Possible mechanism of inhibition of nitrite-induced oxidation of oxyhemoglobin by ergothioneine and uric acid.

The time course of oxyhemoglobin oxidation by nitrite consisted of a kinetic lag followed by a transition phase which progressed into a rapid autocatalytic phase. The imidazolthione and imidazolone derivatives, ergothioneine and uric acid, respectively, caused an increase in the duration of the lag phase in a concentration-dependent manner, without affecting the onset and rate of the autocatalytic phase. Neither compound reacted with H2O2 or nitrite, oxidizing species required in the initiation steps of oxyhemoglobin oxidation. On the other hand, both compounds reduced effectively and at comparable rates the high oxidation state of hemoglobin, i.e., ferrylhemoglobin, which is an intermediate species occurring in the autocatalytic phase. In addition, the rate of ergothioneine oxidation, upon its reaction with ferrylmyoglobin, was accelerated by nitrite, thus suggesting a reaction between the thione and nitrogen dioxide. Nitrogen oxide and ferrylhemoglobin are key species in the free radical chain propagation leading to oxyhemoglobin oxidation by nitrite. These data support the view that ergothioneine and urate delay oxyhemoglobin oxidation by nitrite upon the temporary removal of the propagating species, i.e., nitrogen dioxide and, secondarily, ferrylhemoglobin, and within a mechanism encompassing alterations of the nitrite in equilibrium with nitrogen dioxide and ferrylhemoglobin in equilibrium with methemoglobin redox transitions.     

Catalase-like activity of human methemoglobin: a kinetic and mechanistic study

Hydrogen peroxide triggers a redox cycle between methemoglobin and ferrylhemoglobin, leading to protein inactivation and oxygen evolution. In the present paper, the catalase-like oxygen production by human methemoglobin in the presence of H₂O₂ was kinetically characterized with a Clark-type electrode. Progress curves showed a pseudo-steady state in the first minutes of the reaction, while double-reciprocal plots were upwardly concave, indicating positive co-operativity dependent upon protein concentration, which is a very unusual kinetic behavior. Addition of superoxide radical scavengers slightly increased activity, suggesting that most oxygen was produced biocatalytically. By considering all the experimental data obtained, a possible mechanism was proposed, including: (a) competition between the one-electron and the two-electron reductions of the oxoferryl free radical species of hemoglobin, giving rise to ferrylhemoglobin and methemoglobin, respectively; (b) competition between the superoxide-dependent inactivation of the protein and its reduction back to the met state. Computer simulations of that model have been performed by numerically integrating the differential equations set describing the mechanism, which was seen to yield predictions of the kinetic parameters variation consistently with the kinetic behavior experimentally observed. We suggest that the catalase-like activity of methemoglobin must predominantly be a biocatalytic reaction that protects the protein against H₂O₂-induced suicide inactivation.     

Neutrophil-mediated methemoglobin formation in the erythrocyte. The role of superoxide and hydrogen peroxide

Human neutrophils incubated with phorbol myristate acetate oxidized hemoglobin within the intact erythrocyte by a mechanism dependent on cell-cell contact but independent of phagocytosis. Spectrophotometric examination of the erythrocyte lysates revealed that the major component formed was methemoglobin along with small amounts of a species with spectral characteristics similar to choleglobin. Methemoglobin formation was directly related to the neutrophil concentration and the time of incubation. The addition of superoxide dismutase or catalase modestly inhibited the formation of methemoglobin, while a combination of the enzymes provided the most dramatic protection. Methemoglobin of hydroxyl radical or hypochlorous acid scavengers. Apparently, either O2.- or H2O2 alone was capable of mediating methemoglobin formation in the intact erythrocyte. Maintenance of the intraerythrocytic hemoglobin in its oxygenated state or its derivatization to carbon monoxyhemoglobin markedly inhibited methemoglobin formation. Blockade of the anion channels in the intact erythrocyte with sulfonated stilbenes inhibited O2.- but not H2O2 from oxidizing intracellular hemoglobin. It appears that neutrophil-derived O2.- and H2O2 can cross the erythrocyte membrane through the anion channel or diffuse directly into the intracellular space and react with oxyhemoglobin or deoxyhemoglobin to form a mixture of hemoglobin oxidation products within the intact cell.     

Mechanism of oxyhemoglobin oxidation induced by hydrogen peroxide]

The process of oxyhemoglobin oxidation initiated by hydrogen peroxide in low (10(-7) M) concentrations was investigated. It was found, that H2O2 in this concentration is able to induce the process of chain oxidation of oxyhemoglobin to methemoglobin. The following observations indicate that the process is essentially the chain reaction: 1) The amount of the methemoglobin in haem groups, produced in the reaction, exceed by 20 times the quantity of hydrogen, added initially, to induce the oxidation. 2) Catalase stopped this process at any stage of the reaction. This fact implies that the chain process involves generation of new molecules of H2O2 in the course of oxidation of oxyhemoglobin. The chain reaction proceeded only in the presence of oxygen. But if oxygen was introduced into hemoglobin solution, preincubated with H2O2 in vacuum, than again the oxidation of hemoglobin developed. Apparently, H2O2 in low concentrations appears, mainly, as an inductor of the oxyhemoglobin autooxidation.     

The production of activated oxygen species by an interaction of methemoglobin with ascorbate

Ascorbate reacts with methemoglobin to produce reactive oxygen species, most probably hydroxyl radicals. The main features of this system are: a) disappearance of ascorbate; b) consumption of oxygen with an ascorbate/O2 stoichiometry of 2:1; c) requirement of unliganded heme iron; d) formation of H2O2. The proposed mechanism involves an ascorbate-mediated interconversion of methemoglobin and oxy-hemoglobin, resulting in the production of H2O2. This product is decomposed by hemoglobin to produce hydroxyl radicals according to a Fenton-like reaction in which ascorbate recycles methemoglobin to hemoglobin. Alternative pathways of formation and of decomposition of H2O2 in this system appear to play a minor role.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H(2)O(2); ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

Free radical activation of N-hydroxy-2-acetylaminofluorene by methemoglobin and hydrogen peroxide

We have shown that N-hydroxy-2-acetylaminofluorene, a metabolite of 2-acetylaminofluorene, is converted via a nitroxide free radical into N-acetylaminofluorene and 2-nitrosofluorene by H2O2 in the presence of methemoglobin. Utilizing optical methods, we have demonstrated that the rate of 2-nitrosofluorene production parallels that of N-hydroxy-2-acetylaminofluorene oxidation. This evidence is consistent with a model whereby two molecules of N-hydroxy-2-acetylaminofluorene yield two nitroxide free radicals which then dismutate to form one molecule of N-acetoxy-2-acetylaminofluorene and one molecule of 2-nitrosofluorene. The Km of N-hydroxy-2-acetylaminofluorene for this reaction is 114 microM with a Vmax of 50 microM/min.     

Initiation of membranal lipid peroxidation by activated metmyoglobin and methemoglobin

The interaction of hydrogen peroxide (H2O2) with metmyoglobin (MetMb) led very rapidly to the generation of an active species which could initiate lipid peroxidation. The activity of this prooxidant decreased rapidly during the first minutes, but 50% of its activity remained stable for more than 30 min. In this model system, it was found that small amounts of H2O2 (1-10 microM) could activate MetMb for significant lipid peroxidation. The incubation of the sarcosomal lipids with activated MetMb caused oxygen absorption. No absorption of oxygen was determined in the presence of membrane with MetMb or H2O2 alone. Methemoglobin (MetHb) was also found to be activated by H2O2 and to initiate lipid peroxidation. Membranal lipid peroxidation initiated by activated MetMb was inhibited by several reducing compounds and antioxidants. However, several hydroxyl radical scavengers and catalase failed to inhibit this reaction.     

Peroxynitrite induces long-lived tyrosyl radical(s) in oxyhemoglobin of red blood cells through a reaction involving CO2 and a ferryl species

Peroxynitrite-mediated oxidative chemistry is currently the subject of intense investigation owing to the toxic side effects associated with nitric oxide overproduction. Using direct electron spin resonance spectroscopy (ESR) at 37 degrees C, we observed that in human erythrocytes peroxynitrite induced a long-lived singlet signal at g = 2.004 arising from hemoglobin. This signal was detectable in oxygenated red blood cells and in purified oxyhemoglobin but significantly decreased after deoxygenation. The formation of the g = 2.004 radical required the presence of CO2 and pH values higher than the pKa of peroxynitrous acid (pKa = 6.8), indicating the involvement of a secondary oxidant formed in the interaction of ONOO- with CO2. The g = 2.004 radical yield leveled off at a 1:1 ratio between peroxynitrite and oxyhemoglobin, while CO-hemoglobin formed less radical and methemoglobin did not form the radical at all. These results suggest that the actual oxidant is or is derived from the ONOOCO2- adduct interacting with oxygenated FeII-heme. Spin trapping with 2-methyl-2-nitrosopropane (MNP) of the g = 2.004 radical and subsequent proteolytic digestion of the MNP/hemoglobin adduct revealed the trapping of a tyrosyl-centered radical(s). A similar long-lived unresolved g = 2.004 singlet signal is a common feature of methemoglobin/H2O2 and metmyoglobin/H2O2 systems. We show by spin trapping that these g = 2.004 signals generated by H2O2 also indicated trapping of radicals centered on tyrosine residues. Analysis of visible spectra of hemoglobin treated with peroxynitrite revealed that, in the presence of CO2, oxyhemoglobin was oxidized to a ferryl species, which rapidly decayed to lower iron oxidation states. The g = 2.004 radical may be an intermediate formed during ferrylhemoglobin decay. Our results describe a new pathway of peroxynitrite-dependent hemoglobin oxidation of dominating importance in CO2-containing biological systems and identify the g = 2.004 radical(s) formed in the process as tyrosyl radical(s).     

Metabolic activation of chlorpromazine by stimulated human polymorphonuclear leukocytes. Induction of covalent binding of chlorpromazine to nucleic acids and proteins.

Human polymorphonuclear leukocytes (PMNs) have been stimulated with either phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187 or a combination of both to induce the respiratory burst and myeloperoxidase (MPO) release. Chlorpromazine (CPZ) but not chlorpromazine sulfoxide (CPZSO) inhibited the respiratory burst as measured with lucigenin chemiluminescence. The inhibition was due to interference with processes in the cell leading to the respiratory burst and not to scavenging of produced oxygen radicals that provoke the luminescence. CPZ was metabolized by stimulated PMNs. HPLC analysis revealed formation of CPZSO and an unidentified product. Both products result from decay of chlorpromazine radical cation (CPZ+.), indicating formation of this radical intermediate in CPZSO oxidation by stimulated PMNs. CPZ conversion correlated with H2O2 production and MPO release. The largest CPZ conversion was observed with phorbol ester plus A23187 stimulation. The conversion was reduced by catalase and sodium azide, an inhibitor of MPO, with 70% and 40%, respectively. This indicates only partial involvement of extracellularly released MPO in CPZ metabolism by PMNs. Considerable covalent binding of [3H]CPZ to nucleic acids and proteins of intact stimulated PMNs was observed. This binding was larger upon co-stimulation with phorbol ester and A23187. Azide did not reduce covalent binding. This indicates that covalent binding is not mediated by extracellularly released MPO and that CPZ is probably activated intracellularly. Activation of PMNs and production of H2O2 is a prerequisite for both CPZ conversion and covalent binding. This study demonstrates that phagocytic cells might contribute to drug metabolism and drug-induced toxicity.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

Abstract

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (^*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H₂O₂; ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

Oxidation of 3-hydroxykynurenine catalyzed by methemoglobin with hydrogen peroxide.

Methemoglobin (metHb) with H2O2 catalyzed the oxidation of 3-hydroxykynurenine (3-HKY) in the reaction mixture of metHb, 3-HKY, and H2O2. The spectrophotometric experiments suggest the following mechanism for the 3-HKY oxidation by metHb with H2O2. MetHb first reacts with H2O2 to form the ferryl complex of Hb. This species then oxidizes 3-HKY, while it returns to metHb. 3-HKY was more reactive with the ferryl complex than glutathione but less reactive than ascorbic acid. Scavengers of the hydroxyl radical, dimethyl sulfoxide and ethanol, scarcely inhibited the 3-HKY oxidation by metHb with H2O2. Desferrioxamine, a metal chelator, hardly suppressed the 3-HKY oxidation. These results indicate that the hydroxyl radical is not involved in the 3-HKY oxidation by metHb with H2O2.     

Mechanism of autocatalytic oxidation of oxyhemoglobin by nitrite. An intermediate detected by electron spin resonance

Oxidation of oxyhemoglobin by nitrite is characterized by the presence of a lag phase followed by the autocatalysis. Just before the autocatalysis begins, an asymmetric ESR signal is detected which is similar to that of the methemoglobin radical generated from methemoglobin and H2O2 in shape, g value (2.005), peak-to-peak width (18 G) and other properties, except the difference in the dependence on temperature. Generation of H2O2 is indicated by the prolongation of the lag phase by the addition of catalase. On the other hand, the oxidation is modified by neither superoxide dismutase nor Nitroblue tetrazolium. The oxidation is prolonged in the presence of KCN. The present results indicate a free-radical mechanism for the oxidation in which the asymmetric radical catalyzes the formation of NO2 from NO2- by a peroxidase action and NO2 oxidizes oxyhemoglobin in the autocatalytic phase.     

Role of vitamin E in glutathione-induced oxidant stress: methemoglobin, lipid peroxidation, and hemolysis.

Red blood cells (RBC) from normal and vitamin E-deficient rats were incubated in a hypertonic solution of reduced glutathione adjusted to pH 8. Methemoglobin formation occurred in intact RBC from both normal and vitamin E-deficient rats. Hemolysis was significantly greater in RBC from vitamin E-deficient rats. Experiments with catalase, superoxide dismutase, and methional showed that H(2)O(2) was the primary extracellular source of oxidant stress. Extracellular superoxide and hydroxyl radical were not involved in oxidant stress. Experiments with dimethyl sulfoxide showed that intracellular hydroxyl radical, generated from H(2)O(2), was the hemolytic agent. Neither methemoglobin formation nor lipid peroxidation involved hydroxyl radical. Indeed, lipid peroxidation and hemolysis in RBC from vitamin E-deficient rats were concurrent rather than consecutive events. Phase contrast microscopy showed that rigid, crenated RBC with a precipitate around the interior periphery formed during glutathione-induced oxidant stress. The precipitate dissolved slowly as the crenated RBC were converted to smooth ghosts. It appeared that protein precipitates involving mixed disulfide bonds were reduced and solubilized when extracellular glutathione penetrated the ruptured cell. Comparisons between normal RBC and vitamin E-deficient RBC suggest that vitamin E has little effect on the inward diffusion of extra-cellular H(2)O(2). Vitamin E apparently interacts with different oxidant species derived from intracellular H(2)O(2) in preventing lipid peroxidation and the sulfhydryl group oxidation leading to hemolysis.     

Reaction of human hemoglobin with peroxynitrite. Isomerization to nitrate and secondary formation of protein radicals

Peroxynitrite, a strong oxidant formed intravascularly in vivo, can diffuse onto erythrocytes and be largely consumed via a fast reaction (2 x 10(4) m(-1) s(-1)) with oxyhemoglobin. The reaction mechanism of peroxynitrite with oxyhemoglobin that results in the formation of methemoglobin remains to be elucidated. In this work, we studied the reaction under biologically relevant conditions using millimolar oxyhemoglobin concentrations and a stoichiometric excess of oxyhemoglobin over peroxynitrite. The results support a reaction mechanism that involves the net one-electron oxidation of the ferrous heme, isomerization of peroxynitrite to nitrate, and production of superoxide radical and hydrogen peroxide. Homolytic cleavage of peroxynitrite within the heme iron allows the formation of ferrylhemoglobin in approximately 10% yields, which can decay to methemoglobin at the expense of reducing equivalents of the globin moiety. Indeed, spin-trapping studies using 2-methyl-2-nitroso propane and 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated the formation of tyrosyl- and cysteinyl-derived radicals. DMPO also inhibited covalently linked dimerization products and led to the formation of DMPO-hemoglobin adducts. Hemoglobin nitration was not observed unless an excess of peroxynitrite over oxyhemoglobin was used, in agreement with a marginal formation of nitrogen dioxide. The results obtained support a role of oxyhemoglobin as a relevant intravascular sink of peroxynitrite.     

Iron and dioxygen chemistry is an important route to initiation of biological free radical oxidations: an electron paramagnetic resonance spin trapping study

Iron can be a detrimental catalyst in biological free radical oxidations. Because of the high physiological ratio of [O2]/[H2O2] (> or = 10(3)), we hypothesize that the Fenton reaction with pre-existing H2O2 is only a minor initiator of free radical oxidations and that the major initiators of biological free radical oxidations are the oxidizing species formed by the reaction of Fe2+ with dioxygen. We have employed electron paramagnetic resonance spin trapping to examine this hypothesis. Free radical oxidation of: 1) chemical (ethanol, dimethyl sulfoxide); 2) biochemical (glucose, glyceraldehyde); and 3) cellular (L1210 murine leukemia cells) targets were examined when subjected to an aerobic Fenton (Fe2+ + H2O2 + O2) or an aerobic (Fe2+ + O2) system. As anticipated, the Fenton reaction initiates radical formation in all the above targets. Without pre-existing H2O2, however, Fe2+ and O2 also induce substantial target radical formation. Under various experimental ratios of [O2]/[H2O2] (1-100 with [O2] approximately 250 microM), we compared the radical yield from the Fenton reaction vs. the radical yield from Fe2+ + O2 reactions. When [O2]/[H2O2] < 10, the Fenton reaction dominates target molecule radical formation; however, production of target-molecule radicals via the Fenton reaction is minor when [O2]/[H2O2] > or = 100. Interestingly, when L1210 cells are the oxidation targets, Fe2+ + O2 is observed to be responsible for formation of nearly all of the cell-derived radicals detected, no matter the ratio of [O2]/[H2O2]. Our data demonstrate that when [O2]/[H2O2] > or = 100, Fe2+ + O2 chemistry is an important route to initiation of detrimental biological free radical oxidations.     

Increased phospholipid methylation in the myocardium of alcoholic rats

NAD(P)H is known to be oxidized by singlet molecular oxygen, perhydroxyl radical, and hydroxyl radical. In marked contrast to these reactive oxygen species, NAD(P)H is stable in the presence of micromolar concentrations of H2O2. The experiments herein demonstrate that NADPH is rapidly oxidized by H2O2 in the presence of a heme-peptide. The oxidation product is enzymatically active NADP+. In the absence of NADPH, the heme-peptide undergoes rapid degradation via reaction with H2O2. In the presence of NADPH, the reduced nucleotide is oxidized to NADP and the heme-peptide is partially protected from oxidation. It is suggested that under certain conditions the reduced nucleotides may contribute to the protection of intracellular heme moieties from degradation engendered by endogenous or exogenous H2O2.     

Heme degradation during autoxidation of oxyhemoglobin

Two fluorescent heme degradation compounds are detected during autoxidation of oxyhemoglobin. These fluorescent compounds are similar to fluorescent compounds formed when hydrogen peroxide reacts with hemoglobin [E. Nagababu and J. M. Rifkind, Biochem. Biophys. Res. Commun. 247, 592-596 (1998)]. Low levels of heme degradation in the presence of superoxide and catalase are attributed to a reaction involving the superoxide produced during autoxidation. The inhibition of most of the degradation by catalase suggests that the hydrogen peroxide generated during autoxidation of oxyhemoglobin produces heme degradation by the same mechanism as the direct addition of hydrogen peroxide to hemoglobin. The formation of the fluorescent degradation products was inhibited by the peroxidase substrate, ABTS, which reduces ferrylhemoglobin to methemoglobin, indicating that ferrylhemoglobin is produced during the autoxidation of hemoglobin. It is the transient formation of this highly reactive Fe(IV) hemoglobin, which is responsible for most of the heme degradation during autoxidation.     

Oxidative damage to human red cells induced by copper and iron complexes in the presence of ascorbate

The role of trace metals in the generation of free radical mediated oxidative stress in normal human red cells was studied. Ascorbate and either soluble complexes of Cu(II) or Fe(III) provoked changes in red cell morphology, alteration in the polypeptide pattern of membrane proteins, and significant increases in methemoglobin. Neither ascorbate nor the metal complexes alone caused significant changes to the cells. The rate of methemoglobin formation was a function of ascorbate and metal concentrations, and the chemical nature of the chelate. Cu(II) was about 10-times more effective than Fe(III) in the formation of methemoglobin. Several metals were tested for their ability to compete with Cu(II) and Fe(III). Only zinc caused a significant inhibition of methemoglobin formation by Fe(III)-fructose. These observations suggest that site-specific as well as general free radical damage is induced by redox metals when the metals are either bound to membrane proteins or to macromolecules in the cytoplasm. The Cu(II) and Fe(III) function in two catalytic capacities: (1) oxidation of ascorbate by O2 to yield H2O2, and (2) generation of hydroxyl radicals from H2O2 in a Fenton reaction. These mechanisms are different from the known damage to red cells caused by the binding of Fe(III) or Cu(II) to the thiol groups of glucose-6-phosphate dehydrogenase. Our system may be a useful model for understanding the mechanisms for oxidative damage associated with thalassemia and other congenital hemolytic anemias.     

Green hemoprotein of erythrocytes: methemoglobin superoxide transferase

Influences of base (pH 10), heat (50 degrees C), microwave radiation (2450 MHz, 103 +/- 4 W/kg), and hydrogen peroxide (5.6 mM) generated by glucose oxidase on oxidation of human oxyhemoglobin to methemoglobin were examined. Conversion of oxyhemoglobin to methemoglobin was followed by the difference in absorbancy of 540 or 542 nm and 576 nm wavelength light versus time. Fresh basic hemolysates auto-oxidized on heating with a zero order rate constant, implying that hemoglobin or another protein saturated with oxyhemoglobin catalyzed the oxidation. Simultaneous microwave irradiation inhibited thermally induced auto-oxidation on the average by 28.6%. However, there was great variability among samples and a decrease in auto-oxidation with aging of individual samples. The auto-oxidation rate was independent of initial oxyhemoglobin concentration. Oxidation of partially purified oxyhemoglobin by hydrogen peroxide was not influenced by microwave irradiation. Adding green hemoprotein isolated from human erythrocytes to the oxyhemoglobin/glucose oxidase reaction mixture yielded absorption spectra (500-600 nm) that were a combination of oxyhemoglobin, deoxyhemoglobin, and methemoglobin spectra. Green hemoprotein was labile in hemolysates but stable in a partially purified ferric form. These results imply that thermally unstable reduced green hemoprotein can reverse oxidation of oxyhemoglobin by hydrogen peroxide and could mediate the thermally induced and microwave inhibited auto-oxidation of oxyhemoglobin.