Dikalov S, Khramtsov V, Zimmer G
Institute of Chemical Kinetics and Combustion, Novosibirsk, Russia.
Arch Biochem Biophys. 1996 Feb 15;326(2):207-18. doi: 10.1006/abbi.1996.0067.
Two new EPR approaches were developed for determination of rate constants of reaction glutathione (GSH), N-(2-mercaptopropionyl) glycine (MPG), dihydrolipoic acid (BNL), and tetranor-dihydrolipoic acid (TNL) with superoxide radical. In both cases the competition between thiols and spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) for superoxide radical was used. In the first method the dependence of amplitude of EPR spectrum of DMPO-OOH spin adduct on concentration of thiols in a superoxide-generating system was studied. In the second approach the changes in reduced thiol concentration due to reaction with superoxide radical were measured by nitroxide biradical containing disulfide bond. Observed rate constants were the following: GSH, 1.8 x 10(5) M-1s-1; MPG, 2.2 x 10(5) M-1s-1; TNL, 1.2 x 10(5) M-1s-1; BNL, 2.5 x 10(5) M-1s-1; DHL, 4.8 x 10(5) M-1s-1. The determination of the rate constants of reaction of superoxide radical with thiols by spectrophotometrical cytochrome C assay could result in an underestimation of the values due to the reduction of cytochrome C by thiols. Use of epinephrine for this purpose could lead to an overestimation of experimental rate constants because the adrenochrome formed in the reaction of epinephrine with superoxide radical reacts with thiols.
开发了两种新的电子顺磁共振（EPR）方法来测定谷胱甘肽（GSH）、N-（2-巯基丙酰基）甘氨酸（MPG）、二氢硫辛酸（BNL）和四降二氢硫辛酸（TNL）与超氧自由基反应的速率常数。在这两种情况下，均利用了硫醇与自旋捕捉剂5,5-二甲基-1-吡咯啉-N-氧化物（DMPO）对超氧自由基的竞争。在第一种方法中，研究了DMPO-OOH自旋加合物的EPR谱幅度对超氧生成系统中硫醇浓度的依赖性。在第二种方法中，通过含二硫键的氮氧双自由基测量由于与超氧自由基反应导致的还原型硫醇浓度变化。观察到的速率常数如下：GSH为1.8×10⁵ M⁻¹s⁻¹；MPG为2.2×10⁵ M⁻¹s⁻¹；TNL为1.2×10⁵ M⁻¹s⁻¹；BNL为2.5×10⁵ M⁻¹s⁻¹；DHL为4.8×10⁵ M⁻¹s⁻¹。用分光光度法细胞色素C测定法测定超氧自由基与硫醇反应的速率常数，可能会因硫醇对细胞色素C的还原作用而导致数值被低估。为此使用肾上腺素可能会导致实验速率常数被高估，因为肾上腺素与超氧自由基反应形成的肾上腺色素会与硫醇反应。
