Direct ESR detection or peroxynitrite-induced tyrosine-centred protein radicals in human blood plasma.
Peroxynitrite, the reaction product of O2.- and .NO, is a toxic compound involved in several oxidative processes that modify proteins. The mechanisms of these oxidative reactions are not completely understood. In this study, using direct ESR at 37 degrees C, we observed that peroxynitrite induced in human blood plasma a long-lived singlet signal at g = 2.004 arising from proteins. This signal was not due to a specific plasma protein, because several purified proteins were able to form a peroxynitrite-induced g = 2.004 signal, but serum albumin and IgG showed the most intense signals. Hydroxyurea, a tyrosyl radical scavenger, strongly inhibited the signal, and horseradish peroxidase/H2O2, a radical-generating system known to induce tyrosyl radicals, induced a similar signal. Furthermore peptides containing a Tyr in the central portion of the molecule were able to form a stable peroxynitrite-dependent g = 2.004 signal, whereas peptides in which Tyr was substituted with Gly, Trp or Phe and peptides with Tyr at the N-terminus or near the C-terminus did not form radicals that were stable at 37 degrees C. We suggest that Tyr residues are at least the major radical sources of the peroxynitrite-dependent g = 2.004 signal at 37 degrees C in plasma or in isolated proteins. Although significantly enhanced by CO2/bicarbonate, the signal was detectable in whole plasma at relatively high peroxynitrite concentrations (>2 mM) but, after removal of ascorbate or urate or in dialysed plasma, it was detectable at lower concentrations (100-1000 microM). Our results suggest that the major role of ascorbate and urate is to reduce or 'repair' the radical(s) centred on Tyr residues and not to scavenge peroxynitrite (or nitrosoperoxycarbonate, the oxidant formed in CO2-containing fluids). This mechanism of inhibition by plasma antioxidants may be a means of preserving the physiological functions of peroxynitrite.
Pietraforte
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Laboratorio di Biologia Cellulare, Istituto Superiore di Sanità, V. Regina Elena 299, 00161 Roma, Italy.
Minetti
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D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000097:blood / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000097:blood; D006801:Humans; D008969:Molecular Sequence Data; D009566:Nitrates / Q000097:blood / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000097:blood / Q000737:chemistry; D014443:Tyrosine / Q000097:blood* / Q000737:chemistry; D014527:Uric Acid / Q000097:blood / Q000737:chemistry
D005609:Free Radicals; D009566:Nitrates; D013438:Sulfhydryl Compounds; C039642:peroxynitric acid; D014527:Uric Acid; D014443:Tyrosine; D001205:Ascorbic Acid