Rizzi Cëcile, Samouilov Alexandre, Kutala Vijay Kumar, Parinandi Narasimham L, Zweier Jay L, Kuppusamy Periannan
Center for Biomedical EPR Spectroscopy and Imaging, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA.
Free Radic Biol Med. 2003 Dec 15;35(12):1608-18. doi: 10.1016/j.freeradbiomed.2003.09.014.
The use of triarylmethyl (trityl) free radical, TAM OX063, for detection of superoxide in aqueous solutions by electron paramagnetic resonance (EPR) spectroscopy was investigated. TAM is paramagnetic (EPR active), highly soluble in water and exhibits a single sharp EPR peak in aqueous media. It is also highly stable in presence of many oxidoreductants such as ascorbate and glutathione that are present in the biological systems. TAM reacts with superoxide with an apparent second order rate constant of 3.1 x 10(3) M(-1) s(-1). The specific reactivity of TAM with superoxide, which leads to loss of EPR signal, was utilized to detect the generation of superoxide in various chemical (light/riboflavin/electron/donor), enzymatic (xanthine/xanthine oxidase), and cellular (stimulated neutrophils) model systems. The changes in the EPR line-width, induced by molecular oxygen, were utilized in the simultaneous determination of consumption of oxygen in the model systems. The effects of flux of superoxide and concentration of TAM on the efficiency of detection of superoxide were studied. The use of TAM for detection of superoxide offers unique advantages namely, (i) the utilization of very low concentration of the probe, (ii) its stability to bioreduction, and (iii) its use in the simultaneous determination of concentrations of superoxide and oxygen.
研究了使用三芳基甲基（三苯甲基）自由基TAM OX063，通过电子顺磁共振（EPR）光谱法检测水溶液中的超氧化物。TAM具有顺磁性（EPR活性），高度溶于水，并且在水性介质中表现出单一尖锐的EPR峰。在生物系统中存在的许多氧化还原剂（如抗坏血酸盐和谷胱甘肽）存在下，它也非常稳定。TAM与超氧化物反应，表观二级速率常数为3.1×10³ M⁻¹ s⁻¹。TAM与超氧化物的特异性反应性导致EPR信号丧失，被用于检测各种化学（光/核黄素/电子/供体）、酶促（黄嘌呤/黄嘌呤氧化酶）和细胞（刺激的中性粒细胞）模型系统中超氧化物的产生。由分子氧引起的EPR线宽变化被用于同时测定模型系统中氧气的消耗。研究了超氧化物通量和TAM浓度对超氧化物检测效率的影响。使用TAM检测超氧化物具有独特的优势，即：（i）使用极低浓度的探针；（ii）其对生物还原的稳定性；（iii）用于同时测定超氧化物和氧气的浓度。
