HYPOCHLORITE-INDUCED DAMAGE TO PROTEINS - FORMATION OF NITROGEN-CENTERED RADICALS FROM LYSINE RESIDUES AND THEIR ROLE IN PROTEIN FRAGMENTATION

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 c leavage. Little information is available about the mechanisms and inte rmediates involved in these reactions. EPR spin trapping has been empl oyed to identify radicals on proteins, peptides and amino acids after treatment with HOCl. Reaction with HOCl gives both high- and low-molec ular-mass nitrogen-centred, protein-derived radicals; the yield of the latter increases with both higher HOCl:protein ratios and enzymic dig estion. These radicals, which arise from lysine side-chain amino group s, react with ascorbate, glutathione and Trolox. Reaction of HOCl-trea ted proteins with excess methionine eliminates radical formation, whic h 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 bil irubin do not. Chloramine formation and nitrogen-centred radicals are therefore key species in HOCl-induced protein fragmentation.