Reaction of superoxide with trityl radical: implications for the determination of superoxide by spectrophotometry
Reaction of superoxide with trityl radical: implications for the determination of superoxide by spectrophotometry
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DOI：
10.1016/j.abb.2004.01.020
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发表时间：
2004-04-01
影响因子：
3.9
通讯作者：
Kuppusamy, P
中科院分区：
文献类型：
--
作者：
Kutala, VK;Parinandi, NL;Kuppusamy, P
超氧自由基可以通过利用特定化合物的氧化/还原反应的氧化还原方法来测量。然而，氧化还原方法受到各种干扰，限制了它们在超氧化物测定中的使用。使用自旋陷阱的电子顺磁共振（EPR）光谱已被广泛用作测量超氧自由基的替代和直接技术。在我们最近的研究中，我们展示了通过三芳基甲基（三苯甲基）自由基 TAM Ox063 的 EPR 光谱法检测细胞系统中的超氧化物。 TAM 具有高度水溶性，并且在许多生物氧化剂和还原剂（例如过氧化氢、抗坏血酸和谷胱甘肽）存在下保持稳定。 TAM 与超氧化物发生反应，表观二阶速率常数为 3.1 X 10(3) M-1 s(-1)。在目前的工作中，我们利用与 TAM 和超氧化物之间反应形成的产物相对应的新形成的独特吸收峰，研究了超氧化物分光光度测定的可行性。研究了不同超氧化物通量和TAM浓度对超氧化物定量效率和灵敏度的影响，并与广泛使用的细胞色素c法测定超氧化物进行比较。结果表明，对于超氧化物的测定，TAM 方法与细胞色素 c 方法相当，并进一步表明该测定不受过氧化氢存在的影响。总之，超氧化物的 TAM 分光光度测定为细胞色素 c 测定提供了一种合适的替代方法来测量超氧化物，并进一步补充了我们之前报道的超氧化物 TAM-EPR 测定。 (C) 2004 Elsevier Inc. 保留所有权利。
Superoxide radicals can be measured by redox methods which utilize the oxidation/reduction reactions of specific compounds. The redox methods, however, suffer from various interferences, which limit their use in the assay of superoxide. Electron paramagnetic resonance (EPR) spectroscopy using spin traps has been widely used as an alternative and direct technique to measure superoxide radicals. In our recent study, we have demonstrated the detection of superoxide in cellular system by EPR spectroscopy with triarylmethyl (trityl) free radical, TAM Ox063. TAM is highly water-soluble and stable in the presence of many biological oxidizing and reducing agents such as hydrogen peroxide, ascorbate, and glutathione. TAM reacts with superoxide with an apparent second order rate constant of 3.1 X 10(3) M-1 s(-1). In the present work, we investigated the feasibility of a spectrophotometric assay of superoxide by taking advantage of the newly formed distinct absorption peak corresponding to the product formed from the reaction between TAM and superoxide. The effects of different fluxes of superoxide and concentrations of TAM on the efficiency and sensitivity of quantification of superoxide were investigated and compared with the widely used cytochrome c method of superoxide determination. The results demonstrated that the TAM method is comparable to the cytochrome c method for the assay of superoxide and further revealed that the assay is not affected by the presence of hydrogen peroxide. In summary, the TAM spectrophotometric assay of superoxide provides a suitable alternative method to the cytochrome c assay to measure superoxide and further complements our earlier reported TAM-EPR assay of superoxide. (C) 2004 Elsevier Inc. All rights reserved.