Pulse radiolysis and steady-state analyses of the reaction between hydroethidine and superoxide and other oxidants
Pulse radiolysis and steady-state analyses of the reaction between hydroethidine and superoxide and other oxidants
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DOI：
10.1016/j.abb.2006.09.031
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发表时间：
2006-12-01
影响因子：
3.9
通讯作者：
Kalyanaraman, B.
中科院分区：
文献类型：
--
作者：
Zielonka, Jacek;Sarna, Tadeusz;Kalyanaraman, B.
Hydroethidine（HE）是一种可渗透细胞的探针，用于细胞内超氧化物的检测。本文报道了用脉冲辐解技术直接测定乙氢定与超氧阴离子自由基之间的速率常数。该反应速率常数计算为ca。2 x 10（6）M-1 S-1水：乙醇（1：1）混合液。中间体的光谱特征表明，HE的单电子氧化产物不同于乙锭（E+）的单电子还原产物。在脂肪醇或甲酸盐作为超氧化物生成系统的存在下，含有HE和氧化芬顿试剂（Fe 2 +/DTPA/H2 O2）的孵育混合物的HPLC-电化学测量显示2-OH-E+作为主要产物。无添加剂的芬顿试剂对2-OH-E+的形成具有超氧化物歧化酶的敏感性，我们将超氧阴离子自由基的形成归因于DTPA衍生自由基对氧的单电子还原。在芬顿体系中加入叔丁醇、二甲基亚砜和溴化钾，可抑制2-OH-E+的生成。结果表明，还原性和氧化性自由基对2-OH-E+的形成有不同的影响。(c)2006年爱思唯尔公司All rights reserved.
Hydroethidine (HE) is a cell-permeable probe used for the intracellular detection of superoxide. Here, we report the direct measurement of the rate constant between hydroethidine and superoxide radical anion using the pulse radiolysis technique. This reaction rate constant was calculated to be ca. 2 x 10(6) M-1 S-1 in water:ethanol (1:1) mixture. The spectral characteristics of the intermediates indicated that the one-electron oxidation product of HE was different from the one-electron reduction product of ethidium (E+). The HPLC-electrochemical measurements of incubation mixtures containing HE and the oxygenated Fenton's reagent (Fe2+/DTPA/H2O2) in the presence of aliphatic alcohols or formate as a superoxide generating system revealed 2-OH-E+ as a major product. Formation of 2-OH-E+ by the Fenton's reagent without additives was shown to be superoxide dismutase-sensitive and we attribute the formation of superoxide radical anion to the one-electron reduction of oxygen by the DTPA-derived radical. Addition of tert-butanol, DMSO, and potassium bromide to the Fenton's system caused inhibition of 2-OH-E+ formation. Results indicate that reducing and oxidizing radicals have differential effects on the formation of 2-OH-E+. (c) 2006 Elsevier Inc. All rights reserved.