The mechanism of myeloperoxidase-dependent chlorination of monochlorodimedon

Chlorination of monochlorodimedon is routinely used to measure the production of hypochlorous acid catalysed by myeloperoxidase from H2O2 and Cl-. We have found that the myeloperoxidase/H2O2/Cl- system, at pH 7.8, catalysed the loss of monochlorodimedon with a rapid burst phase followed by a much slower steady-state phase. The loss of monochlorodimedon in the absence of Cl- was only 10% of the steady-state rate in the presence of Cl-, which indicates that the major reaction of monochlorodimedon was with hypochlorous acid. During the steady-state reaction, myeloperoxidase was present as 100% compound II, which cannot participate directly in hypochlorous acid formation. Monochlorodimedon was necessary for formation of compound II, since it was not formed in the presence of methionine. Both the amount of hypochlorous acid formed during the burst phase, and the steady-state rate of hypochlorous acid production, increased with increasing concentrations of myeloperoxidase and with decreasing concentrations of monochlorodimedon. Inhibition by monochlorodimedon was competitive with Cl-. From these results, and the ability of myeloperoxidase to slowly peroxidase monochlorodimedon in the absence of Cl-, we propose that the reaction of monochlorodimedon with the myeloperoxidase/H2O2/Cl- system involves a major pathway due to hypochlorous acid-dependent chlorination and a minor peroxidative pathway. Only a small fraction of compound I needs to react with monochlorodimedon instead of Cl- at each enzyme cycle, for compound II to rapidly accumulate. Monochlorodimedon, therefore, cannot be regarded as an inert detector of hypochlorous acid production by myeloperoxidase, but acts to limit the chlorinating activity of the enzyme. In the presence of reducing species that act like monochlorodimedon, the activity of myeloperoxidase would depend on the rate of turnover of compound II. Components of human serum promoted the conversion of ferric-myeloperoxidase to compound II in the presence of H2O2. We suggest, therefore, that in vivo the rate of turnover of compound II may determine the rate of myeloperoxidase-dependent production of hypochlorous acid by stimulated neutrophils.     

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.     

Inhibition of hyaluronan uptake in lymphatic tissue by chondroitin sulphate proteoglycan.

Stimulated neutrophils discharge large quantities of superoxide (O2.-), which dismutates to form H2O2. In combination with Cl-, H2O2 is converted into the potent oxidant hypochlorous acid (HOCl) by the haem enzyme myeloperoxidase. We have used an H2O2 electrode to monitor H2O2 uptake by myeloperoxidase, and have shown that in the presence of Cl- this accurately represents production of HOCl. Monochlorodimedon, which is routinely used to assay production of HOCl, inhibited H2O2 uptake by 95%. This result confirms that monochlorodimedon inhibits myeloperoxidase, and that the monochlorodimedon assay grossly underestimates the activity of myeloperoxidase. With 10 microM-H2O2 and 100 mM-Cl-, myeloperoxidase had a neutral pH optimum. Increasing the H2O2 concentration to 100 microM lowered the pH optimum to pH 6.5. Above the pH optimum there was a burst of H2O2 uptake that rapidly declined due to accumulation of Compound II. High concentrations of H2O2 inhibited myeloperoxidase and promoted the formation of Compound II. These effects of H2O2 were decreased at higher concentrations of Cl-. We propose that H2O2 competes with Cl- for Compound I and reduces it to Compound II, thereby inhibiting myeloperoxidase. Above pH 6.5, O2.- generated by xanthine oxidase and acetaldehyde prevented H2O2 from inhibiting myeloperoxidase, increasing the initial rate of H2O2 uptake. O2.- allowed myeloperoxidase to function optimally with 100 microM-H2O2 at pH 7.0. This occurred because, as previously demonstrated, O2.- prevents Compound II from accumulating by reducing it to ferric myeloperoxidase. In contrast, at pH 6.0, where Compound II did not accumulate, O2.- retarded the uptake of H2O2. We propose that by generating O2.- neutrophils prevent H2O2 and other one-electron donors from inhibiting myeloperoxidase, and ensure that this enzyme functions optimally at neutral pH.     

Mechanism of reaction of melatonin with human myeloperoxidase

Recently, it was suggested that melatonin (N-acetyl-5-methoxytryptamine) is oxidized by activated neutrophils in a reaction most probably involving myeloperoxidase (Biochem. Biophys. Res. Commun. (2000) 279, 657-662). Myeloperoxidase (MPO) is the most abundant protein of neutrophils and is involved in killing invading pathogens. To clarify if melatonin is a substrate of MPO, we investigated the oxidation of melatonin by its redox intermediates compounds I and II using transient-state spectral and kinetic measurements at 25 degrees C. Spectral and kinetic analysis revealed that both compound I and compound II oxidize melatonin via one-electron processes. The second-order rate constant measured for compound I reduction at pH 7 and pH 5 are (6.1 +/- 0.2) x 10(6) M(-1) s(-1) and (1.0 +/- 0.08) x 10(7) M(-1) s(-1), respectively. The rates for the one-electron reduction of compound II back to the ferric enzyme are (9.6 +/- 0.3) x 10(2) M(-1) s(-1) (pH 7) and (2.2 +/- 0.1) x 10(3) M(-1) s(-1) (pH 5). Thus, melatonin is a much better electron donor for compound I than for compound II. Steady-state experiments showed that the rate of oxidation of melatonin is dependent on the H(2)O(2) concentration, is not affected by superoxide dismutase, and is quickly terminated by sodium cyanide. Melatonin can markedly inhibit the chlorinating activity of MPO at both pH 7 and pH 5. The implication of these findings in the activated neutrophil is discussed.     

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.     

Reactions of pholasin with peroxidases and hypochlorous acid

The ability of myeloperoxidase (MPO) and horseradish peroxidase (HRP) to induce chemiluminescence (CL) in Pholasin (Knight Scientific, Plymouth, UK), the photoprotein of the Common Piddock Pholas dactylus, was studied. The oxidation of Pholasin by compound I or II of HRP induced an intense light emission, whereas native HRP showed only a small effect. The luminescence observed upon incubation of Pholasin with native MPO was diminished by preincubation with catalase. Considering the high instability of diluted MPO, it is concluded that traces of hydrogen peroxide in water converted MPO to its active forms, compound I and/or II, which are able to oxidize Pholasin. Indeed, the addition of hydrogen peroxide to a mixture of MPO and Pholasin induced an intense burst of light. This emission was enhanced in degree and duration in the absence of chloride. Hypochlorous acid, the reaction product of Cl(-) and compound I of MPO, was itself able to elicit a luminescent response in Pholasin and this luminescence was strongly inhibited by methionine and taurine. However, both of these HOCl scavengers only slightly reduced the light emission induced by MPO/H(2)O(2) in both the presence or absence of chloride. Thus, hypochlorous acid produced by the MPO/H(2)O(2)/Cl(-) system, under the conditions described in this study, did not contribute to Pholasin luminescence. The Pholasin luminescence elicited by formyl-leucyl-methionyl-phenylalanine (fMLP)-stimulated neutrophils depends both on superoxide anion radicals and higher oxidation states of myeloperoxidase (but not on hypochlorous acid). This is shown by the inhibition of luminescence with superoxide dismutase and potassium cyanide, together with the lack of effect of both methionine and taurine. The luminescence response is about eight times greater in cells stimulated with fMLP/cytochalasin B than with fMLP alone.     

Effect of dimethylthiourea on the neutrophil myeloperoxidase pathway

The sulfur-centered compound dimethylthiourea (DMTU) affords antioxidant protection in animal models of acute lung injury, an effect that has been attributed to its OH. scavenging properties. Although DMTU can also react with H2O2 in certain experimental systems, the effect of DMTU on the neutrophil myeloperoxidase (MPO) pathway has not been studied. DMTU (1-10 mM) completely blocked stable oxidants and hypochlorous acid formation by phorbol myristate acetate- and zymosan-stimulated neutrophils. DMTU also provided complete inhibition when incubated with cell-free supernatants after the formation of the MPO products. DMTU prevented the oxidative inactivation of alpha 1-antitrypsin by neutrophil-stable oxidants. Evidence that DMTU was oxidized by the MPO products was obtained by titration of oxidized DMTU with reduced glutathione. Surprisingly, supernatants from cells incubated with DMTU (10 mM) consumed two- to threefold higher amounts of reduced glutathione than supernatants from cells incubated with taurine (15 mM). Metabolic studies with stimulated neutrophils and experiments with the MPO enzyme system in a cell-free system suggested that DMTU acts by scavenging the products of the MPO pathway rather than by blocking H2O2 production in the intact cell. These findings demonstrate that DMTU blocks the neutrophil MPO pathway in addition to its known ability to scavenge other reactive O2 species. The capacity of DMTU to scavenge MPO products may explain some of its protective effects in acute lung injury.     

Myeloperoxidase-catalyzed inactivation of alpha 1-protease inhibitor by human neutrophils

We have examined the effect of the myeloperoxidase-hydrogen peroxide-halide system and of activated human neutrophils on the ability of serum alpha 1-protease inhibitor (alpha 1-PI) to bind and inhibit porcine pancreatic elastase. Exposure to the isolated myeloperoxidase system resulted in nearly complete inactivation of alpha 1-PI. Inactivation was rapid (10 to 20 s); required active myeloperoxidase, micromolar concentrations of H2O2 (or glucose oxidase as a peroxide generator), and a halide cofactor (Cl- or I-); and was blocked by azide, cyanide, and catalase. Intact neutrophils similarly inactivated alpha 1-PI over the course of 5 to 10 min. Inactivation required the neutrophils, a halide (Cl-), and a phorbol ester to activate secretory and metabolic activity. It was inhibited by azide, cyanide, and catalase, but not by superoxide dismutase. Neutrophils with absent myeloperoxidase or impaired oxidative metabolism (chronic granulomatous disease) failed to inactivate alpha 1-PI, and these defects were specifically corrected by the addition of myeloperoxidase or H2O2, respectively. Thus, stimulated neutrophils secrete myeloperoxidase and H2O2 which combine with a halide to inactivate alpha 1-PI. We suggest that leukocyte-derived oxidants, especially the myeloperoxidase system, may contribute to proteolytic tissue injury, for example in elastase-induced pulmonary emphysema, by oxidative inactivation of protective antiproteases.     

Reactions of superoxide with myeloperoxidase

When neutrophils ingest bacteria, they discharge superoxide and myeloperoxidase into phagosomes. Both are essential for killing of the phagocytosed micro-organisms. It is generally accepted that superoxide is a precursor of hydrogen peroxide which myeloperoxidase uses to oxidize chloride to hypochlorous acid. Previously, we demonstrated that superoxide modulates the chlorination activity of myeloperoxidase by reacting with its ferric and compound II redox states. In this investigation we used pulse radiolysis to determine kinetic parameters of superoxide reacting with redox forms of myeloperoxidase and used these data in a steady-state kinetic analysis. We provide evidence that superoxide reacts with compound I and compound III. Our estimates of the rate constants for the reaction of superoxide with compound I, compound II, and compound III are 5 x 10(6) M-1 s-1, 5.5 +/- 0.4 x 10(6) M-1 s-1, and 1.3 +/- 0.2 x 10(5) M-1 s-1, respectively. These reactions define new activities for myeloperoxidase. It will act as a superoxide dismutase when superoxide reacts consecutively with ferric myeloperoxidase and compound III. It will also act as a superoxidase by using hydrogen peroxide to oxidize superoxide via compound I and compound II. The favorable kinetics of these reactions indicate that, within the confines of a phagosome, superoxide will react with myeloperoxidase and affect the reactions it will catalyze. These interactions of superoxide and myeloperoxidase will have a major influence on the way neutrophils use oxygen to kill bacteria. Consequently, superoxide should be viewed as a cosubstrate that myeloperoxidase uses to elicit bacterial killing.     

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.     

Oxidation of hydroquinone by myeloperoxidase. Mechanism of stimulation by benzoquinone.

Myeloperoxidase (MPO) is a prime candidate for mediating the inflammatory tissue damage of neutrophils because it converts Cl- to the potent oxidant hypochlorous acid. It also oxidizes xenobiotics to reactive free radicals. We have found that the kinetics of oxidation of hydroquinone by myeloperoxidase are inadequately explained by the classical peroxidase mechanism. Peroxidation of hydroquinone displayed a distinct lag phase, which was practically abolished by excluding O2 and was eliminated by adding benzoquinone at the start of the reaction. Superoxide dismutase increased the rate of peroxidation by 40% but did not eliminate the lag phase. Spectral investigations revealed that during the initial phase of the reaction, MPO was converted to oxy-MPO, or compound III, by a mechanism that was not reliant on superoxide. Benzosemiquinone, however, was able to convert ferric-MPO to compound III. Both compound III and ferro-MPO reacted with benzoquinone to regenerate ferric-MPO. We propose that the lag phase occurs because benzosemiquinone reduces ferric-MPO to ferro-MPO, which rapidly binds O2 to form compound III. Since compound III is outside the peroxidation cycle, conversion of hydroquinone to benzoquinone is retarded. However, as benzoquinone accumulates, it oxidizes ferro-MPO and compound III to ferric-MPO, thereby increasing the rate of peroxidation. There is a minimal lag phase under an atmosphere of N2 because ferro-MPO would be rapidly oxidized by benzoquinone, without formation of compound III. We conclude that when substrates produce radicals capable of reducing ferric-MPO, they will be peroxidized efficiently only if oxy-MPO is readily recycled. Furthermore, these radicals will prevent MP3+ from reacting with H2O2, and thereby prevent the enzyme from oxidizing Cl- to hypochlorous acid. Thus, this mechanism could be exploited to prevent hypochlorous acid-mediated inflammatory tissue damage.     

Inhibition of peroxidase-catalyzed reactions by deferoxamine

Phagocytes generate superoxide (O2-.) and hydrogen peroxide (H2O2) and their interaction in an iron-catalyzed reaction to form hydroxyl radicals (OH.) (Haber-Weiss reaction) has been proposed. Deferoxamine chelates iron in a catalytically inactive form, and thus inhibition by deferoxamine has been employed as evidence for the involvement of OH. generated by the Haber-Weiss reaction. We report here that deferoxamine also inhibits reactions catalyzed by the peroxidases of phagocytes, i.e., myeloperoxidase (MPO) and eosinophil peroxidase (EPO). The reactions inhibited include iodination in the presence and absence of chloride and the oxidation of guaiacol. Iodination by MPO and H2O2 is stimulated by chloride due to the intermediate formation of hypochlorous acid (HOCl). Iodination by reagent HOCl also is inhibited by deferoxamine with the associated consumption of HOCl. Iron saturation of deferoxamine significantly decreased but did not abolish its inhibitory effect on iodination by MPO + H2O2 or HOCl. Deferoxamine did not affect the absorption spectrum of MPO, suggesting that it does not react with or remove the heme iron. The conversion of MPO to Compound II by H2O2 was not seen when H2O2 was added to MPO in the presence of deferoxamine, suggesting either that deferoxamine inhibited the formation of Compound II by acting as an electron donor for MPO Compound I or that deferoxamine immediately reduced the Compound II formed. Iodination by stimulated neutrophils also was inhibited by deferoxamine, suggesting an effect on peroxidase-catalyzed reactions in intact cells. Thus deferoxamine has multiple effects on the formation and activity of phagocyte-derived oxidants and therefore its inhibitory effect on oxidant-dependent damage needs to be interpreted with caution.     

Immunosuppression by activated human neutrophils. Dependence on the myeloperoxidase system

An in vitro model system was used to define the mechanism of interaction between human neutrophils and lymphocytes. Blood mononuclear leukocytes were exposed to purified neutrophils in the presence of a neutrophil-activating agent (phorbol ester, lectin, or opsonized particle). The treated mononuclear cells displayed a marked decrease in both natural killer activity and mitogen-dependent DNA synthesis, but no change in viability. This functional suppression was dependent on neutrophil number, stimulus concentration, and duration of exposure. Lymphocytes were protected by addition of catalase, but not superoxide dismutase. Neutrophils defective in oxidative metabolism (chronic granulomatous disease) failed to suppress lymphocyte function unless an H2O2-generating system, glucose oxidase plus glucose, was added. The patients' neutrophils provided a factor, possibly myeloperoxidase, which interacted with the glucose oxidase system. The immunosuppressive effect of normal neutrophils was diminished when chloride was omitted from the cultures and was enhanced when chloride was replaced by iodide. Myeloperoxidase-deficient neutrophils were partially defective in suppressing lymphocytes and this was corrected by addition of purified myeloperoxidase. Paradoxically, azide caused enhancement of suppression that depended on the neutrophil oxidative burst, but not on myeloperoxidase and was mediated at least in part by an effect of azide on the target mononuclear leukocytes. These data indicate that suppression of lymphocyte function by activated neutrophils is mediated by the secretion of myeloperoxidase and H2O2 that react with halides to form immunosuppressive products. Moreover, the mononuclear leukocytes contain an azide-sensitive factor, probably catalase, which provides partial protection against injury by neutrophil products. These dynamic interactions may be important local determinants of the immune response.     

Role of monochloramine in the oxidation of erythrocyte hemoglobin by stimulated neutrophils

Stimulation of the oxygen (O2) metabolism of isolated human neutrophilic leukocytes resulted in oxidation of hemoglobin of autologous erythrocytes without erythrocyte lysis. Hb oxidation could be accounted for by reduction of O2 to superoxide (O-2) by the neutrophils, dismutation of O-2 to yield hydrogen peroxide (H2O2), myeloperoxidase-catalyzed oxidation of chloride (Cl-) by H2O2 to yield hypochlorous acid (HOCl), the reaction of HOCl with endogenous ammonia (NH+4) to yield monochloramine ( NH2Cl ), and the oxidative attack of NH2Cl on erythrocytes. NH2Cl was detected when HOCl reacted with the NH+4 and other substances released into the medium by neutrophils. The amount of NH+4 released was sufficient to form the amount of NH2Cl required for the observed Hb oxidation. Oxidation was increased by adding myeloperoxidase or NH+4 to increase NH2Cl formation. Due to the volatility of NH2Cl , Hb was oxidized when neutrophils and erythrocytes were incubated separately in a closed container. Oxidation was decreased by adding catalase to eliminate H2O2, dithiothreitol to reduce HOCl and NH2Cl , or taurine to react with HOCl or NH2Cl to yield taurine monochloramine . NH2Cl was up to 50 times more effective than H2O2, HOCl, or taurine monochloramine as an oxidant for erythrocyte Hb, whereas HOCl was up to 10 times more effective than NH2Cl as a lytic agent. NH2Cl contributes to oxidation of erythrocyte components by stimulated neutrophils and may contribute to other forms of neutrophil oxidative cytotoxicity.     

Characterization of nitric oxide consumption pathways by normal,chronic granulomatous disease and myeloperoxidase-deficient human neutrophils

The detailed mechanisms by which acutely activated leukocytes metabolize NO and regulate its bioactivity are unknown. Therefore, healthy, chronic granulomatous disease (CGD) or myeloperoxidase (MPO)-deficient human neutrophils were examined for their ability to consume NO and attenuate its signaling. fMLP or PMA activation of healthy neutrophils caused NO consumption that was fully blocked by NADPH oxidase inhibition, and was absent in CGD neutrophils. Studies using MPO-deficient neutrophils, enzyme inhibitors, and reconstituted NADPH oxidase ruled out additional potential NO-consuming pathways, including Fenton chemistry, PGH synthase, lipoxygenase, or MPO. In particular, the inability of MPO to consume NO resulted from lack of H(2)O(2) substrate since all superoxide (O(2)(-.) reacted to form peroxynitrite. For healthy or MPO-deficient cells, NO consumption rates were 2- to 4-fold greater than O(2)(-.) generation, significantly faster than expected from 1:1 termination of NO with O(2)(-.). Finally, fMLP or PMA-stimulated NO consumption fully blocked NO-dependent neutrophil cGMP synthesis. These data reveal NADPH oxidase as the central regulator of NO signaling in human leukocytes. In addition, they demonstrate an important functional difference between CGD and either normal or MPO-deficient human neutrophils, namely their inability to metabolize NO which will alter their ability to adhere and migrate in vivo.     

Human neutrophils transform prostaglandins by a myeloperoxidase-dependent mechanism

Intact human neutrophils, incubated with the soluble stimulant phorbol myristate acetate, discharge lysosomal components, generate oxygen metabolites, and transform exogenous 6-keto-prostaglandin F1 alpha, prostaglandin E2, and prostaglandin F2 alpha as assessed by thin layer radiochromatography. Neutrophils alone were incapable of transforming the prostaglandins. The addition of catalase or the myeloperoxidase inhibitor, azide, protected all three prostaglandins from the phorbol-stimulated neutrophils. Neither superoxide dismutase, heat-inactivated catalase, nor albumin had any inhibitory effect in this system. A model system consisting of glucose-glucose oxidase, as a source of H2O2, purified myeloperoxidase, and chloride was also able to transform the prostaglandins in an identical fashion. Neither glucose-glucose oxidase alone nor glucose-glucose oxidase and myeloperoxidase under chloride-free conditions were able to mediate this transformation. Thus, it appears that intact human neutrophils can transform prostaglandins by a mechanism dependent on H2O2, the lysosomal enzyme myeloperoxidase, and chloride. Given the importance of prostaglandins in regulating immune function, neutrophil-dependent prostaglandin transformation could play a novel role in modulating the inflammatory response.     

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.     

The interaction of 5,5-dimethyl-1-pyrroline-N-oxide with human myeloperoxidase and its potential impact on spin trapping of neutrophil-derived free radicals

Activation of human neutrophils leads to secretion of myeloperoxidase (MPO) with resulting generation of several oxidant species including OCl-. Spin trapping techniques employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are being applied increasingly to the investigation of free radical production by in vitro and in vivo experimental systems which contain neutrophils. Because such knowledge is critical to the interpretation of these data, we examined the impact of MPO and MPO-derived oxidants on DMPO spin adduct formation and stability. Addition of increasing concentrations of OCl- to DMPO yielded a number of EPR-detectable products including DMPO-OH. However, the concentration of OCl- required was in excess of that expected under physiologic conditions. Addition of purified human MPO and H2O2 to DMPO yielded EPR spectra consisting of small DMPO-OH peaks. The addition of MPO and H2O2 to preformed DMPO-OH and DMPO-CH3 resulted in rapid destruction of these spin adducts. Thus MPO/H2O2 appeared to both generate and destroy DMPO spin adducts. Neutrophils stimulated with phorbol myristate acetate or opsonized zymosan generated large DMPO-OOH and DMPO-OH peaks as well as small DMPO-CH3 peaks. Addition of the MPO inhibitor azide to the reaction mixture had no effecting on resulting DMPO-OH or DMPO-CH3 peak amplitudes but increased that of DMPO-OOH. These data suggest that MPO-derived oxidants likely have little impact on the nature of EPR spectra resulting from DMPO spin trapping of free radical species following neutrophil stimulation. Because MPO oxidants did appear to react with DMPO the ability of DMPO to protect a biologic target from in vitro MPO injury was examined. DMPO (greater than 10 mM) significantly decreased MPO/H2O2/Cl- -mediated erythrocyte hemolysis as assessed by 51Cr release. The experimental and/or pharmacologic implications of this observation require further study.     

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.     

Kinetic studies on the formation and decomposition of compounds II and III. Reactions of lignin peroxidase with H2O2.

The present study characterizes the serial reactions of H2O2 with compounds I and II of lignin peroxidase isozyme H1. These two reactions constitute part of the pathway leading to formation of the oxy complex (compound III) from the ferric enzyme. Compounds II and III are the only complexes observed; no compound III* is observed. Compound III* is proposed to be an adduct of compound III with H2O2, formed from the complexation of compound III with H2O2 (Wariishi, H., and Gold, M. H. (1990) J. Biol. Chem. 265, 2070-2077). We provide evidence that demonstrates that the spectral data, on which the formation of compound III* is based, are merely an artifact caused by enzyme instability and, therefore, rule out the existence of compound III*. The reactions of compounds II and III with H2O2 are pH-dependent, similar to that observed for reactions of compounds I and II with the reducing substrate veratryl alcohol. The spontaneous decay of the compound III of lignin peroxidase results in the reduction of ferric cytochrome c. The reduction is inhibited by superoxide dismutase, indicating that superoxide is released during the decay. Therefore, the lignin peroxidase compound III decays to the ferric enzyme through the dissociation of superoxide. This mechanism is identical with that observed with oxymyoglobin and oxyhemoglobin but different from that for horseradish peroxidase. Compound III is capable of reacting with small molecules, such as tetranitromethane (a superoxide scavenger) and fluoride (a ligand for the ferric enzyme), resulting in ferric enzyme and fluoride complex formation, respectively.