Bonini M G, Radi R, Ferrer-Sueta G, Ferreira A M, Augusto O
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil 05599-970.
J Biol Chem. 1999 Apr 16;274(16):10802-6. doi: 10.1074/jbc.274.16.10802.
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. Peroxynitrite anion (ONOO-) reacts rapidly with carbon dioxide, forming an adduct, nitrosoperoxocarboxylate (ONOOCO2-), whose decomposition has been proposed to produce reactive intermediates such as the carbonate radical (CO-3). Here, by the use of rapid mixing continuous flow electron paramagnetic resonance (EPR), we directly detected the carbonate radical in flow mixtures of peroxynitrite with bicarbonate-carbon dioxide over the pH range of 6-9. The radical was unambiguously identified by its EPR parameters (g = 2.0113; line width = 5.5 G) and by experiments with bicarbonate labeled with 13C. In this case, the singlet EPR signal obtained with 12C bicarbonate splits into the expected doublet because of 13C (a(13C)= 11.7 G). The singlet spectrum of the unlabeled radical was invariant between pH 6 and 9, confirming that in this pH range the detected radical is the carbonate radical anion (CO-3). Importantly, in addition to contributing to the understanding of nitrosoperoxocarboxylate decomposition pathways, this is the first report unambiguously demonstrating the formation of the carbonate radical anion at physiological pHs by direct EPR spectroscopy.
最近人们认为过氧亚硝酸根的生物学效应很大程度上取决于它与二氧化碳的反应，而二氧化碳在细胞内和细胞外区室中都以高浓度存在。过氧亚硝酸根阴离子（ONOO-）与二氧化碳迅速反应，形成加合物亚硝基过氧羧酸盐（ONOOCO2-），有人提出其分解会产生诸如碳酸根自由基（CO-3）等活性中间体。在此，我们通过使用快速混合连续流动电子顺磁共振（EPR），在pH值为6至9的范围内，直接检测了过氧亚硝酸根与碳酸氢根 - 二氧化碳的流动混合物中的碳酸根自由基。通过其EPR参数（g = 2.0113；线宽 = 5.5 G）以及用13C标记的碳酸氢根进行的实验，明确鉴定了该自由基。在这种情况下，由于13C（a(13C)= 11.7 G），用12C碳酸氢根获得的单重态EPR信号分裂为预期的二重态。未标记自由基的单重态光谱在pH 6至9之间不变，这证实了在该pH范围内检测到的自由基是碳酸根自由基阴离子（CO-3）。重要的是，除了有助于理解亚硝基过氧羧酸盐的分解途径外，这是第一份通过直接EPR光谱明确证明在生理pH值下形成碳酸根自由基阴离子的报告。
