Diffusion of extracellular hydrogen peroxide into intracellular compartments of human neutrophils. Studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide

It is well known that catalase is transformed to nitric oxide-Fe2+-catalase by hydrogen peroxide (H2O2) plus azide. In this report, we show that myeloperoxidase is also inactivated by H2O2 plus azide. Utilizing this system, we studied the presence and source of intracellular H2O2 generated by activated neutrophils. Stimulation of neutrophils with phorbol myristate acetate (PMA, 100 ng/ml) plus azide (5 mM) for 30 min completely inactivated intragranular myeloperoxidase and reduced cytosolic catalase to 35% of resting cells. This intracellular inactivation of heme enzymes did not occur in normal neutrophils incubated with either PMA or azide alone or in neutrophils from patients with chronic granulomatous disease (CDG) which cannot produce H2O2 in response to PMA. Incubation of neutrophils with azide and a H2O2 generating system (glucose-glucose oxidase) inactivated 41% of neutrophil myeloperoxidase. Glutathione-glutathione peroxidase (GSH-GSH peroxidase), an extracellular H2O2 scavenger, totally protected neutrophil myeloperoxidase from inactivation by azide plus glucose-glucose oxidase. In addition, when a mixture of normal and CGD cells was stimulated with PMA in the presence of azide, 90% of the myeloperoxidase in CGD neutrophils was inactivated. Therefore, H2O2 released extracellularly from activated neutrophils can diffuse into cells. In contrast, myeloperoxidase in normal polymorphonuclear leukocytes stimulated with PMA in the presence of azide and GSH-GSH peroxidase was 75% inactivated. Thus, the results indicate that a GSH-GSH peroxidase-insensitive pool of H2O2 is also generated, presumably at the plasma membrane, and this pool of H2O2 can undergo direct internal diffusion to inactivate myeloperoxidase.     

Mechanism of reaction of myeloperoxidase with hydrogen peroxide and chloride ion.

The reaction of myeloperoxidase compound I (MPO-I) with chloride ion is widely assumed to produce the bacterial killing agent after phagocytosis. Two values of the rate constant for this important reaction have been published previously: 4.7 x 106 M-1.s-1 measured at 25 degrees C [Marquez, L.A. and Dunford, H.B. (1995) J. Biol. Chem. 270, 30434-30440], and 2.5 x 104 M-1.s-1 at 15 degrees C [Furtmüller, P.G., Burner, U. & Obinger, C. (1998) Biochemistry 37, 17923-17930]. The present paper is the result of a collaboration of the two groups to resolve the discrepancy in the rate constants. It was found that the rate constant for the reaction of compound I, generated from myeloperoxidase (MPO) and excess hydrogen peroxide with chloride, decreased with increasing chloride concentration. The rate constant published in 1995 was measured over a lower chloride concentration range; the 1998 rate constant at a higher range. Therefore the observed conversion of compound I to native enzyme in the presence of hydrogen peroxide and chloride ion cannot be attributed solely to the single elementary reaction MPO-I + Cl- --> MPO + HOCl. The simplest mechanism for the overall reaction which fit the experimental data is the following: MPO+H2O2 ⇄k-1k1 MPO-I+H2O MPO-I+Cl- ⇄k-2k2 MPO-I-Cl- MPO-I-Cl- -->k3 MPO+HOCl where MPO-I-Cl- is a chlorinating intermediate. We can now say that the 1995 rate constant is k2 and the corresponding reaction is rate-controlling at low [Cl-]. At high [Cl-], the reaction with rate constant k3 is rate controlling. The 1998 rate constant for high [Cl-] is a composite rate constant, approximated by k2k3/k-2. Values of k1 and k-1 are known from the literature. Results of this study yielded k2 = 2.2 x 106 M-1.s-1, k-2 = 1.9 x 105 s-1 and k3 = 5.2 x 104 s-1. Essentially identical results were obtained using human myeloperoxidase and beef spleen myeloperoxidase.     

Inactivation of human myeloperoxidase by hydrogen peroxide

Human myeloperoxidase (MPO) uses hydrogen peroxide generated by the oxidative burst of neutrophils to produce an array of antimicrobial oxidants. During this process MPO is irreversibly inactivated. This study focused on the unknown role of hydrogen peroxide in this process. When treated with low concentrations of H₂O₂ in the absence of reducing substrates, there was a rapid loss of up to 35% of its peroxidase activity. Inactivation is proposed to occur via oxidation reactions of Compound I with the prosthetic group or amino acid residues. At higher concentrations hydrogen peroxide acts as a suicide substrate with a rate constant of inactivation of 3.9 × 10⁻³ s⁻¹. Treatment of MPO with high H₂O₂ concentrations resulted in complete inactivation, Compound III formation, destruction of the heme groups, release of their iron, and detachment of the small polypeptide chain of MPO. Ten of the protein’s methionine residues were oxidized and the thermal stability of the protein decreased. Inactivation by high concentrations of H₂O₂ is proposed to occur via the generation of reactive oxidants when H₂O₂ reacts with Compound III. These mechanisms of inactivation may occur inside neutrophil phagosomes when reducing substrates for MPO become limiting and could be exploited when designing pharmacological inhibitors.     

Human neutrophils employ the myeloperoxidase/hydrogen peroxide/chloride system to oxidatively damage apolipoprotein A-I.

The structural integrity of apolipoprotein A-I (apo A-I) is critical to the physiological function of high-density lipoprotein (HDL). Oxidized lipoproteins are thought to be of central importance in atherogenesis, and oxidation products characteristic of myeloperoxidase, a heme protein secreted by activated phagocytes, have been detected in human atherosclerotic tissue. At plasma concentrations of halide ion, hypochlorous acid is a major product of the myeloperoxidase-hydrogen peroxide-chloride system. We therefore investigated the effects of activated human neutrophils, a potent source of myeloperoxidase and hydrogen peroxide, on the protein and lipid components of HDL. Both free and HDL-associated apo A-I exposed to activated human neutrophils underwent extensive degradation as monitored by RP-HPLC and Western blotting with a polyclonal antibody to apo A-I. Replacement of the neutrophils with reagent HOCl resulted in comparable damage (at molar oxidant : HDL subclass 3 ratio = 100) as observed in the presence of activated phagocytes. Apo A-I degradation by activated neutrophils was partially inhibited by the HOCl scavenger methionine, by the heme inhibitor azide, by chloride-free conditions, by the peroxide scavenger catalase, and by a combination of superoxide dismutase (SOD)/catalase, implicating HOCl in the cell-mediated reaction. The addition of a protease inhibitor (3,4-dichloroisocoumarin) further reduced the extent of apo A-I damage. In contrast to the protein moiety, there was little evidence for oxidation of unsaturated fatty acids or cholesterol in HDL3 exposed to activated neutrophils, suggesting that HOCl was selectively damaging apo A-I. Our observations indicate that HOCl generated by myeloperoxidase represents one pathway for protein degradation in HDL3 exposed to activated phagocytes.     

Reaction of human myeloperoxidase with hydrogen peroxide and its true catalase activity

The instability of human myeloperoxidase [EC 1.11.1.7] compound I, which was spontaneously reduced to compound II, and the abnormal stoichiometry of the reaction of myeloperoxidase with H2O2 were investigated. As to the former, a pretreatment of myeloperoxidase with H2O2 did not stabilize compound I, and no difference in its stability was observed between native (alpha 2 beta 2) and hemi (alpha beta) myeloperoxidase. From these results, it was thought that the instability of compound I was caused by neither the presence of endogenous donors nor the intramolecular reduction of compound I to compound II by the other heme in the native enzyme molecule. As for the latter, true catalase activity of myeloperoxidase was demonstrated by monitoring O2 evolution after the injection of H2O2 into the enzyme solution. Myeloperoxidase compound I reacted with H2O2 and returned to the ferric state with concomitant evolution of an O2 molecule. Accordingly, the abnormal stoichiometry of the reaction with H2O2 and a part of the instability of compound I can probably be ascribed to this true catalase activity.     

Oxidative inactivation of pneumolysin by the myeloperoxidase system and stimulated human neutrophils

Pneumolysin, a hemolytic toxin from Streptococcus pneumoniae, is a member of the group of thiol-activated, oxygen-labile cytolysins produced by various Gram-positive bacteria. The toxin activity of pneumolysin, as determined by lysis of 51Cr-labeled human erythrocytes, was destroyed on exposure to the neutrophil enzyme myeloperoxidase, hydrogen peroxide, and a halide (chloride or iodide). Detoxification required each component of the myeloperoxidase system and was prevented by the addition of agents that inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The entire myeloperoxidase system could be replaced by sodium hypochlorite at micromolar concentrations. Toxin inactivation was a function of time of exposure to the myeloperoxidase system (less than 1 min), the rate of formation of H2O2 (0.05 nmol/min), and the concentration of toxin employed. Toxin that had been inactivated by the myeloperoxidase system was reactivated on incubation with the reducing agent dithiothreitol. Pneumolysin was also inactivated when incubated with human neutrophils (10(5)) in the presence of a halide and phorbol myristate acetate, an activator of neutrophil secretion and oxygen metabolism. Toxin inactivation by stimulated neutrophils was blocked by azide, cyanide, or catalase, but not by superoxide dismutase. Neutrophils from patients with impaired oxygen metabolism (chronic granulomatous disease) or absent myeloperoxidase (hereditary deficiency) failed to inactivate the toxin unless they were supplied with an exogenous source of H2O2 or purified myeloperoxidase, respectively. Thus, inactivation of pneumolysin involved the secretion of myeloperoxidase and H2O2, which combined with extracellular halides to form agents (e.g., hypochlorite) capable of oxidizing the toxin. This example of oxidative inactivation of a cytolytic agent may serve as a model for phagocyte-mediated detoxification of microbial products.     

A pulse radiolysis investigation of the reactions of myeloperoxidase with superoxide and hydrogen peroxide

Using pulse radiolysis, the rate constant for the reaction of ferric myeloperoxidase with O2- to give compound III was measured at pH 7.8, and values of 2.1.10(6) M-1.s-1 for equine ferric myeloperoxidase and 1.1.10(6) M-1.s-1 for human ferric myeloperoxidase were obtained. Under the same conditions, the rate constant for the reaction of human ferric myeloperoxidase with H2O2 to give compound I was 3.1.10(7) M-1.s-1. Our results indicate that although the reaction of ferric myeloperoxidase with O2- is an order of magnitude slower than with H2O2, the former reaction is sufficiently rapid to influence myeloperoxidase-dependent production of hypochlorous acid by stimulated neutrophils.     

Tyrosine phosphorylation and its possible role in superoxide production by human neutrophils stimulated with FMLP and IgG.

Superoxide production by human neutrophils stimulated with FMLP and soluble aggregated human IgG were inhibited in a dose dependent manner by two kinds of tyrosine kinase inhibitors, erbstatin and genistein. Superoxide production stimulated with surface bound IgG, however, was scarcely inhibited by either inhibitor. Protein tyrosine phosphorylation studies with immunoblotting revealed specific tyrosine phosphorylation of a 40 Kd protein by soluble aggregated and surface bound IgG, and that of a 39 Kd protein, as well as the 40 Kd protein, by FMLP. These were all inhibited by the tyrosine kinase inhibitors. These data suggest that superoxide production induced by FMLP and soluble aggregated IgG are, at least in part, tyrosine kinase dependent, but the tyrosine kinases and/or substrates of tyrosine kinases involved may be different. In addition, tyrosine kinase independent pathways are also suggested to be involved in superoxide production by stimulation with surface bound IgG.     

Kinetics of superoxide production by stimulated neutrophils.

Oxygen-derived active species and superoxide radical in particular are generated and excreted upon granulocyte activation and are instrumental in host defense against bacterial and fungal infections. Associated with the activation of neutrophils is an apparent transitory oxy-radical production. Evidence from independent methods has previously suggested that radical production peaks shortly following neutrophil stimulation and decays within minutes. However, since neutrophil function in the body might reasonably be expected to last beyond the few minutes following stimulation, cessation of the production of oxy-radicals is unexpected. In an attempt to reconcile this discrepancy, the formation kinetics of superoxide by stimulated human neutrophils was reinvestigated by three independent methods: electron spin resonance, chemiluminescence, and ferricytochrome c reduction. The present results demonstrate that under appropriate experimental conditions stimulated neutrophils have the capacity to produce superoxide for several hours. The reasons for the previously reported "apparent" ephemeral nature of oxy-radical formation are discussed.     

Production of superoxide anion and hydrogen peroxide by capacitating buffalo (Bubalus bubalis) spermatozoa

In the present study attempts were made to detect and quantify the generation of superoxide anion (O(2)(*-)) and hydrogen peroxide (H(2)O(2)) by capacitating buffalo spermatozoa. Ejaculated buffalo spermatozoa were suspended in sp-TALP medium at 50x10(6)mL(-1) and incubated at 38.5 degrees C with 5% CO(2) in air in the absence or presence of heparin (a capacitation inducer) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) or diphenyleneiodonium (DPI, a flavoprotein inhibitor) for 6h. Production rate of O(2)(*-) and H(2)O(2) by spermatozoa at different hours of capacitation were measured by cytochrome c reduction and phenol red oxidation assays, respectively. Spermatozoa generated both O(2)(*-) and H(2)O(2) spontaneously and following stimulation with heparin and a significant increase of O(2)(*-) production was observed in the presence of NADPH. However, DPI inhibited this NADPH-induced O(2)(*-) production and suggested for existence of putative NADPH-oxidase that constitute a specific O(2)(*-) generating systems in buffalo spermatozoa. Results of our study indicated that buffalo spermatozoa generate O(2)(*-) and H(2)O(2) and production of these free radicals is induced during capacitation.     

A kinetic analysis of the interaction of human myeloperoxidase with hydrogen peroxide, chloride ions, and protons

The effect of H2O2, Cl-, and pH on human myeloperoxidase activity has been examined. The Km for H2O2 is shown to be affected by the combined presence of Cl- and acid pH conditions. The Km for H2O2 is independent of pH in the absence of Cl- and dependent on pH in the presence of Cl-. Conversely, the dependence of the Km for H2O2 on Cl- concentration increases as the pH decreases. A model is proposed in which Cl- has a dual role, acting both as a substrate and as an inhibitor. According to this model, the inhibitor Cl- binding site must be protonated prior to the binding of Cl- and is distinct from the substrate Cl- binding site which is unaffected by pH. The rate equation derived from this model is used to further analyze the data presented. The values of Km for H2O2 predicted by the rate equation are in good agreement with the experimentally determined values.     

Oxidation of lysine side-chains of elastin by the myeloperoxidase system and by stimulated human neutrophils

Exposure of [3H]-lysine labeled elastin to either purified myeloperoxidase plus H2O2 and halides or human neutrophils plus phorbol myristate acetate resulted in oxidation of lysine side chains quantitated as 3H2O release. In both the enzyme and cell system oxidation was blocked by azide, cyanide or catalase, but not by beta-aminopropionitrile, an inhibitor of lysyl oxidase. Myeloperoxidase-deficient neutrophils were ineffective unless exogenous myeloperoxidase was added. These data provide a biochemical basis for inflammatory changes in connective tissue proteins mediated by oxidant secretory products of neutrophils.     

Secretion of myeloperoxidase isoforms by human neutrophils.

The human neutrophil lysosomal enzyme, myeloperoxidase (MPO), exists in three major and chromatographically distinct forms, MPO I, MPO II, and MPO III. We used cation-exchange medium-pressure liquid chromatography and kinetic microenzyme assay (or spectrophotometric monitoring) to analyze the secretion of MPO isoforms by neutrophils exposed to N-formylmethionylleucylphenylalanine (FMLP), digitonin, the ionophore A23187, and serum-opsonized zymosan A (SOZ). All three MPO isomers were released into the fluid phase after neutrophils were exposed to these secretagogues. A significant proportional increase in MPO I was released when neutrophils were stimulated with SOZ. MPO I was released in higher proportions than found in the whole cell constituency when neutrophils were stimulated with FMLP + cytochalasin B, A23187, and digitonin, but this was not statistically significant.     

Oxidation of amino acids and peptides in reaction with myeloperoxidase, chloride and hydrogen peroxide

Oxidation was studied of N-acetyl derivatives of cystine, cysteine, methionine and glycyltryptophan employing the myeloperoxidase-Cl--H2O2 system at pH 4.5, 6.0 and 7.0. Moreover, oxidation of pentapeptide composed of Leu-Trp-Met-Arg-Phe-COOH with myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) and hypochlorite was also studied. It was found that amino-acid derivatives having an amino group bound to an acetyl residue react with functional groups of the side-chain. The -SH groups of N-acetylcysteine and the -SS- group of cystine oxidize to cysteic acid. Methionine residues oxidize to methionine sulphoxide, and tryptophan residues to a derivative of 2-oxoindolone. The same reaction products were obtained when respective amounts of hypochlorous acid were used instead of myeloperoxidase, Cl- and H2O2. Differences in the stoichiometry of reactions of myeloperoxidase-mediated oxidation and hypochlorite oxidation suggest differences in the reaction mechanisms of both studied systems. Interaction of the studied pentapeptide with myeloperoxidase-Cl(-)-H2O2 system as well as with hypochlorite showed that in the peptide molecule individual amino acids oxidize consecutively according to their susceptibility to oxidation. No splitting of peptide bonds was observed. Therefore, a modified peptide with methionine sulphoxide and and oxidized tryptophan incorporated into the molecule was obtained.     

Subcellular distribution of superoxide dismutases in human neutrophils. Influence of myeloperoxidase on the measurement of superoxide dismutase activity

We have identified two distinct pools of superoxide dismutase in fractions of human peripheral neutrophils obtained by the isopycnic fractionation of homogenates of the latter with linear sucrose gradients. Superoxide dismutase activity, observed with polyacrylamide gels impregnated with Nitro Blue Tetrazolium, was present in: (1) the mitochondrial fraction [density (rho) 1.169g/ml], containing the high-molecular-weight KCN-resistant enzyme, and (2) the cytoplasm fraction, containing the low-molecular-weight KCN-sensitive enzyme. Superoxide dismutase activity, observed with a quantitative assay involving cytochrome c, was present in: (1) the mitochondria, (2) the cytoplasm, and (3) the azurophil-granule fractions (rho=1.206 and 1.222g/ml). No substantial enzyme activity was observed in specific-granule fractions (rho=1.187g/ml) or in the membranous fraction (rho=1.136g/ml) in either assay. The apparent superoxide dismutase activity observed in the azurophil granules with the cytochrome c assay was attributable not to true superoxide dismutase but to myeloperoxidase, an enzyme found solely in the azurophil granules. In the presence of H(2)O(2), human neutrophil myeloperoxidase oxidized ferrocytochrome c. Thus, in the cytochrome c assay for superoxide dismutase, the oxidation of ferrocytochrome c by myeloperoxidase mimicked the inhibition of reduction of ferricytochrome c by superoxide dismutase. When myeloperoxidase was removed from azurophilgranule fractions by specific immuno-affinity chromatography, both myeloperoxidase and apparent superoxide dismutase activities were removed. It is concluded that there is no detectable superoxide dismutase in either the azurophil or specific granules of human neutrophils. Mitochondrial superoxide dismutase, 15% of the total dismutase activity of the cells, occurred only in fractions of density 1.160g/ml, where isocitrate dehydrogenase and cytochrome oxidase were also observed.     

Studies on the mechanism of superoxide release from human neutrophils stimulated with arachidonate

cis-Unsaturated fatty acids stimulate release of superoxide (O-2) by human neutrophils (Badwey, J. A., Curnutte, J. T., Robinson, J. M., Berde, C. B., Karnovsky, M. J., and Karnovsky, M. L. (1984) J. Biol. Chem. 259, 7870-7877). The rate of O-2 release due to arachidonate (105 +/- 24 S.D., nmol of O-2/min/10(7) cells) was comparable to optimal values obtained with other stimuli. Antagonists of calcium-binding proteins (i.e. phenothiazines, naphthalene sulfonamides) inhibited the release of O-2 in a fashion compatible with the involvement of calmodulin in these phenomena. Synthetic substrates for and an inhibitor of chymotrypsin-like proteases (e.g. N-benzoyl-L-tyrosine ethyl ester, L-1-tosylamido-2-phenylethyl chloromethyl ketone) also blocked O-2 release. Antagonists of calcium-binding proteins and of proteases were effective in this context with neutrophils stimulated with a variety of agents. The implications of these data for recent reports concerning the mechanism of action of cis-unsaturated fatty acids on phagocytes is discussed.     

Reaction of hydrogen peroxide with hexaaquacobalt(III) perchlorate

The reaction of with H₂O₂ in 1.0 M HClO₄/LiClO₄ was found to be first-order in both reactants and the [H⁺] dependence of the second-order rate constant is given by k_2obs = b/[H⁺], b at 25 °C is 26.4 ± 0.5 s⁻¹. The rate law shows a simple inverse dependence on [H⁺] that is consistent with a rapidly maintained equilibrium between and its hydrolyzed form Co(H₂O)₅(OH)²⁺, followed by the rate controlling step, i.e. oxidation of H₂O₂ by Co(H₂O)₅(OH)²⁺.     

Reaction of iron(III)-polyaminocarboxylate complexes with hydrogen peroxide: Correlation between ligand structure and reactivity

Reactions of iron(III) complexes with five polyaminocarboxylates and hydrogen peroxide in an alkaline solution were investigated. Iron(III) complexes of which the ring including two nitrogen and iron atoms is five-membered formed a well-known stable side-on peroxo adduct. On the other hand, iron(III) complexes which have a six-membered ring formed a short-lived side-on peroxo adduct and then changed to iron(II) complex and superoxide. Electrochemical measurements showed that the redox potentials of the iron complexes having a six-membered ring are higher than those of the complexes having a five-membered ring. These results indicate that the chelate size is an important factor for tuning the redox potential of the iron center and for the reactivity toward hydrogen peroxide.     

Endothelin-1 enhances superoxide generation of human neutrophils stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine

Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing the superoxide (O2-) generation of human neutrophils, but it enhanced the O2- generation stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) about 2-fold when the cells had been preincubated with ET-1 for 10 min at 37 degrees C. The concentration at which ET-1 was 50% effective was 1 x 10(-10) M, and the maximal effect was obtained at 1 x 10(-8) M. The enhancement was observed over the range of the effective concentrations of FMLP (10(-8)-10(-6) M). ET-1 did not promote the mobilization of intracellular calcium ions and the enhancing effect of ET-1 did not change when calcium ions were depleted. These findings indicate that ET-1 is a potent modulator of human neutrophils and may thus contribute to the inflammatory process.