Mg. Bonini et al., Direct EPR detection of the carbonate radical anion produced from peroxynitrite and carbon dioxide, J BIOL CHEM, 274(16), 1999, pp. 10802-10806
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.