Determination of Rate Constants of the Reactions of Thiols with Superoxide Radical by Electron Paramagnetic Resonance: Critical Remarks on Spectrophotometric Approaches

Two new EPR approaches were developed for determination of rate constants of reaction of glutathione (GSH),N-(2-mercaptopropionyl)glycine (MPG), dihydrolipoic acid (DHL), bisnor-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 × 105M−1s−1; MPG, 2.2 × 105M−1s−1; TNL, 1.2 × 105M−1s−1; BNL, 2.5 × 105M−1s−1; DHL, 4.8 × 105M−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.