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 center dot-) can be formed biologically by the reaction of (OH)-O-center dot with bicarbonate, the decomposition of the peroxynitritecarbon 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 center dot- to yield transient species to yield finally the DMPO-OH spin adduct. In this study, the kinetics of reaction Of CO3 center dot- 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)-O-center dot that was trapped by DMPO; the presence of 50-500 mM bicarbonate, expected to convert (OH)-O-center dot to CO3 center dot-, markedly inhibited DMPO-OH formation. This was demonstrated to be due mainly to a fast reaction of CO3 center dot- with Fe-II-DTPA (k=6.1 x 10(8) M-1 s(-1)), supported by kinetic analysis. Generation of CO3 center dot- 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)-O-center dot or CO3 center dot-. Similarly, the formation of CO3 center dot- by CuZnSOD/H2O2/bicarbonate and peroxynitritecarbon dioxide was supported by DMPO hydroxylation and kinetic competition data. Finally, the reaction of CO3 center dot- with DMPO to yield DMPO-OH was shown in peroxynitrite-forming macrophages. In conclusion, CO3 center dot- 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 center dot- will be sensitive to the presence of DMPO. (C) 2007 Elsevier Inc. All rights reserved.