Direct EPR detection of the carbonate radical anion produced from peroxynitrite and carbon dioxide
Direct EPR detection of the carbonate radical anion produced from peroxynitrite and carbon dioxide
复制标题
DOI：
10.1074/jbc.274.16.10802
复制
发表时间：
1999-04-16
影响因子：
4.8
通讯作者：
Augusto, O
中科院分区：
文献类型：
--
作者：
Bonini, MG;Radi, R;Augusto, O
过氧亚硝酸盐的生物效应最近被认为在很大程度上依赖于它与二氧化碳的反应，过氧亚硝酸根阴离子(ONOO-)存在于细胞内和胞外，它与二氧化碳迅速反应，形成加合物亚硝酸根过氧羧酸盐(ONOOCO2-)，它的分解被认为是产生活性中间产物，如碳酸盐自由基(CO3自由基阴离子)。本文利用快速混合连续流动电子顺磁共振(EPR)技术，在pH为6-9的范围内，直接检测了过氧亚硝酸根与碳酸氢盐-二氧化碳流动混合物中的碳酸根。通过EPR参数(g=2.0113；线宽=5.5G)和用C-13标记的碳酸氢盐进行的实验，明确地鉴定了该自由基。在这种情况下，由于C-13(a(C-13)=11.7G)，用C-12重碳酸盐获得的单重态EPR信号分裂成预期的双重态。未标记自由基的单线态光谱在pH 6~9范围内不变，证实在此pH范围内检测到的自由基为碳酸盐自由基阴离子(CO3自由基)。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，这是第一个通过直接EPR光谱明确地证明了生理PHS上碳酸根阴离子形成的报告。
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 (CO3radical anion). 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 C-13. In this case, the singlet EPR signal obtained with C-12 bicarbonate splits into the expected doublet because of C-13 (a(C-13)= 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 (CO3radical anion). 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.