Electron paramagnetic resonance spin trapping investigation into the kinetics of glutathione oxidation by the superoxide radical: re-evaluation of the rate constant

The ability of glutathione to scavenge the superoxide radical is a matter of serious contention in the literature: reported values for the second-order rate constant range from 102 to greater than 105 M−1 s−1. The physiological implications of this discrepancy will determine, for example, whether or not glutathione can compete with Mn-superoxide dismutase for reaction with the radical in the mitochondrial matrix, leading to formation of the potentially harmful glutathionyl radical. Several authors have investigated the kinetics of glutathione oxidation by superoxide using spectrophotometric assays, based on competition between either ferricytochrome c or epinephrine for reaction with the radical. However, these approaches have received criticism because the contributions of various secondary reactions to the overall kinetics have been largely overlooked (e.g., the reduction of ferricytochrome c by glutathione). In the present investigation, we have used electron paramagnetic resonance spectroscopy to monitor competition between GSH and the spin trap 5,5-dimethyl-1-pyrroline N-oxide for reaction with superoxide. This method has been used previously and a rate constant of 1.8 × 105 M−1 s−1 obtained (Dikalov, S.; Khramtsov, V.; Zimmer, G. Arch. Biochem. Biophys. 326:207–218; 1996). However, we demonstrate that this value is a gross overestimation because the spectrum of the hydroxyl radical adduct of the spin trap was incorrectly assigned to the glutathionyl radical adduct. The relatively high yield of the DMPO hydroxyl radical adduct is shown to be due to the two-electron reduction of the corresponding superoxide radical adduct by glutathione. Taking these factors into consideration, we estimate the second order rate constant for the oxidation of glutathione by superoxide to be not, vert, similar 200 M−1 s−1.