Reaction of hydrogen peroxide with ferrylhemoglobin: superoxide production and heme degradation

The reaction of Fe(II) hemoglobin (Hb) but not Fe(III) hemoglobin (metHb) with hydrogen peroxide results in degradation of the heme moiety. The observation that heme degradation was inhibited by compounds, which react with ferrylHb such as sodium sulfide, and peroxidase substrates (ABTS and o-dianisidine), demonstrates that ferrylHb formation is required for heme degradation. A reaction involving hydrogen peroxide and ferrylHb was demonstrated by the finding that heme degradation was inihibited by the addition of catalase which removed hydrogen peroxide even after the maximal level of ferrylHb was reached. The reaction of hydrogen peroxide with ferrylHb to produce heme degradation products was shown by electron paramagnetic resonance to involve the one-electron oxidation of hydrogen peroxide to the oxygen free radical, superoxide. The inhibition by sodium sulfide of both superoxide production and the formation of fluorescent heme degradation products links superoxide production with heme degradation. The inability to produce heme degradation products by the reaction of metHb with hydrogen peroxide was explained by the fact that hydrogen peroxide reacting with oxoferrylHb undergoes a two-electron oxidation, producing oxygen instead of superoxide. This reaction does not produce heme degradation, but is responsible for the catalytic removal of hydrogen peroxide. The rapid consumption of hydrogen peroxide as a result of the metHb formed as an intermediate during the reaction of reduced hemoglobin with hydrogen peroxide was shown to limit the extent of heme degradation.     

Hydrogen-peroxide-induced heme degradation in red blood cells: the protective roles of catalase and glutathione peroxidase

Catalase and glutathione peroxidase (GSHPX) react with red cell hydrogen peroxide. A number of recent studies indicate that catalase is the primary enzyme responsible for protecting the red cell from hydrogen peroxide. We have used flow cytometry in intact cells as a sensitive measure of the hydrogen-peroxide-induced formation of fluorescent heme degradation products. Using this method, we have been able to delineate a unique role for GSHPX in protecting the red cell from hydrogen peroxide. For extracellular hydrogen peroxide, catalase completely protected the cells, while the ability of GSHPX to protect the cells was limited by the availability of glutathione. The effect of endogenously generated hydrogen peroxide in conjunction with hemoglobin autoxidation was investigated by in vitro incubation studies. These studies indicate that fluorescent products are not formed during incubation unless the glutathione is reduced to at least 40% of its initial value as a result of incubation or by reacting the glutathione with iodoacetamide. Reactive catalase only slows down the depletion of glutathione, but does not directly prevent the formation of these fluorescent products. The unique role of GSHPX is attributed to its ability to react with hydrogen peroxide generated in close proximity to the red cell membrane in conjunction with the autoxidation of membrane-bound hemoglobin.     

The origin of red cell fluorescence caused by hydrogen peroxide treatment

Fluorescence in red cells following hydrogen peroxide treatment has been attributed to lipid peroxidation of the membrane. The putative relationship between lipid peroxidation and fluorescence was questioned by the finding that BHT and alpha-tocopherol, which are thought to inhibit lipid peroxidation, do not inhibit the fluorescence detected by flow cytometry. Furthermore, lipid peroxidation induced in red cells by the Fe(III)-ADP-ascorbate system did not produce fluorescence. These results require an alternative explanation for the hydrogen peroxide-induced fluorescence. A role for reduced hemoglobin is indicated by the inhibition of fluorescence by pretreatment of cells with CO that binds strongly to ferrohemoglobin and nitrite that oxidizes ferrohemoglobin. Our earlier studies have shown the formation of fluorescent heme degradation products during the reaction of purified hemoglobin with hydrogen peroxide, which was also inhibited by CO and nitrite pretreatment. The fluorescence produced in red cells after the addition of hydrogen peroxide can, therefore, be attributed to fluorescent heme degradation products.     

Unusual susceptibility of heme proteins to damage by glucose during non-enzymatic glycation

Glucose modifies the amino groups of proteins by a process of non-enzymatic glycation, leading to potentially deleterious effects on structure and function that have been implicated in the pathogenesis of diabetic complications. These changes are extremely complex and occur very slowly. We demonstrate here that hemoglobin and myoglobin are extremely susceptible to damage by glucose in vitro through a process that leads to complete destruction of the essential heme group. This process appears in addition to the expected formation of so-called advanced glycation end products (AGEs) on lysine and other side-chains. AGE formation is enhanced by the iron released. In contrast, the heme group is not destroyed during glycation of cytochrome c, where the sixth coordination position of the heme iron is not accessible to solvent ligands. Glycation leads to reduction of ferricytochrome c in this case. Since hydrogen peroxide is known to destroy heme, and the destruction observed during glycation of hemoglobin and myoglobin is sensitive to catalase, we propose that the degradation process is initiated by hydrogen peroxide formation. Damage may then occur through reaction with superoxide generated (a reductant of ferricytochrome c), or hydroxyl radicals, or with both.     

Purification, partial characterization, and possible role of catalase in the bacterium Vitreoscilla

Vitreoscilla is a gram-negative bacterium that contains a unique bacterial hemoglobin that is relatively autoxidizable. It also contains a catalase whose primary function may be to remove hydrogen peroxide produced by this autoxidation. This enzyme was purified and partially characterized. It is a protein of 272,000 Da with a probable A2B2 subunit structure, in which the estimated molecular size of A is 68,000 Da and that of B, 64,000 Da, and an average of 1.6 molecules of protoheme IX per tetramer. The turnover number for its catalase activity was 27,000 s-1 and the Km for hydrogen peroxide was 16 mM. The peroxidase activity measured using o-dianisidine was 0.6% that of the catalase activity. Cyanide, which inhibited both catalase and peroxidase activities, bound the heme in a noncooperative manner. Azide inhibited the catalase activity but stimulated the peroxidase activity. An apparent compound II was formed by the reaction of the enzyme with ethyl hydrogen peroxide. The enzyme was reducible by dithionite, and the ferrous enzyme reacted with CO. The cellular content of Vitreoscilla hemoglobin varies during the growth cycle and in cells grown under different conditions, but the ratio of hemoglobin to catalase activity remained relatively constant, indicating possible coordinated biosynthesis and supporting the putative role of Vitreoscilla catalase as a scavenger of peroxide generated by Vitreoscilla hemoglobin.     

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.     

Autoxidation reactions of hemoglobin A free from other red cell components: a minimal mechanism

Rates of autoxidation reactions are determined for normal human hemoglobin A preparations which are extensively purified to remove all other redox active red cell components. The effects of superoxide dismutase, catalase, and hydroxyl radical scavengers on the reaction provide evidence for superoxide formation as the rate determing step followed by fast reactions that involve peroxide and hydroxyl radical. These results support a minimum overall mechanism for heme iron(II) oxidation and dioxygen reduction to water. Side reactions also occur that result in the modification and precipitation of the protein moiety; catalase and hydroxyl radical scavengers reduce the extent of the side reactions. These studies provide insight into the basis of oxidant stress in the red cell.     

A stoichiometric study of heme degradation catalyzed by the reconstituted heme oxygenase system with special consideration of the production of hydrogen peroxide during the reaction

In heme degradation catalyzed by the reconstituted heme oxygenase system, 8 to 9 mol of dioxygen and 11 to 12 mol of NADPH were consumed per mol of hemin lost, and about half the amount of dioxygen consumed could be accounted for by the production of hydrogen peroxide, which accumulated in the reaction mixture. Production of hydrogen peroxide in the heme oxygenase reaction did not appear to be due to the bimolecular dismutation of superoxide anions but rather seemed to be due to dissociation of a "peroxo" species formed on heme or intermediates of heme degradation. The hydrogen peroxide produced appeared to cause a considerable degree of non-specific degradation of heme (not leading to the formation of biliverdin) and also caused an inactivation of heme oxygenase. By taking into account the amount of dioxygen incorporated into hydrogen peroxide and some other factors, it could be deduced that 3 mol of dioxygen is consumed for the formation of 1 mol of biliverdin in the heme oxygenase 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.     

Production of superoxide anion during the oxidation of hemoglobin by menadione

Menadione in the presence of oxyhemoglobin will accelerate the formation of methomoglobin 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.     

Immuno-spin trapping of hemoglobin and myoglobin radicals derived from nitrite-mediated oxidation

The reaction of nitrite with hemoglobin has become of increasing interest due to the realization that plasma nitrite may act as an NO congener that is activated by interaction with red blood cells. Using a combination of spectrophotometry, immuno-spin trapping, and EPR, we have examined the formation of radicals during the oxidation of oxyhemoglobin (oxyHb) and oxymyoglobin (oxyMb) by inorganic nitrite. The proposed intermediacy of ferryl species during this oxidation was confirmed by spectrophotometry using multiple linear regression analysis of kinetic data. Using EPR/spin trapping, a protein radical was observed in the case of oxyMb, but not oxyHb, and was inhibited by catalase. When DMPO spin trapping was combined with Western blot analysis using an anti-DMPO-nitrone antibody, globin/DMPO adducts of both oxyHb and oxyMb were detected, and their formation was inhibited by catalase. Catalase effects confirm the intermediacy of hydrogen peroxide as a heme oxidant in this system. Spectrophotometric kinetic studies revealed that the presence of DMPO elongated the lag phase and decreased the maximal rate of oxidation of both oxyHb and oxyMb, which suggests that the globin radical plays an active role in the mechanism of autocatalysis. Interestingly, the oxidation of oxyHb or oxyMb by nitrite, but not by hydrogen peroxide, produced a diffusible radical that was able to generate spin adducts on a bystander protein. This indicates that the oxidation of oxyhemeproteins by nitrite may cause more widespread oxidative damage than the corresponding oxidation by hydrogen peroxide. The immuno-spin trapping technique represents an important new development for the study of the range and extent of protein oxidation by free radicals and oxidants.     

Formation and reactions of the Wurster's blue radical cation during the reaction of N,N,N',N'-tetramethyl-p-phenylenediamine with oxyhemoglobin.

The aromatic amine N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) reacted directly with oxyhemoglobin in a catalytic reaction resulting in formation of ferrihemoglobin. The second order rate constant of the reaction was found to be 5.5 M-1.s-1. The stable Wurster's blue radical cation produced ferrihemoglobin at rates greater 10(3) M-1.s-1, i.e. more than two orders of magnitude faster than the parent amine. In contrast to the reactions of aminophenols with hemoglobin, free hydrogen peroxide was formed which additionally contributed to ferrihemoglobin formation. Since ferrihemoglobin formation proceeded by two orders of magnitude faster than autoxidation of TMPD, oxyhemoglobin itself acted as an oxidase/peroxidase resulting in electron abstraction from the amino alone pair electrons.     

Mechanism of dismutation of superoxide produced during autoxidation of melanin pigments

The hydrogen peroxide produced during the autoxidation of melanin pigments has been measured using an oxidase electrode. The autoxidation has been shown to occur via the superoxide intermediate. The melanin pigment competes with superoxide dismutase for the scavenging of superoxide radicals. However, superoxide dismutase at high concentrations caused a substantial increase in the production of hydrogen peroxide, formed during melanin autoxidation. The implications of this finding are discussed in light of melanin's ability to function as a pseudo-dismutase.     

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.     

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.     

Pyridoxalated hemoglobin polyoxyethylene: a nitric oxide scavenger with antioxidant activity for the treatment of nitric oxide-induced shock

Hemoglobins modified for therapeutic use as either hemoglobin-based oxygen carriers or scavengers of nitric oxide are currently being evaluated in clinical trials. One such product, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), is a human-derived and chemically modified hemoglobin that has yielded promising results in Phase II clinical trials, and is entering a pivotal Phase III clinical trial for the treatment of shock associated with systemic inflammatory response syndrome (SIRS). Shock associated with SIRS is a NO-induced shock. PHP, a new mechanism-based therapy, has been demonstrated in clinical trials to have the expected hemodynamic activity of raising blood pressure and reducing catecholamine use, consistent with its mechanism of action as a NO scavenger. PHP is conjugated with polyoxyethylene, which results in a surface-decorated molecule with enhanced circulation time and stability as well as in attachment of soluble red blood cell enzymes, including catalase and superoxide dismutase. PHP thus contains an antioxidant profile similar to the intact red blood cell and is therefore resistant to both initial oxidative modification by oxidants such as hydrogen peroxide and subsequent ferrylhemoglobin formation. These studies suggest both that the redox activity of modified hemoglobins can be attenuated and that modified hemoglobins containing endogenous antioxidants, such as PHP, may have reduced pro-oxidant potential. These antioxidant properties, in addition to the NO-scavenging properties, may allow the use of PHP in other indications in which excess NO, superoxide, or hydrogen peroxide is involved, including ischemia-reperfusion injury and hemorrhagic shock.     

Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase.

Accompanying the autoxidation of hydroxylamine at pH 10.2, nitroblue tetrazolium was reduced and nitrite was produced in the presence of EDTA. The rate of autoxidation was negligible below pH 8.0, but sharply increased with increasing pH. The reduction of nitroblue tetrazolium was inhibited by superoxide dismutase, indicating the participation of superoxide anion radical in the autoxidation. Hydrogen peroxide stimulated the autoxidation and superoxide dismutase inhibited the hydrogen peroxide-induced oxidation, results which suggest the participation of hydrogen peroxide in autoxidation and in the generation of superoxide radical. An assay for superoxide dismutase using autoxidation of hydroxylamine is described.     

Mechanism of reaction of 3-hydroxyanthranilic acid with molecular oxygen

The autoxidation of the tryptophan metabolite, 3-hydroxyanthranilic acid, at pH 7 gives rise to a p-quinone dimer and cinnabarinic acid. A novel dimer formed by radical-radical coupling of 3-hydroxyanthranilic acid is also produced. Labelling studies have shown that the C-2 oxygen in the p-quinone dimer is derived from molecular oxygen. A product versus time study of this reaction has revealed that, in the absence of catalase, cinnabarinic acid is formed but undergoes decomposition by hydrogen peroxide. At pH 7, in the presence of catalase, both the p-quinone dimer and cinnabarinic acid are formed at approximately the same rate and this rate of formation increases with increasing pH. Inclusion of superoxide dismutase was found to increase the rate of formation of cinnabarinic acid, suggesting that superoxide ions may also cause decomposition of cinnabarinic acid. This was confirmed by treating cinnabarinic acid with superoxide. A mechanism involving a common anthranilyl radical intermediate is proposed to account for the formation of the different oxidation products.     

Stabilizing effect of organic solvents on oxyhemoglobin

The role of hemoglobin solutions as oxygen carriers in biotechnology are numerous, such as in the oxygen supply to biocatalysts or in the preparation of blood substitutes. However, the major barrier to the successful use of hemoglobin in biological and medical engineering is the autoxidation of heme iron during preparation, storage, and utilization. Fifty-six solvents, chosen among the group of Parker's classification, were studied with regard to the autoxidation kinetics of oxyhemoglobin under nondenaturant conditions. Among these solvents 27 present a concentration range in which the autoxidation rates were reduced compared to autoxidation in water. Three groups of solvent have been observed: one exhibiting only a destabilizing effect regardless of the solvent proportion, a second showing a strong stabilizing effect (k(H2O)/k(solvent) greater than 20) and a third showing a low stabilization (k(H2O)/k(solvent) less than 20). The most effective stabilizing solvents were glycerol, glycols, and alcohols. The effect of hydroorganic solvents could be explained by taking into account the globin solvation by water molecules. The solvents that enhance the structure of the water and form few hydrophobic interactions with globin prevent oxyhemoglobin autoxidation.     

Reaction of autoxidation products of penicillamine with myeloperoxidase

Spectral evidence is presented which shows that penicillamine is able to initiate the formation of the oxidized intermediates of myeloperoxidase in the absence of exogenous hydrogen peroxide. The autoxidation of penicillamine presumably produces superoxide which dismutates spontaneously to form hydrogen peroxide. Thus, the formation of both compounds II and III of myeloperoxidase was observed. We also report that penicillamine can directly reduce cytochrome c and therefore, it could possibly act as a one-electron donor to myeloperoxidase.