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.
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.
Alvarez
María Noel
MN
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.
Peluffo
Gonzalo
G
Folkes
Lisa
L
Wardman
Peter
P
Radi
Rafael
R
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
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