Direct EPR detection of the carbonate radical anion produced from peroxynitrite and carbon dioxide

The biological effects of peroxynitrite have been recently considered to be largely dependent on its reaction with carbon dioxide, which is present in high concentrations in intra- and extracellular compartments, Peroxynitrit e anion (ONOO-) reacts rapidly with carbon dioxide, forming an adduct, nitr osoperoxocarboxylate (ONOOCO2-), whose decomposition has been proposed to p roduce reactive intermediates such as the carbonate radical (CO3radical ani on). Here, by the use of rapid mixing continuous flow electron paramagnetic resonance (EPR), we directly detected the carbonate radical in flow mixtur es of peroxynitrite with bicarbonate-carbon dioxide over the pH range of 6- 9. The radical was unambiguously identified by its EPR parameters (g = 2.01 13; line width = 5.5 G) and by experiments with bicarbonate labeled with C- 13. In this case, the singlet EPR signal obtained with C-12 bicarbonate spl its into the expected doublet because of C-13 (a(C-13)= 11.7 G). The single t spectrum of the unlabeled radical was invariant between pH 6 and 9, confi rming that in this pH range the detected radical is the carbonate radical a nion (CO3radical anion). Importantly, in addition to contributing to the un derstanding of nitrosoperoxocarboxylate decomposition pathways, this is the first report unambiguously demonstrating the formation of the carbonate ra dical anion at physiological pHs by direct EPR spectroscopy.