Hydrogen Exchange Equilibria in Glutathione Radicals: Rate Constants

[Image: see text] The reduction of oxidized glutathione GSSG by hydrated electrons and hydrogen atoms to form GSSG(•−) is quantitative. The radical anion dissociates into GS(•) and GS(−), and the S-centered radical subsequently abstracts a hydrogen intramolecularly. We observe sequential development of UV absorbance signatures that indicate the formation of both α- and β-carbon-centered radicals. From experiments performed at pH 2 and pH 11.8, we determined forward and reverse rate constants for the overall equilibrium between sulfur-centered and carbon-centered radicals: k(forward) = 3·10(5) s(−1), k(reverse) = 7·10(5) s(−1), and K = 0.4. Furthermore, on the basis of the differences between the kinetics traces at 240 and 280 nm, we estimate that α- and β-carbon-centered radicals are formed at a surprising ratio of 1:3. The ratios found at pH 2 also apply to pH 7, with the conclusion that the equilibrium ratio of S-centered:β-centered:α-centered radicals is, very approximately, 8:3:1. The formation of carbon-centered radicals could lead to irreversible damage in proteins via the formation of carbon−carbon bonds or backbone fragmentation.