Tempol diverts peroxynitrite/carbon dioxide reactivity toward albumin and cells from protein-tyrosine nitration to protein-cysteine nitrosation.
Tempol has been shown to protect experimental animals from injuries associated with excessive nitric oxide production. In parallel, tempol decreased the levels of protein-3-nitrotyrosine in the injured tissues, suggesting that it interacted with nitric oxide-derived oxidants such as nitrogen dioxide and peroxynitrite. Relevantly, a few recent studies have shown that tempol catalytically diverts peroxynitrite/carbon dioxide reactivity toward phenol from nitration to nitrosation. To examine whether this shift occurs in biological environments, we studied the effects of tempol (10-100 microM) on peroxynitrite/carbon dioxide (1 mM/2 mM) reactivity toward proteins, native bovine serum albumin (BSA) (0.5-0.7 cys/mol) and reductively denatured BSA (7-19 cys/mol), and cells (J774 macrophages). Although not a true catalyst, tempol strongly inhibited protein-tyrosine nitration (70-90%) and protein-cysteine oxidation (20-50%) caused by peroxynitrite/carbon dioxide in BSA, denatured BSA, and cells while increasing protein-cysteine nitrosation (200-400%). Tempol consumption was attributed mainly to its reaction with protein-cysteinyl radicals. Most of the tempol, however, reacted with the radicals produced from peroxynitrite/carbon dioxide, that is, nitrogen dioxide and carbonate radical anion. Accordingly, tempol decreased the yields of BSA-cysteinyl and BSA-tyrosyl/tryptophanyl radicals, as well their decay products such as protein-3-nitrotyrosine. The parallel increase in protein-nitrosocysteine yields demonstrated that part of the peroxynitrite is oxidized to nitric oxide by the oxammonium cation produced from tempol oxidation by peroxynitrite/carbon dioxide-derived radicals. Protein-nitrosocysteine formation was shown to occur by radical and nonradical mechanisms in studies with a protein-cysteinyl radical trapper. These studies may contribute to the understanding of the protective effects of tempol in animal models of inflammation.
Fernandes
Denise C
DC
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 26077, CEP 05513-970, São Paulo, SP, Brazil.
Medinas
Danilo B
DB
Alves
Maria Júlia M
MJ
Augusto
Ohara
O
D000418:Albumins / Q000378:metabolism*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002384:Catalysis; D003497:Cyclic N-Oxides / Q000494:pharmacology*; D003545:Cysteine / Q000737:chemistry*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D016166:Free Radical Scavengers / Q000494:pharmacology; D005609:Free Radicals; D015151:Immunoblotting; D007150:Immunohistochemistry; D007249:Inflammation; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D008956:Models, Chemical; D009569:Nitric Oxide / Q000737:chemistry; D009573:Nitrites / Q000737:chemistry; D009584:Nitrogen / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry; D030421:Peroxynitrous Acid / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013113:Spin Labels; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry*
D000418:Albumins; D000975:Antioxidants; D003497:Cyclic N-Oxides; D016166:Free Radical Scavengers; D005609:Free Radicals; D009573:Nitrites; D013113:Spin Labels; D030421:Peroxynitrous Acid; D012710:Serum Albumin, Bovine; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D003545:Cysteine; D009584:Nitrogen; D010100:Oxygen; C001803:tempol