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

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• 作者：Marí ; a Noel Alvarez ; Gonzalo Peluffo ; Lisa Folkes ; Peter Wardman ; Rafael Radi
• 关键词：Carbonate radical ; Fenton reaction ; CuZnSOD ; Peroxynitrite ; MPO ; 5 ; 5-dimethyl-1-pyrroline-N-oxide ; DTPA ; Free radicals ; Reaction kinetics
• 刊名：Free Radical Biology and Medicine
• 出版年：2007
• 期刊代码：105_08915849
• 类别：med
• 出版时间：1 December 2007
• 卷：43
• 期：11
• 页码：1523-1533
• 文件大小：636 K

摘要

Carbonate radicals (CO₃^{radical dot" border=0>−}) can be formed biologically by the reaction of ^{radical dot" border=0>}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^{radical dot" border=0>−} to yield transient species to yield finally the DMPO–OH spin adduct. In this study, the kinetics of reaction of CO₃^{radical dot" border=0>−} with DMPO were studied by pulse radiolysis, yielding a second-order rate constant of 2.5 × 10⁶ M^{− 1} s^{− 1}. A Fenton system, composed of Fe^II–DTPA plus H₂O₂, generated ^{radical dot" border=0>}OH that was trapped by DMPO; the presence of 50–500 mM bicarbonate, expected to convert ^{radical dot" border=0>}OH to CO₃^{radical dot" border=0>−}, markedly inhibited DMPO–OH formation. This was demonstrated to be due mainly to a fast reaction of CO₃^{radical dot" border=0>−} with Fe^II–DTPA (k = 6.1 × 10⁸ M^{− 1} s^{− 1}), supported by kinetic analysis. Generation of CO₃^{radical dot" border=0>−} 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 ^{radical dot" border=0>}OH or CO₃^{radical dot" border=0>−}. Similarly, the formation of CO₃^{radical dot" border=0>−} by CuZnSOD/H₂O₂/bicarbonate and peroxynitrite–carbon dioxide was supported by DMPO hydroxylation and kinetic competition data. Finally, the reaction of CO₃^{radical dot" border=0>−} with DMPO to yield DMPO–OH was shown in peroxynitrite-forming macrophages. In conclusion, CO₃^{radical dot" border=0>−} 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^{radical dot" border=0>−} will be sensitive to the presence of DMPO.