The oxidizing power of the glutathione thiyl radical as measured by its electrode potential at physiological pH

The oxidizing power of the thiyl radical (GS) produced on oxidation of glutathione (GSH) was determined as the mid-point electrode potential (reduction potential) of the one-electron couple Em(GS,H+/GSH) in water, as a function of pH over the physiological range. The method involved measuring the equilibrium constants for electron-transfer equilibria with aniline or phenothiazine redox indicators of known electrode potential. Thiyl and indicator radicals were generated in microseconds by pulse radiolysis, and the position of equilibrium measured by fast kinetic spectrophotometry. The electrode potential Em(GS,H+/GSH) showed the expected decrease by ∼0.06 V/pH as pH was increased from ∼6 to 8, reflecting thiol/thiolate dissociation and yielding a value of the reduction potential of GS = 0.92 ± 0.03 V at pH 7.4. An apparently almost invariant potential between pH ∼3 and 6, with potentials significantly lower than expected, is ascribed at least in part to errors arising from radical decay during the approach to the redox equilibrium and slow electron transfer of thiol compared to thiolate.