Reversible reduction of nitroxides to hydroxylamines: Roles for ascorbate and glutathione
Reversible reduction of nitroxides to hydroxylamines: Roles for ascorbate and glutathione
复制标题
DOI：
10.1016/j.freeradbiomed.2006.11.007
复制
发表时间：
2007-02-01
影响因子：
7.4
通讯作者：
Khramtsov, Valery V.
中科院分区：
文献类型：
--
作者：
Bobko, Andrey A.;Kirilyuk, Igor A.;Khramtsov, Valery V.
稳定的硝酰基自由基NR的生物学应用包括其用作磁共振成像的造影剂、自旋标记物、超氧化物歧化酶模拟物和抗氧化剂。生物样品中的NR快速还原为羟胺（HA）显著限制了它们的应用。反过来，HA再氧化回NR已被用于检测活性氧（ROS）。在这项工作中，吡咯烷，咪唑啉，咪唑烷NR抗坏血酸的还原进行了比较研究，利用最近合成的四乙基取代的NR，具有更高的稳定性，减少在体外和体内。令人惊讶的是，这些NR在100倍过量的抗坏血酸存在下保持10-50%的电子顺磁共振信号的初始强度约1小时。为了解释这些数据，再氧化相应的HA抗坏血酸自由基和脱氢抗坏血酸回NR的建议。这一假设得到了支持，直接测量的NR外观从HA抗坏血酸自由基生成抗坏血酸氧化酶，或在脱氢抗坏血酸的存在下。NR和抗坏血酸之间的可逆反应，观察到的各种类型的NR，并确定了直接和逆反应的速率常数。发现四乙基取代的NR被抗坏血酸盐单电子还原的平衡常数在2.65 × 10（-6）至10（-5）的范围内，这显著低于四甲基取代的NR的相应值（大于或约10（-4））。这解释了在过量抗坏血酸存在下，EPR可检测的四乙基取代NR的准平衡水平的建立。还原反应的NR-HA对在抗坏血酸的介质中被发现显着影响谷胱甘肽（GSH）。这一效应归因于GSH对抗坏血酸自由基的还原作用，发现该反应的速率常数等于10 M-1 s（-1）。总之，这些数据提供了新的见解NR和HA的氧化还原化学，并显着影响其作为氧化还原和ROS敏感的探针，或作为抗氧化剂使用的解释和策略。(c)2006年爱思唯尔公司All rights reserved.
Biological applications of stable nitroxyl radicals, NR, include their use as contrast agents for magnetic resonance imaging, spin labels, superoxide dismutase mimics, and antioxidants. The rapid reduction of NR in biological samples into hydroxylamines (HA) significantly limits their application. In turn, reoxidation of HA back to the NR has been used for detection of reactive oxygen species (ROS). In this work comparative studies of the reduction of pyrrolidine, imidazoline, and imidazolidine NR by ascorbate were performed taking advantage of recently synthesized tetraethyl-substituted NR with much higher stability toward reduction both in vitro and in vivo. Surprisingly, these NR kept 10-50% of initial intensity of electron paramagnetic resonance signal for about 1 h in the presence of 100-fold excess of ascorbate. To explain these data, reoxidation of the corresponding HA by ascorbate radical and dehydroascorbic acid back to the NR was proposed. This hypothesis was supported by direct measurement of the NR appearance from the HA on ascorbate radical generation by ascorbate oxidase, or in the presence of the dehydroascorbic acid. The reversible reaction between NR and ascorbate was observed for the various types of NR, and the rate constants for direct and reverse reactions were determined. The equilibrium constants for one-electron reduction of the tetraethyl-substituted NR by ascorbate were found to be in the range from 2.65 x 10(-6) to 10(-5) which is significantly lower than corresponding values for the tetramethyl-substituted NR (more or about 10(-4)). This explains the establishment of an EPR-detectable quasi-equilibrium level of tetraethyl-substituted NR in the presence of an excess of ascorbate. The redox reactions of the NR-HA couple in ascorbate-containing media were found to be significantly affected by glutathione (GSH). This effect was attributed to the reduction of ascorbate radicals by GSH, and the rate constant of this reaction was found to be equal to 10 M-1 s(-1). In summary, the data provide new insight into the redox chemistry of NR and HA, and significantly affect interpretation and strategy of their use as redox- and ROS-sensitive probes, or as antioxidants. (c) 2006 Elsevier Inc. All rights reserved.