Myeloperoxidase Compound Research Articles

Rapid, Reversible Oxygen Atom Transfer between an Oxomanganese(V) Porphyrin and Bromide: A Haloperoxidase Mimic with Enzymatic Rates.

The oxygen atom donor ability of the oxomanganese(V) porphyrin species 1 is comparable to that of myeloperoxidase compound I. This is one of the key findings of the reversible oxygen atom transfer reaction between 1 and bromide ion [Eq. (1)]. The well-behaved equilibrium 1⇌2 allows definition of the free energy of the oxoMnV species.

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.

Kinetics of oxidation of serotonin by myeloperoxidase compounds I and II

The oxidation of serotonin (5-hydroxytryptamine) by the myeloperoxidase intermediates compounds I and II was investigated by using transient-state spectral and kinetic measurements at 25.0 +/- 0.1 degrees C. Rapid scan spectra demonstrated that both compound I and compound II oxidize serotonin via one-electron processes. Rate constants for these reactions were determined using both sequential-mixing and single-mixing stopped-flow techniques. The second order rate constant obtained for the one-electron reduction of compound I to compound II by serotonin is (1.7 +/- 0.1) x 10(7) M(-1) x s(-1), and that for compound II reduction to native enzyme is (1.4 +/- 0.1) x 10(6) M(-1) x s(-1) at pH 7.0. The maximum pH of the compound I reaction with serotonin occurs in the pH range 7.0-7.5. At neutral pH, the rate constant for myeloperoxidase compound I reacting with serotonin is an order of magnitude larger than for its reaction with chloride, (2.2 +/- 0.2) x 10(6) M(-1) x s(-1). A direct competition of serotonin with chloride for myeloperoxidase compound I oxidation was observed. Our results suggest that serotonin may have a role to protect lipoproteins from oxidation and to prevent enzymes from inactivation caused by the potent oxidants HOCl and active oxygen species.

Kinetics of oxidation of aliphatic and aromatic thiols by myeloperoxidase compounds I and II

Myeloperoxidase (MPO) is the most abundant protein in neutrophils and plays a central role in microbial killing and inflammatory tissue damage. Because most of the non-steroidal anti-inflammatory drugs and other drugs contain a thiol group, it is necessary to understand how these substrates are oxidized by MPO. We have performed transient kinetic measurements to study the oxidation of 14 aliphatic and aromatic mono- and dithiols by the MPO intermediates, Compound I ( k 3) and Compound II ( k 4), using sequential mixing stopped-flow techniques. The one-electron reduction of Compound I by aromatic thiols (e.g. methimidazole, 2-mercaptopurine and 6-mercaptopurine) varied by less than a factor of seven (between 1.39±0.12×10 5 M −1 s −1 and 9.16±1.63×10 5 M −1 s −1), whereas reduction by aliphatic thiols was demonstrated to depend on their overall net charge and hydrophobic character and not on the percentage of thiol deprotonation or redox potential. Cysteamine, cysteine methyl ester, cysteine ethyl ester and α-lipoic acid showed k 3 values comparable to aromatic thiols, whereas a free carboxy group (e.g. cysteine, N-acetylcysteine, glutathione) diminished k 3 dramatically. The one-electron reduction of Compound II was far more constrained by the nature of the substrate. Reduction by methimidazole, 2-mercaptopurine and 6-mercaptopurine showed second-order rate constants ( k 4) of 1.33±0.08×10 5 M −1 s −1, 5.25±0.07×10 5 M −1 s −1 and 3.03±0.07×10 3 M −1 s −1. Even at high concentrations cysteine, penicillamine and glutathione could not reduce Compound II, whereas cysteamine (4.27±0.05×10 3 M −1 s −1), cysteine methyl ester (8.14±0.08×10 3 M −1 s −1), cysteine ethyl ester (3.76±0.17×10 3 M −1 s −1) and α-lipoic acid (4.78±0.07×10 4 M −1 s −1) were demonstrated to reduce Compound II and thus could be expected to be oxidized by MPO without co-substrates.

Kinetics of oxidation of serotonin by myeloperoxidase compounds I and II

Kinetics of oxidation of serotonin by myeloperoxidase compounds I and II

Reaction of myeloperoxidase compound I with chloride, bromide, iodide, and thiocyanate.

Myeloperoxidase plays a fundamental role in oxidant production by neutrophils. The enzyme uses hydrogen peroxide to oxidize chloride (Cl-), bromide (Br-), iodide (I-), and the pseudohalide thiocyanate (SCN-) to their respective hypohalous acids. This study for the first time presents transient kinetic measurements of the oxidation of these halides and thiocyanate by the myeloperoxidase intermediate compound I, using the sequential mixing stopped-flow technique. At pH 7 and 15 degrees C, the two-electron reduction of compound I to the native enzyme by Cl- has a second-order rate constant of (2.5 +/- 0.3) x 10(4) M(-1) s(-1), whereas reduction of compound I by SCN- has a second-order rate constant of (9.6 +/- 0.5) x 10(6) M(-1) s(-1). Iodide [(7.2 +/- 0.7) x 10(6) M(-1) s(-1)] is shown to be a better electron donor for compound I than Br- [(1.1 +/- 0.1) x 10(6) M(-1) s(-1)]. The pH dependence studies suggest that compound I reduction by (pseudo-)halides is controlled by a residue with a pKa of about 4.6. The protonation of this group is necessary for optimum (pseudo-)halide anion oxidation. These transient kinetic results are underlined by steady-state spectral and kinetic investigations. SCN- is shown to be most effective in shifting the system myeloperoxidase/hydrogen peroxide from the peroxidatic cycle to the halogenation cycle, whereas iodide is shown to be more effective than bromide which in turn is much more effective than chloride. Decreasing pH increases the rate of this transition. Our results show that thiocyanate is an important substrate of myeloperoxidase in most environments and that hypothiocyanate is likely to contribute to leukocyte antimicrobial activity.

Formation of Reactive Nitrogen Species during Peroxidase-catalyzed Oxidation of Nitrite: A POTENTIAL ADDITIONAL MECHANISM OF NITRIC OXIDE-DEPENDENT TOXICITY

Involvement of peroxynitrite (ONOO-) in inflammatory diseases has been implicated by detection of 3-nitrotyrosine, an allegedly characteristic protein oxidation product, in various inflamed tissues. We show here that nitrite (NO2-), the primary metabolic end product of nitric oxide (NO.), can be oxidized by the heme peroxidases horseradish peroxidase, myeloperoxidase (MPO), and lactoperoxidase (LPO), in the presence of hydrogen peroxide (H2O2), to most likely form NO.2, which can also contribute to tyrosine nitration during inflammatory processes. Phenolic nitration by MPO-catalyzed NO2- oxidation is only partially inhibited by chloride (Cl-), the presumed major physiological substrate for MPO. In fact, low concentrations of NO2- (2-10 microM) catalyze MPO-mediated oxidation of Cl-, indicated by increased chlorination of monochlorodimedon or 4-hydroxyphenylacetic acid, most likely via reduction of MPO compound II. Peroxidase-catalyzed oxidation of NO2-, as indicated by phenolic nitration, was also observed in the presence of thiocyanate (SCN-), an alternative physiological substrate for mammalian peroxidases. Collectively, our results suggest that NO2-, at physiological or pathological levels, is a substrate for the mammalian peroxidases MPO and lactoperoxidase and that formation of NO2. via peroxidase-catalyzed oxidation of NO2- may provide an additional pathway contributing to cytotoxicity or host defense associated with increased NO. production.

Kinetics of Oxidation of Tyrosine and Dityrosine by Myeloperoxidase Compounds I and II: IMPLICATIONS FOR LIPOPROTEIN PEROXIDATION STUDIES

The oxidation of lipoproteins is considered to play a key role in atherogenesis, and tyrosyl radicals have been implicated in the oxidation reaction. Tyrosyl radicals are generated in a system containing myeloperoxidase, H2O2, and tyrosine, but details of this enzyme-catalyzed reaction have not been explored. We have performed transient spectral and kinetic measurements to study the oxidation of tyrosine by the myeloperoxidase intermediates, compounds I and II, using both sequential mixing and single-mixing stopped-flow techniques. The one-electron reduction of compound I to compound II by tyrosine has a second order rate constant of (7.7 +/- 0.1) x 10(5) M-1 s-1. Compound II is then reduced by tyrosine to native enzyme with a second order rate constant of (1.57 +/- 0.06) x 10(4) M-1 s-1. Our study further revealed that, compared with horseradish peroxidase, thyroid peroxidase, and lactoperoxidase, myeloperoxidase is the most efficient catalyst of tyrosine oxidation at physiological pH. The second order rate constant for the myeloperoxidase compound I reaction with tyrosine is comparable with that of its compound I reaction with chloride: (4.7 +/- 0.1) x 10(6) M-1 s-1. Thus, although chloride is considered the major myeloperoxidase substrate, tyrosine is able to compete effectively for compound I. Steady state inhibition studies demonstrate that chloride binds very weakly to the tyrosine binding site of the enzyme. Coupling of tyrosyl radicals yields dityrosine, a highly fluorescent stable compound that had been identified as a possible marker for lipoprotein oxidation. We present spectral and kinetic data showing that dityrosine is further oxidized by both myeloperoxidase compounds I and II. The second order rate constants we determined for dityrosine oxidation are (1.12 +/- 0.01) x 10(5) M-1 s-1 for compound I and (7.5 +/- 0.3) x 10(2) M-1 s-1 for compound II. Therefore, caution must be exercised when using dityrosine as a quantitative index of lipoprotein oxidation, particularly in the presence of myeloperoxidase and H2O2.

Spectral and Kinetic Studies on the Formation of Myeloperoxidase Compounds I and II: Roles of Hydrogen Peroxide and Superoxide

The conversion of myeloperoxidase to compounds I and II in the presence of H2O2 has been reinvestigated in order to explain the abnormal stoichiometry of compound I formation and the fast spontaneous decay of compound I to compound II. Rapid-scan studies show that at least a 20-fold excess of H2O2 is required to obtain a good spectrum of relatively pure compound I; a further increase in H2O2 concentration causes compound I to be reduced to compound II, which is a very stable intermediate. Compound I formation is reversible, with an apparent second-order forward rate constant of (1.8 +/- 0.1) x 10(7) M-1 s-1 and a reverse rate constant of 58 +/- 4 s-1, giving a constant of 3.2 microM for the dissociation of compound I to native enzyme and H2O. This reversibility is one factor that can explain the large excess of H2O2 required to form compound I. The apparent second-order rate constant for compound II formation from compound I and H2O2 is (8.2 +/- 0.2) x 10(4) M-1 s-1. We confirm pH dependence studies, which suggest that the formation of compounds I and II is controlled by a residue in the enzyme with a pKa of about 4.0. Excess H2O2 is also converted to O2 via catalase activity of the enzyme. However, we do not consider this a dominant pathway because it fails to account for the fast spontaneous reduction of compound I to compound II. The time courses for both the decay of compound I and the formation of compound II are biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic studies on the reaction of compound II of myeloperoxidase with ascorbic acid. Role of ascorbic acid in myeloperoxidase function.

Ascorbic acid is known to stimulate leukocyte functions. In a recent publication it was suggested that the role of ascorbic acid is to reduce compound II of myeloperoxidase back to the native enzyme (Bolscher, B. G. J. M., Zoutberg, G. R., Cuperus, R. A., and Wever, R. (1984) Biochim. Biophys. Acta 784, 189-191). In this paper we report rapid spectral scan and transient state kinetic results on the reaction of three myeloperoxidase compounds II, namely, human neutrophil myeloperoxidase, canine myeloperoxidase, and bovine spleen heme protein with ascorbate. We show by rapid scan spectra that compound II does not pass through any other intermediate when ascorbic acid reduces it back to native form. We also show that the reactions of all three compounds II involve a simple binding interaction before enzyme reduction with an apparent dissociation constant of 6.3 +/- 0.9 x 10(-4) to 2.0 +/- 0.3 x 10(-3)M and a first-order rate constant for reduction of 12.6 +/- 0.6 to 18.8 +/- 1.3 s-1. The optimum pH is 4.5, and at this pH the activation energy for the reaction is 13.2 kJ mol-1. Results of this work lend further evidence that the spleen green heme protein is very similar if not identical to leukocyte myeloperoxidase based on a comparison of spectral scans, pH-rate profiles, and kinetic parameters. We demonstrate that chloride cannot reduce compound II whereas iodide reduces compound II to native enzyme at a rate comparable to that of ascorbate. This explains why ascorbate accelerates chlorination but inhibits iodination. Formation of compound II is a dead end for the generation of hypochlorous acid; ascorbate regenerates more native enzyme to enhance the chlorination reaction namely: myeloperoxidase + peroxide----compound I followed by compound I + chloride----HOCl. On the other hand, ascorbate is a competitor with iodide for both compounds I and II and so inhibits iodination.

Inhibition of myeloperoxidase by salicylhydroxamic acid

Salicylhydroxamic acid inhibited the luminol-dependent chemiluminescence of human neutrophils stimulated by phorbol 12-myristate 13-acetate or the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). This compound had no inhibitory effect on the kinetics of O2.- generation or O2 uptake during the respiratory burst, but inhibited both the peroxidative activity of purified myeloperoxidase and the chemiluminescence generated by a cell-free myeloperoxidase/H2O2 system. The concentration of salicylhydroxamic acid necessary for complete inhibition of myeloperoxidase activity was 30-50 microM (I50 values of 3-5 microM) compared with the non-specific inhibitor NaN3, which exhibited maximal inhibition at 100-200 microM (I50 values of 30-50 microM). Whereas taurine inhibited the luminol chemiluminescence of an H2O2/HOC1 system by HOC1 scavenging, this compound had little effect on myeloperoxidase/H2O2-dependent luminol chemiluminescence; in contrast, 10 microM-salicylhydroxamic acid did not quench HOC1 significantly but greatly diminished myeloperoxidase/H2O2-dependent luminol chemiluminescence, indicating that its effects on myeloperoxidase chemiluminescence were largely due to peroxidase inhibition rather than non-specific HOC1 scavenging. Salicylhydroxamic acid prevented the formation of myeloperoxidase Compound II, but only at low H2O2 concentrations, suggesting that it may compete for the H2O2-binding site on the enzyme. These data suggest that salicylhydroxamic acid may be used as a potent inhibitor to delineate the function of myeloperoxidase in neutrophil-mediated inflammatory events.

Inhibition of peroxidase-catalyzed reactions by deferoxamine

Phagocytes generate superoxide (O ⨪ 2) and hydrogen peroxide (H 2O 2) 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 H 2O 2 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 + H 2O 2 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 H 2O 2 was not seen when H 2O 2 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 oxidantdependent damage needs to be interpreted with caution.

Identification and properties of an oxoferryl structure in myeloperoxidase compound II.

Myeloperoxidase compound II has been characterized by using optical absorption and resonance Raman spectroscopies. Compared to compounds II in other peroxidases, the electronic and vibrational properties of this intermediate are strongly perturbed due to the unusual active-site iron chromophore that occurs in myeloperoxidase. Despite this difference in prosthetic group, however, other properties of myeloperoxidase compound II are similar to those observed for this intermediate in the more common peroxidases (horseradish peroxidase in particular). Two forms of the myeloperoxidase intermediate species, each with distinct absorption spectra, are recognized as a function of pH. We present evidence consistent with interconversion of these two forms via a heme-linked ionization of a distal amino acid residue with a pKa congruent to 9. From resonance Raman studies of isotopically labeled species at pH 10.7, we identify an iron-oxygen stretching frequency at 782 cm-1, indicating the presence of an oxoferryl (O = FeIV) group in myeloperoxidase compound II. We further conclude that the oxo ligand is not hydrogen bonded above the pKa but possibly exhibits oxygen exchange with the medium at pH values below the pKa due to hydrogen bonding of the oxo ligand to the distal protein group.

Spectral properties of myeloperoxidase Compounds II and III

In order to resolve the confusion about the spectral properties of myeloperoxidase Compound II and Compound III (myeloperoxidase is donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7), the absorbance spectra in the visible and ultraviolet regions were measured under conditions where either Compound II or Compound III was present. Peak positions, isosbestic points and absorption coefficients are presented. We conclude that in most studies on Compound II or Compound III, mixtures of these compounds had been present. Our data indicate that the relative contributions of Compound II and Compound III in a sample can be determined from the absorbance ratio A 625 nm/ A 456 nm. The optical absorbance spectrum of myeloperoxidase compound III was not affected by pH (pH 3–8). The absorbance spectrum of Compound II, however, was dependent on pH. The absorbance spectrum of Compound II at high pH is described.

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.

Formation of myeloperoxidase compound II during aerobic stimulation of rat neutrophils.

We have made simultaneous spectrophotometric and O2 measurements on suspensions of rat neutrophils during activation of the respiratory burst. Under aerobic conditions an absorption increase attributable to myeloperoxidase compound II was observed in parallel with the rapid phase of O2 uptake. Identification of this compound was confirmed by analysis of a spectrum obtained with purified myeloperoxidase and H2O2. Whereas a second addition of stimulus did not increase O2 uptake any further, a second phase of myeloperoxidase release and compound II formation was observed. These results suggest that in vivo myeloperoxidase reacts with H2O2 generated via the respiratory burst to form compound II under conditions in which the chlorination reaction would be the expected major pathway.

発光化合物を用いるか粒球，マクロファージの酸化種生成能の測定法

Chemicals emitting light in visible region, when they are oxidized at physiological pH, have been used for testing the generation of active oxygen species and related oxidants. Of these chemicals, luminol has been widely used for testing the function of phagocytizing monocytes and granulocytes. Luminol is known to be oxidized to aminophthalic dianion which in turn emits a light with maximum at 425 nm in aqueous media. The various oxidants generated during phagoctizing, such as O-2, -O Cl and myeloperoxidase compound III, are involved in luminol dependent luminescences.Lucigenin, also a chemiluminescence compound, emits a light in visible region during phagocytizing, but produce much move weaker light compared with luminol in phagocytizing granulocytes. Lucigenin-dependent luminescence, however, participates in the generation of O-2 (probably also H2O2) but not in the myeloperoxidase-mediated reactions.Very sensitive and specific method for testing an ability of O- generation in phagocitizing granulocytes and macrophages is considered to be a chemilunescence probe with a crypridina luciferin analog. The compound is, in the main, oxidized to an excited carbonyl in singlet state by O-, but not by H2O2. Under specified conditions, O generating ability of macrophages and granulocytes can be calculated and expressed as xanthine oxidase units.

Myeloperoxidase of the leukocyte of normal blood III. The reaction of ferric myeloperoxidase with superoxide anion

Myeloperoxidase (donor:H 2O 2 oxidoreductase, EC 1.11.1.7) Compound III was formed by the addition of superoxide anions (O 2 −), which had been produced from the electrolytic reduction of molecular oxygen in an aprotic solvent, to a solution of ferric myeloperoxidase. The formation of Compound III was inhibited by superoxide dismutase from pig erythrocytes. It is concluded that myeloperoxidase Compound III can be formed from the direct reaction between ferric myeloperoxidase and (O 2 −. By the use of electron spin resonance the concentration of O 2 − in a cathodic chamber was found to reach approx. 10 mM.

Myeloperoxidase of the leukocyte of normal blood. II. The oxidation-reduction reaction mechanism of the myeloperoxidase system

The initial reaction of the aerobic oxidation of NADH in the myeloperoxidase (donor: H 2O 2 oxidoreductase, EC 1.11.1.7) system was inhibited by superoxide dismutase when the latter had been added to the reaction mixture previously, but the oxidation of NADH was not inhibited when it was added halfway during the reaction. The formation of myeloperoxidase compound III in the aerobic solution containing NADH and 2,4-dichlorophenol was also inhibited by superoxide dismutase. From these results it was concluded that a superoxide anion radical and myeloperoxidase compound III are very important active intermediates for the aerobic myeloperoxidase-catalyzed oxidation of NADH. Myeloperoxidase compound III can be formed by direct reaction between ferric myeloperoxidase and O 2 −, and the electronic structure of myeloperoxidase compound III can be considered to be Fe 3+O 2 −. Cytochrome b 5 prepared from thrombocytes was effectively reduced by the NADH-myeloperoxidase system. Myeloperoxidase catalyzed a reaction between epinephrine and H 2O 2. The light absorption maxima of a product of the reaction between epinphrine and H 2O 2 corresponds to those of adrenochrome. Myeloperoxidase also catalyzed the reaction between epinephrine analogues (norepinephrine, dopamine, DOPA, tyrosine and phenylalanine) and H 2O 2.

Myeloperoxidase of the leukocyte of normal blood. I. Reaction of myeloperoxidase with hydrogen peroxide

Myeloperoxidase (donor:H 2O 2 oxidoreductase, EC 1.11.1.7; also known as verdoperoxidase, neutrophil peroxidase) prepared from normal peripheral blood leukocyte of pig reacted with hydrogen peroxide to form three compounds which correspond to Compounds I, II and III, well characterized in the reaction between hydrogen peroxide and peroxidases of horseradish and milk. The light absorption spectra of these compounds were found to be quite peculiar. Myeloperoxidase Compound III was formed during the aerobic oxidation of NADH and also from a direct reaction between ferrous myeloperoxidase and molecular oxygen. Ferrous myeloperoxidase formed a complex with CO under restricted conditions. From the results obtained it is concluded that, in spite of the remarkable anomaly in the absorption spectra, the reaction mechanism of myeloperoxidase is similar to that of peroxidases of horseradish and milk.