title: One-electron oxidation pathway of peroxynitrite decomposition in human blood plasma: evidence for the formation of protein tryptophan-centred radicals.
abstract: Exposure of human blood plasma to peroxynitrite in the presence of 3,5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS) resulted in the trapping of a strongly immobilized nitroxide radical adduct. The adduct was due to protein-centred radicals derived not only from serum albumin but also from other major plasma proteins (fibrinogen, IgG, alpha1-antitrypsin and transferrin). Urate significantly protected plasma from the peroxynitrite-induced DBNBS-plasma protein adduct, whereas ascorbate and glutathione were protective at concentrations exceeding those usually found in plasma. Alkylation of plasma -SH groups did not affect the intensity of DBNBS-plasma protein adduct, whereas bicarbonate increased its formation, thus showing a pro-oxidant effect. The DBNBS-plasma protein adduct provided little structural information, but subsequent non-specific-protease treatment resulted in the detection of an isotropic three-line spectrum, indicating the trapping of radicals centred on a tertiary carbon. The nitrogen hyperfine coupling constant of this adduct and its superhyperfine structure were similar to those of DBNBS-tryptophan peptides with the alpha-amino group of tryptophan linked in the amide bond, consistent with a radical adduct formed at C-3 of the indole ring of tryptophan-containing peptides. DBNBS was unable to trap radicals derived from peroxynitrite-treated tyrosine or tyrosine-containing peptides. Methionine treated with peroxynitrite resulted in the trapping of at least two DBNBS-methionine adducts with hyperfine structures different from that of protease-treated DBNBS-plasma proteins. These results demonstrate that peroxynitrite induced in blood plasma the formation of protein radicals centred on tryptophan residues and underline the relevance of the one-electron oxidation pathway of peroxynitrite decomposition in biological fluids.
authors.affiliation: Laboratorio di Biologia Cellulare, Istituto Superiore di Sanita, Roma, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Pietraforte
authors.forename: M
authors.initials: M
authors.lastname: Minetti
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism; D001557:Benzenesulfonates / Q000378:metabolism; D001639:Bicarbonates / Q000494:pharmacology; D001798:Blood Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005609:Free Radicals / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D008715:Methionine / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism*; D009603:Nitroso Compounds / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011402:Pronase / Q000378:metabolism; D019097:Spin Trapping; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism*; D014527:Uric Acid / Q000378:metabolism
chemical_list: D001557:Benzenesulfonates; D001639:Bicarbonates; D001798:Blood Proteins; D005609:Free Radicals; D009566:Nitrates; D009603:Nitroso Compounds; D010455:Peptides; D013438:Sulfhydryl Compounds; C039642:peroxynitric acid; D014527:Uric Acid; C048757:3,5-dibromo-4-nitrosobenzenesulfonate; D014364:Tryptophan; D008715:Methionine; D011402:Pronase; D005978:Glutathione; D001205:Ascorbic Acid