title: Reaction of the carbonate radical with the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide in chemical and cellular systems: pulse radiolysis, electron paramagnetic resonance, and kinetic-competition studies.
abstract: Carbonate radicals (CO3-) can be formed biologically by the reaction of OH with bicarbonate, the decomposition of the peroxynitrite-carbon dioxide adduct (ONOOCO2-), and enzymatic activities, i.e., peroxidase activity of CuZnSOD and xanthine oxidase turnover in the presence of bicarbonate. It has been reported that the spin-trap DMPO reacts with CO3(-) to yield transient species to yield finally the DMPO-OH spin adduct. In this study, the kinetics of reaction of CO3(-) with DMPO were studied by pulse radiolysis, yielding a second-order rate constant of 2.5 x 10(6) M(-1) s(-1). A Fenton system, composed of Fe(II)-DTPA plus H2O2, generated OH that was trapped by DMPO; the presence of 50-500 mM bicarbonate, expected to convert OH to CO3(-), markedly inhibited DMPO-OH formation. This was demonstrated to be due mainly to a fast reaction of CO3(-) with FeII-DTPA (k=6.1 x 10(8) M(-1) s(-1)), supported by kinetic analysis. Generation of CO3(-) by the Fenton system was further proved by analysis of tyrosine oxidation products: the presence of bicarbonate caused a dose-dependent inhibition of 3,4-dihydroxiphenylalanine with a concomitant increase of 3,3'-dityrosine yields, and the presence of DMPO inhibited tyrosine oxidation, in agreement with the rate constants with OH or CO3(-). Similarly, the formation of CO3(-) by CuZnSOD/H(2)O(2)/bicarbonate and peroxynitrite-carbon dioxide was supported by DMPO hydroxylation and kinetic competition data. Finally, the reaction of CO3(-) with DMPO to yield DMPO-OH was shown in peroxynitrite-forming macrophages. In conclusion, CO3(-) reacts quite rapidly with DMPO and may contribute to DMPO-OH yields in chemical and cellular systems; in turn, the extent of oxidation of other target molecules (such as tyrosine) by CO3(-) will be sensitive to the presence of DMPO.
authors.affiliation: Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, 11800 Montevideo, Uruguay.
authors.forename: María Noel
authors.initials: MN
authors.lastname: Alvarez
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Peluffo
authors.forename: Lisa
authors.initials: L
authors.lastname: Folkes
authors.forename: Peter
authors.initials: P
authors.lastname: Wardman
authors.forename: Rafael
authors.initials: R
authors.lastname: Radi
mesh_terms: D000818:Animals; D002460:Cell Line; D003497:Cyclic N-Oxides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D017665:Hydroxyl Radical / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D011675:Pulse Radiolysis; D013113:Spin Labels; D013482:Superoxide Dismutase / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
chemical_list: D003497:Cyclic N-Oxides; C045076:Fenton's reagent; D005609:Free Radicals; D013113:Spin Labels; D017665:Hydroxyl Radical; D014443:Tyrosine; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D006861:Hydrogen Peroxide; D007501:Iron; D013482:Superoxide Dismutase