Affiliation:
1. Laboratorio di Biologia Cellulare, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy
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, α1-antitrypsin and transferrin). Urate significantly protected plasma from the peroxynitrite-induced DBNBSŐplasma protein adduct, whereas ascorbate and glutathione were protective at concentrations exceeding those usually found in plasma. Alkylation of plasma ŐSH groups did not affect the intensity of DBNBSŐplasma protein adduct, whereas bicarbonate increased its formation, thus showing a pro-oxidant effect. The DBNBSŐplasma 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 DBNBSŐtryptophan peptides with the α-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 DBNBSŐmethionine adducts with hyperfine structures different from that of protease-treated DBNBSŐplasma 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.
Subject
Cell Biology,Molecular Biology,Biochemistry
Cited by
63 articles.
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