ESR Spin Trapping Detection of Hydroxyl Radicals in the Reactions of Cr(V) Complexes with Hydrogen Peroxide

Electron spin resonance (ESR) measurments provide direct evidence for the involvement of Cr(V) in the reduction of Cr(VI) by NAD(P)H. Addition of hydrogen peroxide (H₂O₂) to NAD(P)H-Cr(VI) reaction mixtures suppresses the Cr(V) signal and generates hydroxyl (OH) radicals (as detected via spin trapping), suggesting that Cr(V) reacts with H₂O₂ to generate the OH radicals. Reaction between H₂O₂ and a Cr(V)-glutathione complex. and between H₂O₂ and several Cr(V)-cdrboxylato complexes also produces OH radicals. These results suggest that Cr(V) complexes catalyze the generation of OH radicals from H₂O₂, and that OH radicals might play a significant role in the mechanism of Cr(VI) cytotoxicity.     

Deferoxamine inhibition of Cr(V)-mediated radical generation and deoxyguanine hydroxylation: ESR and HPLC evidence.

Electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques were utilized to investigate the effect of deferoxamine on free radical generation in the reaction of Cr(V) with H2O2 and organic hydroperoxides. ESR measurements demonstrated that deferoxamine can efficiently reduce the concentration of the Cr(V) intermediate as formed in the reduction of Cr(VI) by NAD(P)H or a flavoenzyme glutathione reductase/NADH. ESR spin trapping studies showed that deferoxamine also inhibits Cr(V)-mediated .OH radical generation from H2O2, as well as Cr(V)-mediated alkyl and alkoxy radical formation from t-butyl hydroperoxide and cumene hydroperoxide. HPLC measurements showed that .OH radicals generated by the Cr(VI)/flavoenzyme/NAD(P)H enzymatic system react with 2'-deoxyguanine to form 8-hydroxy-2'-deoxyguanine (8-OHdG), a DNA damage marker. Deferoxamine effectly inhibited the formation of 8-OHdG also.     

Formation of Hydroxyl Radicals in Biological Systems. Does Myoglobin Stimulate Hydroxyl Radical Formation from Hydrogen Peroxide?

Incubation of horse-heart oxymyoglobin or metmyoglobin with excess H₂O₂ causes formation of myoglobin(IV), followed by haem degradation. At the time when haem degradation is observed, hydroxyl radicals (.OH) can be detected in the reaction mixture by their ability to degrade the sugar deoxyribose. Detection of hydroxyl radicals can be decreased by transferrin or by OH scavengers (mannitol, arginine, phenylalanine) but not by urea. Neither transferrin nor any of these scavengers inhibit the haem degradation. It is concluded that intact oxymyoglobin or metmyoglobin molecules do not react with H₂O₂ to form OH detectable by deoxyribose, but that H₂O₂ eventually leads to release of iron ions from the proteins. These released iron ions can react to form OH outside the protein or close to its surface. Salicylate and the iron chelator desferrioxamine stabilize myoglobin and prevent haem degradation. The biological importance of OH generated using iron ions released from myoglobin by H₂O₂ is discussed in relation to myocardial reoxygenation injury.     

Comparative study of the formation of oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct from the nucleoside 2'-deoxyguanosine by transition metals and suspensions of particulate matter in relation to metal content and redox reactivity

An association between exposure to ambient particulate matter (PM) and increased incidence of mortality and morbidity due to lung cancer and cardiovascular diseases has been demonstrated by recent epidemiological studies. Reactive oxygen species (ROS), especially hydroxyl radicals, generated by PM, have been suggested by many studies as an important factor in the oxidative damage of DNA by PM. The purpose of this study was to characterize quantitatively hydroxyl radical generation by various transition metals in the presence of H₂O₂ in aqueous buffer solution (pH 7.4) and hydroxylation of 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG) under similar conditions. The order of metals' redox reactivity and hydroxyl radical production was Fe(II), V(IV), Cu(I), Cr(III), Ni(II), Co(II), Pb(II), Cd(II). Then, we investigated the generation of hydroxyl radicals in the presence of H₂O₂ by various airborne PM samples, such as total suspended particulate (TSP), PM₁₀, PM_2.5 (PM with aerodynamic diameter 10 and 2.5 μm), diesel exhaust particles (DEP), gasoline exhaust particles (GEP) and woodsmoke soot under the same conditions. When suspensions of PMs were incubated with H₂O₂ and dG at pH 7.4, all particles induced hydroxylation of dG and formation of 8-OHdG in a dose-dependent increase. Our findings demonstrated that PM's hydroxyl radical (HO√) generating ability and subsequent dG hydroxylation is associated with the concentration of water-soluble metals, especially Fe and V and other redox or ionizable transition metals and not their total metal content, or insoluble metal oxides, via a Fenton-driven reaction of H₂O₂ with metals. Additionally, we observed, by Electron paramagnetic resonance (EPR), that PM suspensions in the presence of H₂O₂ generated radical species with dG, which were spin-trapped by 2-methyl-2-nitroso-propane (MNP).     

Role of Hydroxyl Radicals in Escherzchza Colz Killing Induced by Hydrogen Peroxide

Escherichia coli lethality by hydrogen peroxide is characterized by two modes of killing. In this paper we have found that hydroxyl radicals (OH -) generated by H₂O₂ and intracellular divalent iron are not involved in the induction of mode one lethality (i.e. cell killing produced by concentrations of H₂O₂ lower than 2.5 mM). In fact, the OH radical scavengers, thiourea, ethanol and dimethyl sulfoxide, and the iron chelator, desferrioxarnine, did not affect the survival of cells exposed to 2.5mM H₂O₂. In addition cell vulnerability to the same H₂O₂ concentration was independent on the intracellular iron content. In contrast, mode two lethality (i.e. cell killing generated by concentrations of H₂O₂ higher than 10mM) was markedly reduced by OH radical scavengers and desferrioxamine and was augmented by increasing the intracellular iron content.

It is concluded that OH. are required for mode two killing of E. coli by hydrogen peroxide.     

Inactivation of Yeast Glutathione Reductase by Fenton Systems: Effect of Metal Chelators, Catecholamines and Thiol Compounds

Oxygen radical generating systems, namely, Cu(II)/ H₂O₂, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H₂O₂/catecholamine and Cu(II)/H₂O₂/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H₂O₂ enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H₂O₂ depended on Cu(II) and H₂O₂ concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO^˙ scavengers prevented GR inactivation by Cu(II)/H₂O₂ and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropi-onylglycine and penicillamine enhanced the effect of Cu(II)/H₂O₂ in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H₂O₂ effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and try-panothione disulfide effectively protected GR against Cu(II)/H₂O₂ inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS*⁺. GR inactivation by the systems assayed correlated with their capability for HO* radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.     

Radical-Induced Damage to Proteins: E.S.R. Spin-Trapping Studies

The reactions of hydroxyl radicals generated from Fe¹¹/H₂O₂ and Cu¹¹/H₂O₂ redox couples with a variety of proteins (BSA, histones, cytochrome c, lysozyme and protamine) have been investigated by e.s.r. spin trapping. The signals obtained, which are generally anisotropic in nature, characterize the formation of partially-immobilized spin-adducts resulting from attack of the HO- radicals on the protein and subsequent reaction of the protein-derived radicals with the spin trap. Similar spin adducts are observed on incubation of two haem-proteins (haemoglobin and myoglobin) with H₂O₂ in the absence of added metal ions implying a reaction at the haem centre followed by internal electron transfer reactions.

Two strategies have been employed to obtain information about the site(s) of radical damage in these proteins. The first involves the use of a variety of spin traps and in particular DMPO: with this particular trap the broad spectra from largely immobilized radicals show characteristic a(β-H) values which enable carbon-, oxygen- and sulphur-centred radicals to be distinguished. The second involves the use of enzymatic cleavage of first-formed adducts to release smaller nitroxides, with isotropic spectra, which allow the recognition of β-proton splittings and hence information about the sites of radical damage to be obtained. These results, which allows backbone and side-chain attack to be distinguished, are in agreement with random attack of the HO^. radical on the protein and are in accord with studies carried out on model peptides. In contrast the use of less reactive attacking radicals [N₃·, ·CH(CH₃)OH] and oxidising agents (Ce⁴⁺) provides evidence for selective attack on these proteins at particular residues.     

Hydroxyl Radical Generation in the Nadh/Microsomal Reduction of vanadate

ESR spin trapping measurements demonstrate generation of hydroxyl (.OH) radical from reduction of vanadate by rat liver microsomes/NADH without exogenous H₂O₂. Catalase decreases the .OH signal while increasing a vanadium(4+) signal. Addition of superoxide dismutase (SOD) or measurements under an argon atmosphere show decreased .OH radical production. The results suggest that during the one-electron vanadate reduction process by microsomes/NADH, molecular oxygen is reduced to H₂O₂, which then reacts with vanadium (4+) to generate .OH radical via a Fenton-like mechanism.     

Effect of hydrogen peroxide in redox status estimation using nitroxyl spin probe

A procedure for estimating in vivo redox status using EPR and a hydrogen peroxide (H₂O₂)-dependent spin probe method is described. The mechanism of decreasing spin clearance in the selenium-deficient (SeD) rat is discussed. The in vivo decay constant of the nitroxyl spin probe in the liver region of SeD rats appeared to be slightly lower that of the selenium-adequate control (SeC) group, and was significantly smaller than that of normal rats. Bile H₂O₂ levels in normal rats were significantly lower than those in SeD rats. The in vivo decay constant of the spin probe in SeD rats depended on the bile H₂O₂ level. Furthermore, H₂O₂ was detected in the bile in all SeD rats, whereas bile H₂O₂ could be detected in only half of the normal rats. It was found that the in vivo decay constant of the spin probe in normal rats also depended on whether bile H₂O₂ was detected or not. In vivo decay constants were smaller in rats subjected to the surgical operation than in the nonoperated groups. The EPR signal of the nitroxyl radical in the liver homogenate was increased by addition of H₂O₂, which was administered 30 min before the rat was killed. It appears that H₂O₂ can oxidize the hydroxylamine formed following reduction of the spin probe in the liver.     

Direct NAD(P)H hydrolysis into ADP-ribose(P) and nicotinamide induced by reactive oxygen species: a new mechanism of oxygen radical toxicity

The effect of different oxygen radical-generating systems on NAD(P)H was determined by incubating the reduced forms of the pyridine coenzymes with either Fe²⁺-H₂O₂ or Fe³⁺-ascorbate and by analyzing the reaction mixtures using a HPLC separation of adenine nucleotide derivatives. The effect of the azo-initiator 2,2'-azobis(2-methylpropionamidine)dihydrochloride was also tested. Results showed that, whilst all the three free radical-producing systems induced, with different extent, the oxidation of NAD(P)H to NAD(P)⁺, only Fe²⁺-H₂O₂ also caused the formation of equimolar amounts of ADP-ribose(P) and nicotinamide. Dose-dependent experiments, with increasing Fe²⁺ iron (concentration range 3-180 μM) or H₂O₂ (concentration range 50-1000 μM), were carried out at pH 6.5 in 50 mM ammonium acetate. NAD(P)⁺, ADP-ribose(P) and nicotinamide formation increased by increasing the amount of hydroxyl radicals produced in the medium. Under such incubation conditions NAD(P)⁺/ADP-ribose(P) ratio was about 4 at any Fe²⁺ or H₂O₂ concentration. By varying pH to 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 7.4, NAD(P)⁺/ADP-ribose(P) ratio changed to 5.5, 3.2, 1.8, 1.6, 2.0, 2.5, 3.0, 5.4 and 6.5, respectively. Kinetic experiments indicated that 90-95% of all compounds were generated within 5s from the beginning of the Fenton reaction. Inhibition of ADP-ribose(P), nicotinamide and NAD(P)⁺ production of Fe²⁺-H₂O₂-treated NAD(P)H samples, was achieved by adding mannitol (10-50 mM) to the reaction mixture. Differently, selective and total inhibition of ADP-ribose(P) and nicotinamide formation was obtained by performing the Fenton reaction in an almost completely anhydrous medium, i.e. in HPLC-grade methanol. Experiments carried out in isolated postischemic rat hearts perfused with 50 mM mannitol, showed that, with respect to values of control hearts, this hydroxyl radical scavenger prevented reperfusion-associated pyridine coenzyme depletion and ADP-ribose formation. On the basis of these results, a possible mechanism of action of ADP-ribose(P) and nicotinamide generation through the interaction between NAD(P)H and hydroxyl radical (which does not involve the C-center where “conventional” oxidation occurs) is presented. The implication of this phenomenon in the pyridine coenzyme depletion observed in postischemic tissues is also discussed.     

Influence of chelators and iron ions on the production and degradation of H2O2 by beta-amyloid-copper complexes

β-Amyloid peptide (Aβ) 1–42, involved in the pathogenesis of Alzheimer’s disease, binds copper ions to form Aβ · Cu_n complexes that are able to generate H₂O₂ in the presence of a reductant and O₂. The production of H₂O₂ can be stopped with chelators. More reactive than H₂O₂ itself, hydroxyl radicals HO (generated when a reduced redox active metal complex interacts with H₂O₂) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H₂O₂ associated to a pre-incubation step between ascorbate and Aβ · Cu_n allows to study the formation of H₂O₂ but also, at the same time, its transformation by the copper complexes. Aβ · Cu_n peptides produce but do not efficiently degrade H₂O₂. The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H₂O₂ production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from Aβ · Cu_n, whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with Aβ · Cu_n. The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.     

Singlet Oxygen-Trapping Reaction as a Method of 1O2 Detection: Role of Some Reducing Agents

The production of singlet oxygen by H₂O₂ disproportionation and via the oxidation of H₂O₂ by NaOCl in a neutral medium was monitored by spin trapping with 2,2,6,6 tetramethyl-4-piperidone (TMPone). The singlet oxygen formed in both reactions oxidized 2,2,6,6 tetramethyl-4-piperidone to give nitroxide radicals. However the production of nitroxide radicals was relatively small considering the concentrations of H₂O₂ and NaOCl used in the reaction systems. Addition of electron donating agents: ascorbate, Fe²⁺ and desferrioxamine leads to an increase in the production of nitroxide radicals. We assumed that a very slow step of the reaction sequence, the homolytic breaking of the O-O bond of N-hydroperoxide (formed as an intermediate product during the reaction of ¹O₂ with TMPone) could be responsible for the relatively small production of nitroxide radicals. Electron donating agents added to the reaction system probably raise the rate of the hydroperoxide decomposition by allowing a more rapid heterolytic cleavage of the O-O bond leading to a greater production of nitroxide radicals. The largest effect was observed in the presence of desferrioxamine. Its participation in this process is proved by the concomitant appearance of desferrioxamine nitroxide radicals. The results obtained demonstrate that the method proposed by several authors and tested in this study to detect singlet oxygen is not convenient for precise quantitative studies. The reactivity of TMPone towards O₂^-7HO₂' and 'OH has been also investigated. It has been found that both O₂^-7HO₂' and 'OH radicals formed in a phosphate buffer solution (pH 7.4, 37°C), respectively by a xanthine-oxidase/hypoxanthine system and via H₂O₂ UV irradiation, do not oxidize 2,2,6,6 tetramethyl-4-piperidone to nitroxide radicals.     

Structural determinations, magnetic and EPR studies of complexes involving the Cr(OH)2Cr unit

Five heterometallic compounds with formulae [Ba(H₂O)₄Cr₂(μ-OH)₂(nta)₂] · 3H₂O (I), [M(bpy)₂(H₂O)₂] [Cr₂(OH)₂(nta)₂] · 7H₂O, where M²⁺ = Zn, (II); Ni, (III); Co, (IV) and [Mn(H₂O)₃(bpy)Cr₂(OH)₂(nta)₂] · (bpy) · 5H₂O (V); bpy = 2,2′-bipyridine, (nta = nitrilotriacetate ion) have been prepared by reaction of I with the corresponding M^II-sulfates in the presence of 2,2′-bipyridine. Substances I–V have been characterized by magnetic susceptibility measurements, EPR and X-ray determinations. I represents a 2D coordination polymer formed by coordination of centrosymmetrical dimeric chromium(III) units and Barium cations. The 10-coordinate Ba polyhedron is completed by four water molecules. Compounds II–IV are isostructural and consist of non-centrosymmetric dimeric anions [Cr₂(μ-OH)₂(nta)₂]²⁻, complex cations [M^II(bpy)₂(H₂O)₂]²⁺ and solvate water molecules. The octahedral coordination of chromium atoms implies four donor atoms of the nta³⁻ ligands and two bridging OH groups. Multiple hydrogen bonds of coordinated and solvate water molecules link anions and cations in a 3D network. A similar [Cr₂(μ-OH)₂(nta)₂]²⁻ unit is found in V. The bridging function is performed by a carboxylate oxygen atom of the nta ligand that leads to the formation of a trinuclear complex [Mn(bpy)(H₂O)₂Cr₂(μ-OH)₂(nta)₂]. Experimental and calculated frequency and temperature dependences of EPR spectra of these compounds are presented. The fine structure appearing on the EPR spectra of compound V is analyzed in detail at different temperatures. It is established that the main part of the EPR signals is due to the transitions in the spin states of a spin multiplet with S = 2. Analyses of experimental and calculated spectra confirm the absence of interaction between metal ions (M^II) and Cr-dimers in complexes III and IV and the presence of weak Mn–Cr interactions in V. The temperature dependence of magnetic susceptibilities for I–V was fitted on the basis of the expression derived from isotropic Hamiltonian including a bi-quadratic exchange term.     

Protein radicals in the reaction between H2O2-activated metmyoglobin and bovine serum albumin

Hydrogen peroxide activation of MMb with and without the presence of BSA gave rise to rapid formation of hyper-valent myoglobin species, myoglobin ferryl radical (·MbFe(IV)=O) and/or ferrylmyoglobin (MbFe(IV)=O). Reduction of MbFe(IV)=O showed first-order kinetics for a 1-2 times stoichiometric excess of H₂O₂ to MMb while a 3-10 times stoichiometric excess of H₂O₂ resulted in a biphasic reaction pattern. Radical species formed in the reaction between MMb, H₂O₂ and BSA were influenced by [H₂O₂] as measured by electron spin resonance (ESR) spectroscopy and resulted in the formation of cross-linking between BSA and myoglobin which was confirmed by SDS-PAGE and subsequent amino acid sequencing. Moreover, dityrosine was formed in the initial phases of the reaction for all concentrations of H₂O₂. However, initially formed dityrosine was subsequently utilized in reactions employing stoichiometric excess of H₂O₂ to MMb. The observed breakdown of dityrosine was ascribed to additional radical species formed from the interaction between H₂O₂ and the hyper-valent iron-center of H₂O₂-activated MMb.     

Photo-Fenton氧化法处理废水的原理及影响因素

Photo-Fenton高级氧化技术是处理难降解有毒有机废水的一种有效的方法。本文阐述了该氧化法的原理及其影响因素,photo-Fenton氧化法在反应中会产生大量羟自由基(·OH),它是一种非常活泼及非选择性物种,其氧化电位为2.8V,氧化能力很强,能够引发水溶液中大部分有机物的氧化还原反应。其优点是操作简便及无二次污染等,反应产物Fe³⁺可与OH反应形成Fe(OH)₃沉淀而对环境无害。缺点是反应必须在pH≤3条件下进行,且H₂O₂消耗量大而导致价格昂贵,处理成本较高等。     

PI3P在暗诱导的蚕豆气孔关闭中的作用及与H2O2和NO的关系

以蚕豆(Vicia faba)表皮条为材料, 利用磷脂酰肌醇3-激酶(PI3K)的抑制剂沃曼青霉素(WM)和LY294002 (LY)抑制磷脂酰肌醇3-磷酸(PI3P)的形成, 并结合气孔开度分析及激光扫描共聚焦显微镜技术, 探讨暗诱导蚕豆气孔关闭过程中PI3P与过氧化氢(H₂O₂)和一氧化氮(NO)之间的相互关系。结果表明, WM和LY显著抑制暗诱导的保卫细胞H₂O₂和NO的形成以及气孔的关闭, 但不能抑制外源H₂O₂和NO诱导的气孔关闭, 外源H₂O₂和NO处理能完全逆转WM和LY对暗诱导的气孔关闭的抑制效应。实验结果暗示, 在暗诱导的气孔关闭的信号转导途径中PI3P在信号分子H₂O₂和NO的上游起作用。     

Influence of different site symmetries of Eu centers on the luminescence properties of Anderson-based compounds

Two compounds, [Eu(H₂O)₇][Al(OH)₆Mo₆O₁₈] · 4H₂O (1) and {(C₂H₅NO₂)₂[Eu(H₂O)₅]}[Al(OH)₆Mo₆O₁₈] · 10H₂O (2), have been synthesized by conventional solution method and determined by single-crystal X-ray diffraction. Compound 1 shows a 1D chain structure built up of alternating Anderson-type polyanions [Al(OH)₆Mo₆O₁₈]³⁻ and hydrated rare-earth ions Eu³⁺. Compound 2 displays a 3D supramolecular network structure containing 1D sandglass-like channels along c axis, which were occupied by repetitive array of (H₂O)₈ clusters. Extensive hydrogen bonds play an important role in the formation of the 3D structures of 1 and 2. Luminescence measurements reveal that 1 and 2 exhibit intense red and orange fluorescent emission at room temperature, respectively. Origin of the distinct emission can be assigned to the different site symmetries of Eu³⁺ centers in the two compounds. These results are consistent with the crystal structures of the two compounds.     

Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement

The deleterious effects of H₂O₂ on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. Exposure to H₂O₂ resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. The effect of H₂O₂ on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-DCIP oxidoreductase activities. Inhibitory effect of H₂O₂ on respiratory complexes was almost completely recovered by β-mercaptoethanol treatment. H₂O₂ treatment resulted in full resistance to Q_O site inhibitor myxothiazol and thus it is suggested that the quinol oxidase site (Q_O) of complex III is the target for H₂O₂. H₂O₂ did not modify basal levels of lipid peroxidation in yeast mitochondria. However, H₂O₂ addition to rat brain and liver mitochondria induced an increase in lipid peroxidation. These results are discussed in terms of the known physiological differences between mammalian and yeast mitochondria.     

When Do Metal Complexes Protect the Biological System from Superoxide Toxicity and When Do They Enhance It?

Many copper and iron complexes can be reduced by O^-₂ as well as by H₂O₂. According to the rates of reduction and the concentration of O^-₂ and H₂O₂, the metal complexes may serve either as catalyst of O^-₂ dismutation or as catalysts of the reaction between O^-₂ and H₂O₂ to form OH' radical (Haber-Weiss reaction). Various factors which influence whether metal complexes protect the biological systems from superoxide toxicity or enhance it are discussed.     

Decay Studies of Dmpo-Spin Adducts of Free Radicals Produced by Reactions of Metmyoglobin and Methemoglobin with Hydrogen Peroxide

The 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adduct of myoglobin (Mb) or hemoglobin (Hb) was formed when metmyoglobin (MetMb) or methemoglobin (MetHb) reacted with H₂O₂ in the presence of DMPO, and both decayed with half-life of a few minutes. The DMPO spin adduct of Mb decayed with biphasic kinetics with k₁ = 0.645 min^-1 and k₂ = 0.012 min^-1, indicating that the spin adduct consisted of two kinetically heterogeneous species, stable and unstable ones. The DPMO spin adduct of Hb, however, was homogeneous. Decay of both spin adducts was accelerated in the presence of tyrosine, tryptophan or cysteine, but not phenylalanine, methionine or histidine. The decay obeyed the first order kinetics at varying concentrations of the spin adducts. The decay was accelerated by denaturation and proteolysis of protein moiety. The decay rate was not affected by the extra addition of MetMb or MetHb to each spin adduct. The decay rate of the spin adduct of Mb was increased by hematin in the presence of H₂O₂ and decreased by catalase. Decay of stable spin adduct of Mb, however, was not significantly changed under any experimental conditions used. These results led us to conclude that instability of the DMPO-spin adducts of Mb and Hb is due to intramolecular redox reactions between the spin adducts and amino acid residues and/or products of the reaction between heme and H₂O₂.