Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Jun 15;332(Pt 3):617–625. doi: 10.1042/bj3320617

Hypochlorite-induced damage to proteins: formation of nitrogen-centred radicals from lysine residues and their role in protein fragmentation.

C L Hawkins 1, M J Davies 1
PMCID: PMC1219520  PMID: 9620862

Abstract

Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl damages proteins by reaction with amino acid side-chains or backbone cleavage. Little information is available about the mechanisms and intermediates involved in these reactions. EPR spin trapping has been employed to identify radicals on proteins, peptides and amino acids after treatment with HOCl. Reaction with HOCl gives both high- and low-molecular-mass nitrogen-centred, protein-derived radicals; the yield of the latter increases with both higher HOCl:protein ratios and enzymic digestion. These radicals, which arise from lysine side-chain amino groups, react with ascorbate, glutathione and Trolox. Reaction of HOCl-treated proteins with excess methionine eliminates radical formation, which is consistent with lysine-derived chloramines (via homolysis of N-Cl bonds) being the radical source. Incubation of HOCl-treated proteins, after removal of excess oxidant, gives rise to both nitrogen-centred radicals, over a period of hours, and time-dependent fragmentation of the protein. Treatment with excess methionine or antioxidants (Trolox, ascorbate, glutathione) protects against fragmentation; urate and bilirubin do not. Chloramine formation and nitrogen-centred radicals are therefore key species in HOCl-induced protein fragmentation.

Full Text

The Full Text of this article is available as a PDF (563.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beck-Speier I., Leuschel L., Luippold G., Maier K. L. Proteins released from stimulated neutrophils contain very high levels of oxidized methionine. FEBS Lett. 1988 Jan 18;227(1):1–4. doi: 10.1016/0014-5793(88)81401-7. [DOI] [PubMed] [Google Scholar]
  2. Bellomo G., Thor H., Orrenius S. Modulation of cellular glutathione and protein thiol status during quinone metabolism. Methods Enzymol. 1990;186:627–635. doi: 10.1016/0076-6879(90)86158-r. [DOI] [PubMed] [Google Scholar]
  3. Bernofsky C., Bandara B. M., Hinojosa O., Strauss S. L. Hypochlorite-modified adenine nucleotides: structure, spin-trapping, and formation by activated guinea pig polymorphonuclear leukocytes. Free Radic Res Commun. 1990;9(3-6):303–315. doi: 10.3109/10715769009145689. [DOI] [PubMed] [Google Scholar]
  4. Birnbaumer M., Seibold A., Gilbert S., Ishido M., Barberis C., Antaramian A., Brabet P., Rosenthal W. Molecular cloning of the receptor for human antidiuretic hormone. Nature. 1992 May 28;357(6376):333–335. doi: 10.1038/357333a0. [DOI] [PubMed] [Google Scholar]
  5. Buettner G. R. Spin trapping: ESR parameters of spin adducts. Free Radic Biol Med. 1987;3(4):259–303. doi: 10.1016/s0891-5849(87)80033-3. [DOI] [PubMed] [Google Scholar]
  6. Candeias L. P., Stratford M. R., Wardman P. Formation of hydroxyl radicals on reaction of hypochlorous acid with ferrocyanide, a model iron(II) complex. Free Radic Res. 1994 Apr;20(4):241–249. doi: 10.3109/10715769409147520. [DOI] [PubMed] [Google Scholar]
  7. Clark R. A., Stone P. J., El Hag A., Calore J. D., Franzblau C. Myeloperoxidase-catalyzed inactivation of alpha 1-protease inhibitor by human neutrophils. J Biol Chem. 1981 Apr 10;256(7):3348–3353. [PubMed] [Google Scholar]
  8. Clark R. A., Szot S., Williams M. A., Kagan H. M. Oxidation of lysine side-chains of elastin by the myeloperoxidase system and by stimulated human neutrophils. Biochem Biophys Res Commun. 1986 Mar 13;135(2):451–457. doi: 10.1016/0006-291x(86)90015-x. [DOI] [PubMed] [Google Scholar]
  9. Daugherty A., Dunn J. L., Rateri D. L., Heinecke J. W. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J Clin Invest. 1994 Jul;94(1):437–444. doi: 10.1172/JCI117342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Davies J. M., Horwitz D. A., Davies K. J. Potential roles of hypochlorous acid and N-chloroamines in collagen breakdown by phagocytic cells in synovitis. Free Radic Biol Med. 1993 Dec;15(6):637–643. doi: 10.1016/0891-5849(93)90167-s. [DOI] [PubMed] [Google Scholar]
  11. Davies K. J., Lin S. W., Pacifici R. E. Protein damage and degradation by oxygen radicals. IV. Degradation of denatured protein. J Biol Chem. 1987 Jul 15;262(20):9914–9920. [PubMed] [Google Scholar]
  12. Davies M. J., Forni L. G., Willson R. L. Vitamin E analogue Trolox C. E.s.r. and pulse-radiolysis studies of free-radical reactions. Biochem J. 1988 Oct 15;255(2):513–522. [PMC free article] [PubMed] [Google Scholar]
  13. Davies M. J., Gilbert B. C., Haywood R. M. Radical-induced damage to bovine serum albumin: role of the cysteine residue. Free Radic Res Commun. 1993;18(6):353–367. doi: 10.3109/10715769309147502. [DOI] [PubMed] [Google Scholar]
  14. Davies M. J., Gilbert B. C., Haywood R. M. Radical-induced damage to proteins: e.s.r. spin-trapping studies. Free Radic Res Commun. 1991;15(2):111–127. doi: 10.3109/10715769109049131. [DOI] [PubMed] [Google Scholar]
  15. Dean R. T., Fu S., Stocker R., Davies M. J. Biochemistry and pathology of radical-mediated protein oxidation. Biochem J. 1997 May 15;324(Pt 1):1–18. doi: 10.1042/bj3240001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Duling D. R. Simulation of multiple isotropic spin-trap EPR spectra. J Magn Reson B. 1994 Jun;104(2):105–110. doi: 10.1006/jmrb.1994.1062. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Hawkins C. L., Davies M. J. Oxidative damage to collagen and related substrates by metal ion/hydrogen peroxide systems: random attack or site-specific damage? Biochim Biophys Acta. 1997 Feb 27;1360(1):84–96. doi: 10.1016/s0925-4439(96)00069-5. [DOI] [PubMed] [Google Scholar]
  19. Hazell L. J., Arnold L., Flowers D., Waeg G., Malle E., Stocker R. Presence of hypochlorite-modified proteins in human atherosclerotic lesions. J Clin Invest. 1996 Mar 15;97(6):1535–1544. doi: 10.1172/JCI118576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hazell L. J., Stocker R. Oxidation of low-density lipoprotein with hypochlorite causes transformation of the lipoprotein into a high-uptake form for macrophages. Biochem J. 1993 Feb 15;290(Pt 1):165–172. doi: 10.1042/bj2900165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hazell L. J., van den Berg J. J., Stocker R. Oxidation of low-density lipoprotein by hypochlorite causes aggregation that is mediated by modification of lysine residues rather than lipid oxidation. Biochem J. 1994 Aug 15;302(Pt 1):297–304. doi: 10.1042/bj3020297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kettle A. J. Neutrophils convert tyrosyl residues in albumin to chlorotyrosine. FEBS Lett. 1996 Jan 22;379(1):103–106. doi: 10.1016/0014-5793(95)01494-2. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Leeuwenburgh C., Rasmussen J. E., Hsu F. F., Mueller D. M., Pennathur S., Heinecke J. W. Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques. J Biol Chem. 1997 Feb 7;272(6):3520–3526. doi: 10.1074/jbc.272.6.3520. [DOI] [PubMed] [Google Scholar]
  25. Matheson N. R., Travis J. Differential effects of oxidizing agents on human plasma alpha 1-proteinase inhibitor and human neutrophil myeloperoxidase. Biochemistry. 1985 Apr 9;24(8):1941–1945. doi: 10.1021/bi00329a021. [DOI] [PubMed] [Google Scholar]
  26. McKenna S. M., Davies K. J. The inhibition of bacterial growth by hypochlorous acid. Possible role in the bactericidal activity of phagocytes. Biochem J. 1988 Sep 15;254(3):685–692. doi: 10.1042/bj2540685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pereira W. E., Hoyano Y., Summons R. E., Bacon V. A., Duffield A. M. Chlorination studies. II. The reaction of aqueous hypochlorous acid with alpha-amino acids and dipeptides. Biochim Biophys Acta. 1973 Jun 20;313(1):170–180. doi: 10.1016/0304-4165(73)90198-0. [DOI] [PubMed] [Google Scholar]
  28. Prütz W. A. Hypochlorous acid interactions with thiols, nucleotides, DNA, and other biological substrates. Arch Biochem Biophys. 1996 Aug 1;332(1):110–120. doi: 10.1006/abbi.1996.0322. [DOI] [PubMed] [Google Scholar]
  29. Test S. T., Lampert M. B., Ossanna P. J., Thoene J. G., Weiss S. J. Generation of nitrogen-chlorine oxidants by human phagocytes. J Clin Invest. 1984 Oct;74(4):1341–1349. doi: 10.1172/JCI111544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thomas E. L., Bozeman P. M., Jefferson M. M., King C. C. Oxidation of bromide by the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase. Formation of bromamines. J Biol Chem. 1995 Feb 17;270(7):2906–2913. doi: 10.1074/jbc.270.7.2906. [DOI] [PubMed] [Google Scholar]
  31. Thomas E. L., Grisham M. B., Jefferson M. M. Preparation and characterization of chloramines. Methods Enzymol. 1986;132:569–585. doi: 10.1016/s0076-6879(86)32042-1. [DOI] [PubMed] [Google Scholar]
  32. Thomas E. L., Jefferson M. M., Grisham M. B. Myeloperoxidase-catalyzed incorporation of amines into proteins: role of hypochlorous acid and dichloramines. Biochemistry. 1982 Nov 23;21(24):6299–6308. doi: 10.1021/bi00267a040. [DOI] [PubMed] [Google Scholar]
  33. Thomas E. L. Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli. Infect Immun. 1979 Feb;23(2):522–531. doi: 10.1128/iai.23.2.522-531.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vissers M. C., Winterbourn C. C. Oxidative damage to fibronectin. I. The effects of the neutrophil myeloperoxidase system and HOCl. Arch Biochem Biophys. 1991 Feb 15;285(1):53–59. doi: 10.1016/0003-9861(91)90327-f. [DOI] [PubMed] [Google Scholar]
  35. Wellman P. J., Davies B. T., Morien A., McMahon L. Modulation of feeding by hypothalamic paraventricular nucleus alpha 1- and alpha 2-adrenergic receptors. Life Sci. 1993;53(9):669–679. doi: 10.1016/0024-3205(93)90243-v. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Wright N. C. The Action of Hypochlorites on Amino-Acids and Proteins. Biochem J. 1926;20(3):524–532. doi: 10.1042/bj0200524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yang C. Y., Gu Z. W., Yang H. X., Yang M., Gotto A. M., Jr, Smith C. V. Oxidative modifications of apoB-100 by exposure of low density lipoproteins to HOCL in vitro. Free Radic Biol Med. 1997;23(1):82–89. doi: 10.1016/s0891-5849(96)00624-7. [DOI] [PubMed] [Google Scholar]
  39. Zgliczyński J. M., Stelmaszyńska T., Domański J., Ostrowski W. Chloramines as intermediates of oxidation reaction of amino acids by myeloperoxidase. Biochim Biophys Acta. 1971 Jun 16;235(3):419–424. doi: 10.1016/0005-2744(71)90281-6. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES