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. 1989 Nov 1;263(3):823–828. doi: 10.1042/bj2630823

Influence of superoxide on myeloperoxidase kinetics measured with a hydrogen peroxide electrode.

A J Kettle 1, C C Winterbourn 1
PMCID: PMC1133504  PMID: 2557013

Abstract

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.

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Selected References

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  1. Andrews P. C., Krinsky N. I. A kinetic analysis of the interaction of human myeloperoxidase with hydrogen peroxide, chloride ions, and protons. J Biol Chem. 1982 Nov 25;257(22):13240–13245. [PubMed] [Google Scholar]
  2. BEERS R. F., Jr, SIZER I. W. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem. 1952 Mar;195(1):133–140. [PubMed] [Google Scholar]
  3. Bakkenist A. R., de Boer J. E., Plat H., Wever R. The halide complexes of myeloperoxidase and the mechanism of the halogenation reactions. Biochim Biophys Acta. 1980 Jun 13;613(2):337–348. doi: 10.1016/0005-2744(80)90088-1. [DOI] [PubMed] [Google Scholar]
  4. Bolscher B. G., Wever R. A kinetic study of the reaction between human myeloperoxidase, hydroperoxides and cyanide. Inhibition by chloride and thiocyanate. Biochim Biophys Acta. 1984 Jul 17;788(1):1–10. doi: 10.1016/0167-4838(84)90290-5. [DOI] [PubMed] [Google Scholar]
  5. Bolscher B. G., Zoutberg G. R., Cuperus R. A., Wever R. Vitamin C stimulates the chlorinating activity of human myeloperoxidase. Biochim Biophys Acta. 1984 Jan 31;784(2-3):189–191. doi: 10.1016/0167-4838(84)90127-4. [DOI] [PubMed] [Google Scholar]
  6. Harrison J. E., Schultz J. Studies on the chlorinating activity of myeloperoxidase. J Biol Chem. 1976 Mar 10;251(5):1371–1374. [PubMed] [Google Scholar]
  7. Hoogland H., Dekker H. L., van Riel C., van Kuilenburg A., Muijsers A. O., Wever R. A steady-state study on the formation of Compounds II and III of myeloperoxidase. Biochim Biophys Acta. 1988 Aug 10;955(3):337–345. doi: 10.1016/0167-4838(88)90213-0. [DOI] [PubMed] [Google Scholar]
  8. Iwamoto H., Kobayashi T., Hasegawa E., Morita Y. Reaction of human myeloperoxidase with hydrogen peroxide and its true catalase activity. J Biochem. 1987 Jun;101(6):1407–1412. doi: 10.1093/oxfordjournals.jbchem.a122010. [DOI] [PubMed] [Google Scholar]
  9. Kettle A. J., Winterbourn C. C. Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid. Biochem J. 1988 Jun 1;252(2):529–536. doi: 10.1042/bj2520529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kettle A. J., Winterbourn C. C. The mechanism of myeloperoxidase-dependent chlorination of monochlorodimedon. Biochim Biophys Acta. 1988 Nov 23;957(2):185–191. doi: 10.1016/0167-4838(88)90271-3. [DOI] [PubMed] [Google Scholar]
  11. Klebanoff S. J. Myeloperoxidase-halide-hydrogen peroxide antibacterial system. J Bacteriol. 1968 Jun;95(6):2131–2138. doi: 10.1128/jb.95.6.2131-2138.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Klebanoff S. J., Rosen H. The role of myeloperoxidase in the microbicidal activity of polymorphonuclear leukocytes. Ciba Found Symp. 1978 Jun 6;(65):263–284. doi: 10.1002/9780470715413.ch15. [DOI] [PubMed] [Google Scholar]
  13. Rossi F. The O2- -forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim Biophys Acta. 1986 Nov 4;853(1):65–89. doi: 10.1016/0304-4173(86)90005-4. [DOI] [PubMed] [Google Scholar]
  14. Stelmaszyńska T., Zgliczyński J. M. Myeloperoxidase of human neutrophilic granulocytes as chlorinating enzyme. Eur J Biochem. 1974 Jun 1;45(1):305–312. doi: 10.1111/j.1432-1033.1974.tb03555.x. [DOI] [PubMed] [Google Scholar]
  15. Test S. T., Weiss S. J. Assay of the extracellular hydrogen peroxide pool generated by phagocytes. Methods Enzymol. 1986;132:401–406. doi: 10.1016/s0076-6879(86)32025-1. [DOI] [PubMed] [Google Scholar]
  16. Tsan M. F. Myeloperoxidase-mediated oxidation of methionine and amino acid decarboxylation. Infect Immun. 1982 Apr;36(1):136–141. doi: 10.1128/iai.36.1.136-141.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Weiss S. J., LoBuglio A. F. Phagocyte-generated oxygen metabolites and cellular injury. Lab Invest. 1982 Jul;47(1):5–18. [PubMed] [Google Scholar]
  18. Winterbourn C. C. Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride, and similarity of the oxidant to hypochlorite. Biochim Biophys Acta. 1985 Jun 18;840(2):204–210. doi: 10.1016/0304-4165(85)90120-5. [DOI] [PubMed] [Google Scholar]
  19. Winterbourn C. C., Garcia R. C., Segal A. W. Production of the superoxide adduct of myeloperoxidase (compound III) by stimulated human neutrophils and its reactivity with hydrogen peroxide and chloride. Biochem J. 1985 Jun 15;228(3):583–592. doi: 10.1042/bj2280583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yamazaki I., Tamura M., Nakajima R., Nakamura M. Physiological aspects of free-radical reactions. Environ Health Perspect. 1985 Dec;64:331–342. doi: 10.1289/ehp.8564331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zgliczynski J. M., Selvaraj R. J., Paul B. B., Stelmaszynska T., Poskitt P. K., Sbarra A. J. Chlorination by the myeloperoxidase-H2O2-Cl- antimicrobial system at acid and neutral pH. Proc Soc Exp Biol Med. 1977 Mar;154(3):418–422. doi: 10.3181/00379727-154-39684. [DOI] [PubMed] [Google Scholar]

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