone absorbs at 300 nm rather than at 400 nm. The ratio of the rate constants with .OH and O-2 of 100 again demonstrates that any oxidation reaction by the latter radical is unspecific due to the more efficient reaction of .OH radicals, leading to the same products with catechol compounds.     

Generation of nitro radical anions of some 5-nitrofurans, 2- and 5-nitroimidazoles by norepinephrine, dopamine, and serotonin. A possible mechanism for neurotoxicity caused by nitroheterocyclic drugs

Catecholamine neurotransmitters such as norepinephrine, dopamine, and related catecholamine derivatives reduce nitroheterocyclic drugs such as nitrofurantoin, nifurtimox, nifuroxime, nitrofurazone, misonidazole, and metronidazole in slightly alkaline solutions. Drugs which contain 5-nitrofurans are reduced at lower pH than drugs which contain 2- and 5-nitroimidazoles. 5-Nitroimidazole derivatives such as metronidazole and ronidazole are known to be more difficult to reduce than 2-nitroimidazole derivatives, due to their lower redox potential. Catecholamines, when reducing nitro drugs, undergo concomitant oxidation to form semiquinone radicals. Both semiquinone radicals and nitro anion radicals formed in a reaction of nitro drug and catecholamine derivative were detected by electron spin resonance spectroscopy. Oxygen consumption studies in solutions containing nitro drug and catecholamine derivative showed that nitro anion radicals formed under aerobic conditions reduce oxygen to form the superoxide radical and hydrogen peroxide. Quinones formed in the reaction of catecholamine and nitro drug were detected by optical spectroscopy. Biosynthetic precursors and some metabolic products of catecholamines were also used in these studies, and they all exhibited reactions similar to catecholamines. Bovine chromaffin granules which synthesize and store catecholamines produced the nitrofurantoin anion radical when intact granules were treated with nitrofurantoin. These radicals formed inside the granules were observed by ESR spectroscopy. The formation of nitrofurantoin radical, semiquinone radicals of catecholamines, and oxygen-derived radicals by chromaffin granules is proposed to cause damage to adrenal medulla, and this process may lead to neurotoxicity.     

Properties of the radical intermediate obtained on oxidation of 2',7'-dichlorodihydrofluorescein, a probe for oxidative stress

Reduced "leuco" dyes such as dichlorodihydrofluorescein (DCFH(2)) are widely used as profluorescent probes for oxidative stress, although they require a catalyst to be oxidized by hydrogen peroxide and react indiscriminately with oxidizing radicals and the fluorescent product (DCF) is a potential photosensitizer of superoxide generation. In this study, key properties of the radical intermediate in oxidation ("semiquinone," DCFH(.-)/DCF(.)(-)) were measured, to help understand the reactions that can occur in biological systems. The intermediate was generated by oxidizing DCFH(2) or reducing DCF by radiolytically generated radicals and monitoring the reactions using kinetic spectrophotometry. The semiquinone showed pH-sensitive absorption spectral changes, decay kinetics (both in the absence and in the presence of oxygen), and reduction potential, all corresponding to prototropic dissociations with pK(a)'s of approximately 7.1 and 9.0. DCFH(2) has pK(a)'s in a similar region (8-9) and hence pH variations are potentially important in the use of this probe. The rate constant for reaction of the semiquinone with oxygen at pH 7.4 is 5.3 x 10(8) M(-1) s(-1): this reaction, rather than disproportionation of DCFH(.-)/DCF(.)(-), generates DCF in biological systems, concomitantly forming superoxide and hence H(2)O(2) to cycle the catalyst. The midpoint reduction potential of the couple DCF,H(+)/DCFH() is approximately -0.75 V vs. NHE at pH 7.4; DCF is unlikely to be reduced rapidly by common flavoprotein reductases.     

Kinetic studies on spin trapping of superoxide and hydroxyl radicals generated in NADPH-cytochrome P-450 reductase-paraquat systems. Effect of iron chelates

Electron spin resonance (ESR) studies on spin trapping of superoxide and hydroxyl radicals by 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were performed in NADPH-cytochrome P-450 reductase-paraquat systems at pH 7.4. Spin adduct concentrations were determined by comparing ESR spectra of the adducts with the ESR spectrum of a stable radical solution. Kinetic analysis in the presence of 100 microM desferrioxamine B (deferoxamine) showed that: 1) the oxidation of 1 mol of NADPH produces 2 mol of superoxide ions, all of which can be trapped by DMPO when extrapolated to infinite concentration; 2) the rate constant for the reaction of superoxide with DMPO was 1.2 M-1 s-1; 3) the superoxide spin adduct of DMPO (DMPO-OOH) decays with a half-life of 66 s and the maximum level of DMPO-OOH formed can be calculated by a simple steady state equation; and 4) 2.8% or less of the DMPO-OOH decay occurs through a reaction producing hydroxyl radicals. In the presence of 100 microM EDTA, 5 microM Fe(III) ions nearly completely inhibited the formation of the hydroxyl radical adduct of DMPO (DMPO-OH) as well as the formation of DMPO-OOH and, when 100 microM hydrogen peroxide was present, produced DMPO-OH exclusively. Fe(III)-EDTA is reduced by superoxide and the competition of superoxide and hydrogen peroxide in the reaction with Fe(II)-EDTA seems to be reflected in the amounts of DMPO-OOH and DMPO-OH detected. These effects of EDTA can be explained from known kinetic data including a rate constant of 6 x 10(4) M-1 s-1 for reduction of DMPO-OOH by Fe(II)-EDTA. The effect of diethylenetriamine pentaacetic acid (DETAPAC) on the formation of DMPO-OOH and DMPO-OH was between deferoxamine and EDTA, and about the same as that of endogenous chelator (phosphate).     

Effects of superoxide dismutase on the autoxidation of 1,4-hydroquinone

During autoxidation of 1,4-hydroquinone (H2Q, less than 1 mM) at pH 7.4 and 37 degrees C, stoichiometric amounts of 1,4-benzoquinone (Q) and hydrogen peroxide were formed during the initial reaction. The reaction kinetics showed a significant induction period which was abolished by minute amounts of Q. Hydrogen peroxide and catalase were without effect on the autoxidation process. Transition metals apparently were not involved, since chelators like EDTA, DETAPAC, and desferrioxamine or FeSO4 had no influence on the autoxidation kinetics. Superoxide dismutase (SOD) did not abolish the induction period but dramatically enhanced the autoxidation rate by more than two orders of magnitude. The stimulatory effect was first-order in SOD concentration but showed saturation kinetics. The dependence of Q and hydrogen peroxide formation rates on H2Q concentration shows a biphasic behaviour: dependence on the square at low H2Q, but on the square root at high H2Q concentration. As revealed by calculatory simulations the results can be adequately described by the known reaction rate constants. The reaction starts with the comproportionation of H2Q and Q to yield two semiquinone molecules which autoxidize to give two superoxide radicals and two molecules of Q which enter into a new cycle of comproportionation. Because of unfavourable equilibria the autocatalytic reaction soon comes to steady state, and the further reaction is governed by the rate of superoxide removal. At excess SOD, the comproportionation reaction is rate-limiting, thus explaining the saturation effects of SOD. The experiments do not allow a decision between the two functions of SOD; the conventional action as a superoxide:superoxide oxidoreductase or as a semiquinone:superoxide oxidoreductase. In the latter reaction SOD is thought to be reduced by semiquinone with Q formation. In the second step the reduced enzyme would be re-oxidized by a superoxide radical which is formed during autoxidation of the second semiquinone molecule generated in the comproportionation reaction. From thermodynamic considerations, the latter function of SOD appears to be plausible.     

Lipid peroxidation and the generation of free radicals, superoxide anion, and hydrogen peroxide in beta-lapachone-treated Trypanosoma cruzi epimastigotes.

β-Lapachone, an antimicrobial agent, was reduced by Trypanosoma cruzi epimastigotes to a semiquinone radical. It markedly increased the generation of superoxide anion and hydrogen peroxide in intact cells. Using NADH as electron donor, β-lapachone also increased significantly the rate of H₂O₂ generation in epimastigote homogenates. Incubation of epimastigotes with β-lapachone stimulated lipid peroxidation.     

Catalysis of the Haber-Weiss reaction by iron-diethylenetriaminepentaacetate.

To help settle controversy as to whether the chelating agent diethylenetriaminepentaacetate (DTPA) supports or prevents hydroxyl radical production by superoxide/hydrogen peroxide systems, we have reinvestigated the question by spectroscopic, kinetic, and thermodynamic analyses. Potassium superoxide in DMSO was found to reduce Fe(III)DTPA. The rate constant for autoxidation of Fe(II)DTPA was found (by electron paramagnetic resonance spectroscopy) to be 3.10 M-1 s-1, which leads to a predicted rate constant for reduction of Fe(III)DTPA by superoxide of 5.9 x 10(3) M-1 s-1 in aqueous solution. This reduction is a necessary requirement for catalytic production of hydroxyl radicals via the Fenton reaction and is confirmed by spin-trapping experiments using DMPO. In the presence of Fe(III)DTPA, the xanthine/xanthine oxidase system generates hydroxyl radicals. The reaction is inhibited by both superoxide dismutase and catalase (indicating that both superoxide and hydrogen peroxide are required for generation of HO.). The generation of hydroxyl radicals (rather than oxidation side-products of DMPO and DMPO adducts) is attested to by the trapping of alpha-hydroxethyl radicals in the presence of 9% ethanol. Generation of HO. upon reaction of H2O2 with Fe(II)DTPA (the Fenton reaction) can be inhibited by catalase, but not superoxide dismutase. The data strongly indicate that iron-DTPA can catalyze the Haber-Weiss reaction.     

Enzymic formation of glycolate in Chromatium. Role of superoxide radical in a transketolase-type mechanism.

Chromatophores prepared from Chromatium exhibit a light-dependent O2 uptake in the presence of reduced 2,6-dichlorophenolindophenol, the maximum rate observed being 10.8 micronmol (mg of Bchl)-1 h-1 (air-saturated condition). As it was found that the uptake of O2 was markedly inhibited by superoxide dismutase, it is suggested that molecular oxygen is subject to light-dependent monovalent reduction, resulting in the formation of the superoxide anion radical (O2-). By coupling baker's yeast transketolase with illuminated chromatophore preparations, it was demonstrated that [U-14C]-fructose 6-phosphate (6-P) is oxidatively split to produce glycolate, and that the reaction was markedly inhibited by superoxide dismutase and less strongly by catalase. A coupled system containing yeast transketolase and xanthine plus xanthine oxidase showed a similar oxidative formation of glycolate from [U-14C] fructose 6-P. It is thus suggested that photogenerated O2- serves as an oxidant in the transketolase-catalyzed formation of glycolate from the alpha, beta-dihydroxyethyl (C2) thiamine pyrophosphate complex, whereas H2O2 is not an efficient oxidant. The rate of glycolate formation in vitro utilizing O2- does not account for the in vivo rate of glycolate photosynthesis in Chromatium cells exposed to an O2 atmosphere (10 micronmol (mg of Bchl)-1 h-1). However, the enhancement of glycolate formation by the autoxidizable electron acceptor methyl viologen in Chromatium cells in O2, as well as the strong suppression by 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an O2- scavenger, suggest that O2- is involved in the light-dependent formation of glycolate in vivo.     

Mechanisms of hydroxyl radical formation and ethanol oxidation by ethanol-inducible and other forms of rabbit liver microsomal cytochromes P-450

The hydroxyl radical-mediated oxidation of 5,5-dimethyl-1-pyrroline N-oxide, benzene, ketomethiolbutyric acid, deoxyribose, and ethanol, as well as superoxide anion and hydrogen peroxide formation was quantitated in reconstituted membrane vesicle systems containing purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochromes P-450 LM2, P-450 LMeb , or P-450 LM4, and in vesicle systems devoid of cytochrome P-450. The presence of cytochrome P-450 in the membranes resulted in 4-8-fold higher rates of O-2, H2O2, and hydroxyl radical production, indicating that the oxycytochrome P-450 complex constitutes the major source for superoxide anions liberated in the system, giving as a consequence hydrogen peroxide and also, subsequently, hydroxyl radicals formed in an iron-catalyzed Haber-Weiss reaction. Depletion of contaminating iron in the incubation systems resulted in small or negligible rates of cytochrome P-450-dependent ethanol oxidation. However, small amounts (1 microM) of chelated iron (e.g. Fe3+-EDTA) enhanced ethanol oxidation specifically when membranes containing the ethanol and benzene-inducible form of cytochrome P-450 (cytochrome P-450 LMeb ) were used. Introduction of the Fe-EDTA complex into P-450 LMeb -containing incubation systems caused a decrease in hydrogen peroxide formation and a concomitant 6-fold increase in acetaldehyde production; consequently, the rate of NADPH consumption was not affected. In iron-depleted systems containing cytochrome P-450 LM2 or cytochrome P-450 LMeb , an appropriate stoichiometry was attained between the NADPH consumed and the sum of hydrogen peroxide and acetaldehyde produced. Horseradish peroxidase and scavengers of hydroxyl radicals inhibited the cytochrome P-450 LMeb -dependent ethanol oxidation both in the presence and in the absence of Fe-EDTA. The results are not consistent with a specific mechanism for cytochrome P-450-dependent ethanol oxidation and indicate that hydroxyl radicals, formed in an iron-catalyzed Haber-Weiss reaction and in a Fenton reaction, constitute the active oxygen species. Cytochrome P-450-dependent ethanol oxidation under in vivo conditions would, according to this concept, require the presence of non-heme iron and endogenous iron chelators.     

An electron paramagnetic resonance study of the interactions between the adriamycin semiquinone, hydrogen peroxide, iron-chelators, and radical scavengers.

Anaerobic reduction of hydrogen peroxide in a xanthine/xanthine oxidase system by adriamycin semiquinone in the presence of chelators and radical scavengers was investigated by direct electron paramagnetic resonance and spin trapping techniques. Under these conditions, adriamycin semiquinone appears to react with hydrogen peroxide forming the hydroxyl radical in the presence of chelators such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid. In the absence of chelators, a related, but unknown oxidant is formed. In the presence of desferrioxamine, adriamycin semiquinone does not disappear in the presence of hydrogen peroxide at a detectable rate. The presence of adventitious iron is therefore implicated during adriamycin semiquinone-catalyzed reduction of hydrogen peroxide. Formation of alpha-hydroxyethyl radical and carbon dioxide radical anion from ethanol and formate, respectively, was detected by spin trapping. Both the hydroxyl radical and the related oxidant react with these scavengers, forming the corresponding radical. In the presence of scavengers from which reducing radicals are formed, the rate of consumption of hydrogen peroxide in this system is increased. This result can be explained by a radical-driven Fenton reaction.     

Production of superoxide anion during the oxidation of hemoglobin by menadione.

Menadione in the presence of oxyhemoglobin will accelerate the formation of methemoglobin and result in the generation of superoxide anion. Menadione appears to oxidize slowly ferrohemoglobin to ferrihemoglobin, while forming menadione semiquinone in the process. Menadione semiquinone is known to react with molecular oxygen to yield superoxide anion. The superoxide anion appears to be the source of hydrogen peroxide which accounts for most of the observed methemoglobin formation when hemoglobin is reacted with menadione.     

Free radicals in iron-containing systems

All oxidative damage in biological systems arises ultimately from molecular oxygen. Molecular oxygen can scavenge carbon-centered free radicals to form organic peroxyl radicals and hence organic hydroperoxides. Molecular oxygen can also be reduced in two one-electron steps to hydrogen peroxide in which case superoxide anion is an intermediate; or it can be reduced enzymatically so that no superoxide is released. Organic hydroperoxides or hydrogen peroxide can diffuse through membranes whereas hydroxyl radicals or superoxide anion cannot. Chain reactions, initiated by chelated iron and peroxides, can cause tremendous damage. Chain carriers are chelated ferrous ion; hydroxyl radical .OH, or alkoxyl radical .OR, and superoxide anion O2-. or organic peroxyl radical RO2.. Of these free radicals .OH and RO2. appear to be most harmful. All of the biological molecules containing iron are potential donors of iron as a chain initiator and propagator. An attacking role for superoxide dismutase is proposed in the phagocytic process in which it may serve as an intermediate enzyme between NADPH oxidase and myeloperoxidase. The sequence of reactants is O2----O2-.----H2O2----HOCl.     

Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.

Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively, at pH 7.4 and 37 degrees C. These rate constants are 3 orders of magnitude greater than the corresponding rate constants for the reaction of hydrogen peroxide with sulfhydryls at pH 7.4. Unlike hydrogen peroxide, which oxidizes thiolate anion, peroxynitrite anion reacts preferentially with the undissociated form of the thiol group. Peroxynitrite oxidizes cysteine to cystine and the bovine serum albumin thiol group to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol-oxidizing agent than its anion, with oxidation presumably mediated by the decomposition products, hydroxyl radical and nitrogen dioxide. The reactive peroxynitrite anion may exert cytotoxic effects in part by oxidizing tissue sulfhydryls.     

Production of hydroxyl radical by the synergistic action of fungal laccase and aryl alcohol oxidase

A mechanism for the production of hydroxyl radical (*OH) during the oxidation of hydroquinones by laccase, the ligninolytic enzyme most widely distributed among white-rot fungi, has been demonstrated. Production of Fenton reagent (H2O2 and ferrous ion), leading to *OH formation, was found in reaction mixtures containing Pleurotus eryngii laccase, lignin-derived hydroquinones, and chelated ferric ion. The semiquinones produced by laccase reduced both ferric to ferrous ion and oxygen to superoxide anion radical (O2*-). Dismutation of the latter provided the H2O2 for *OH generation. Although O2*- could also contribute to ferric ion reduction, semiquinone radicals were the main agents accomplishing the reaction. Due to the low extent of semiquinone autoxidation, H2O2 was the limiting reagent in Fenton reaction. The addition of aryl alcohol oxidase and 4-methoxybenzyl alcohol (the natural H2O2-producing system of P. eryngii) to the laccase reaction greatly increased *OH generation, demonstrating the synergistic action of both enzymes in the process.     

Phenoxyl free radical formation during the oxidation of the fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements.

The oxidation of the fluorescent dye 2',7'-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF(.)). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF(.) oxidizes GSH, generating the glutathione thiyl radical (GS(.)), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS(.) radical. Similarly, DCF(.) oxidized NADH, generating the NAD(.) radical that reduced oxygen to superoxide (O-(2)), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H(2)O(2), which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H(2)O(2) production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.