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 (CO 3 − ) can be formed biologically by the reaction of OH with bicarbonate, the decomposition of the peroxynitrite–carbon dioxide adduct (ONOOCO 2 −), 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 CO 3 − to yield transient species to yield finally the DMPO–OH spin adduct. In this study, the kinetics of reaction of CO 3 − with DMPO were studied by pulse radiolysis, yielding a second-order rate constant of 2.5 × 10 6 M − 1 s − 1 . A Fenton system, composed of Fe II–DTPA plus H 2O 2, generated OH that was trapped by DMPO; the presence of 50–500 mM bicarbonate, expected to convert OH to CO 3 − , markedly inhibited DMPO–OH formation. This was demonstrated to be due mainly to a fast reaction of CO 3 − with Fe II–DTPA ( k = 6.1 × 10 8 M − 1 s − 1 ), supported by kinetic analysis. Generation of CO 3 − 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 CO 3 − . Similarly, the formation of CO 3 − by CuZnSOD/H 2O 2/bicarbonate and peroxynitrite–carbon dioxide was supported by DMPO hydroxylation and kinetic competition data. Finally, the reaction of CO 3 − with DMPO to yield DMPO–OH was shown in peroxynitrite-forming macrophages. In conclusion, CO 3 − 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 CO 3 − will be sensitive to the presence of DMPO.