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. 1997 Oct 1;327(Pt 1):275–281. doi: 10.1042/bj3270275

Oxidation of neutrophil glutathione and protein thiols by myeloperoxidase-derived hypochlorous acid.

A C Carr 1, C C Winterbourn 1
PMCID: PMC1218791  PMID: 9355763

Abstract

Neutrophils, when stimulated, generate reactive oxygen species including myeloperoxidase-derived HOCl. There is an associated decrease in reduced glutathione (GSH) concentration. We have shown that neutrophil GSH levels decrease on exposure to reagent HOCl, whereas the equivalent concentration of H2O2 had no effect. GSH loss occurred without cell lysis, was not reversible, and was accompanied by the loss of an equivalent proportion of the total protein thiols. No glutathione disulphide was formed. Studies with 35S-labelled cells indicated that much of the GSH lost was accounted for by mixed disulphides with protein and a product that co-migrated on HPLC with a novel compound formed in the reaction of HOCl and pure GSH. The properties of this compound are consistent with an intramolecular sulphonamide. Neutrophils stimulated with PMA lost 30-40% of their GSH and a similar proportion of protein thiols. Little glutathione disulphide was formed and the products were the same as seen with HOCl-treated cells. From the results and studies with inhibitors and scavengers, we conclude that HOCl was responsible for the GSH loss. Propargylglycine and buthionine sulphoximine, inhibitors of glutathione synthesis, enhanced GSH loss, but their effects were due to the production of long-lived chloramines that oxidized GSH with greater efficiency than HOCl, rather than to the inhibition of GSH synthesis. The lack of thiol selectivity by HOCl and irreversibility of oxidation means that GSH will provide limited antioxidant protection for thiol enzymes in stimulated neutrophils.

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Selected References

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  1. Bilzer M., Lauterburg B. H. Glutathione metabolism in activated human neutrophils: stimulation of glutathione synthesis and consumption of glutathione by reactive oxygen species. Eur J Clin Invest. 1991 Jun;21(3):316–322. doi: 10.1111/j.1365-2362.1991.tb01376.x. [DOI] [PubMed] [Google Scholar]
  2. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  3. Chai Y. C., Ashraf S. S., Rokutan K., Johnston R. B., Jr, Thomas J. A. S-thiolation of individual human neutrophil proteins including actin by stimulation of the respiratory burst: evidence against a role for glutathione disulfide. Arch Biochem Biophys. 1994 Apr;310(1):273–281. doi: 10.1006/abbi.1994.1167. [DOI] [PubMed] [Google Scholar]
  4. Clancy R. M., Levartovsky D., Leszczynska-Piziak J., Yegudin J., Abramson S. B. Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: evidence for S-nitrosoglutathione as a bioactive intermediary. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3680–3684. doi: 10.1073/pnas.91.9.3680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cotgreave I. A., Moldéus P. Methodologies for the application of monobromobimane to the simultaneous analysis of soluble and protein thiol components of biological systems. J Biochem Biophys Methods. 1986 Nov;13(4-5):231–249. doi: 10.1016/0165-022x(86)90102-8. [DOI] [PubMed] [Google Scholar]
  6. Cross A. R., Jones O. T. The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutrophils. Specific labelling of a component polypeptide of the oxidase. Biochem J. 1986 Jul 1;237(1):111–116. doi: 10.1042/bj2370111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fliss H., Ménard M., Desai M. Hypochlorous acid mobilizes cellular zinc. Can J Physiol Pharmacol. 1991 Nov;69(11):1686–1691. doi: 10.1139/y91-250. [DOI] [PubMed] [Google Scholar]
  8. Folkes L. K., Candeias L. P., Wardman P. Kinetics and mechanisms of hypochlorous acid reactions. Arch Biochem Biophys. 1995 Oct 20;323(1):120–126. doi: 10.1006/abbi.1995.0017. [DOI] [PubMed] [Google Scholar]
  9. Grisham M. B., Jefferson M. M., Melton D. F., Thomas E. L. Chlorination of endogenous amines by isolated neutrophils. Ammonia-dependent bactericidal, cytotoxic, and cytolytic activities of the chloramines. J Biol Chem. 1984 Aug 25;259(16):10404–10413. [PubMed] [Google Scholar]
  10. Kettle A. J., Gedye C. A., Winterbourn C. C. Superoxide is an antagonist of antiinflammatory drugs that inhibit hypochlorous acid production by myeloperoxidase. Biochem Pharmacol. 1993 May 25;45(10):2003–2010. doi: 10.1016/0006-2952(93)90010-t. [DOI] [PubMed] [Google Scholar]
  11. Kettle A. J., Winterbourn C. C. Assays for the chlorination activity of myeloperoxidase. Methods Enzymol. 1994;233:502–512. doi: 10.1016/s0076-6879(94)33056-5. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Martin J., White I. N. Fluorimetric determination of oxidised and reduced glutathione in cells and tissues by high-performance liquid chromatography following derivatization with dansyl chloride. J Chromatogr. 1991 Jul 17;568(1):219–225. doi: 10.1016/0378-4347(91)80356-h. [DOI] [PubMed] [Google Scholar]
  14. Nauseef W. M., Metcalf J. A., Root R. K. Role of myeloperoxidase in the respiratory burst of human neutrophils. Blood. 1983 Mar;61(3):483–492. [PubMed] [Google Scholar]
  15. Radi R., Beckman J. S., Bush K. M., Freeman B. A. Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J Biol Chem. 1991 Mar 5;266(7):4244–4250. [PubMed] [Google Scholar]
  16. Roos D., Weening R. S., Voetman A. A., van Schaik M. L., Bot A. A., Meerhof L. J., Loos J. A. Protection of phagocytic leukocytes by endogenous glutathione: studies in a family with glutathione reductase deficiency. Blood. 1979 May;53(5):851–866. [PubMed] [Google Scholar]
  17. Seres T., Ravichandran V., Moriguchi T., Rokutan K., Thomas J. A., Johnston R. B., Jr Protein S-thiolation and dethiolation during the respiratory burst in human monocytes. A reversible post-translational modification with potential for buffering the effects of oxidant stress. J Immunol. 1996 Mar 1;156(5):1973–1980. [PubMed] [Google Scholar]
  18. Spielberg S. P., Boxer L. A., Oliver J. M., Allen J. M., Schulman J. D. Oxidative damage to neutrophils in glutathione synthetase deficiency. Br J Haematol. 1979 Jun;42(2):215–223. doi: 10.1111/j.1365-2141.1979.tb01126.x. [DOI] [PubMed] [Google Scholar]
  19. Stamler J. S., Singel D. J., Loscalzo J. Biochemistry of nitric oxide and its redox-activated forms. Science. 1992 Dec 18;258(5090):1898–1902. doi: 10.1126/science.1281928. [DOI] [PubMed] [Google Scholar]
  20. Stuehr D. J., Fasehun O. A., Kwon N. S., Gross S. S., Gonzalez J. A., Levi R., Nathan C. F. Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs. FASEB J. 1991 Jan;5(1):98–103. doi: 10.1096/fasebj.5.1.1703974. [DOI] [PubMed] [Google Scholar]
  21. Tatsumi T., Fliss H. Hypochlorous acid and chloramines increase endothelial permeability: possible involvement of cellular zinc. Am J Physiol. 1994 Oct;267(4 Pt 2):H1597–H1607. doi: 10.1152/ajpheart.1994.267.4.H1597. [DOI] [PubMed] [Google Scholar]
  22. Turkall R. M., Tsan M. F. Oxidation of glutathione by the myeloperoxidase system. J Reticuloendothel Soc. 1982 Apr;31(4):353–360. [PubMed] [Google Scholar]
  23. Vissers M. C., Winterbourn C. C. Oxidation of intracellular glutathione after exposure of human red blood cells to hypochlorous acid. Biochem J. 1995 Apr 1;307(Pt 1):57–62. doi: 10.1042/bj3070057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Voetman A. A., Loos J. A., Roos D. Changes in the levels of glutathione in phagocytosing human neutrophils. Blood. 1980 May;55(5):741–747. [PubMed] [Google Scholar]
  25. Wefers H., Sies H. Oxidation of glutathione by the superoxide radical to the disulfide and the sulfonate yielding singlet oxygen. Eur J Biochem. 1983 Dec 1;137(1-2):29–36. doi: 10.1111/j.1432-1033.1983.tb07791.x. [DOI] [PubMed] [Google Scholar]
  26. Weis M., Cotgreave I. C., Moore G. A., Norbeck K., Moldéus P. Accessibility of hepatocyte protein thiols to monobromobimane. Biochim Biophys Acta. 1993 Mar 10;1176(1-2):13–19. doi: 10.1016/0167-4889(93)90171-k. [DOI] [PubMed] [Google Scholar]
  27. Winterbourn C. C., Brennan S. O. Characterization of the oxidation products of the reaction between reduced glutathione and hypochlorous acid. Biochem J. 1997 Aug 15;326(Pt 1):87–92. doi: 10.1042/bj3260087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Winterbourn C. C., Metodiewa D. The reaction of superoxide with reduced glutathione. Arch Biochem Biophys. 1994 Nov 1;314(2):284–290. doi: 10.1006/abbi.1994.1444. [DOI] [PubMed] [Google Scholar]

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