Hydrogen exchange equilibria in glutathione radicals: rate constants.
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) = 3·10⁵ s⁻¹, k(reverse) = 7·10⁵ 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.
Hofstetter
Dustin
D
Laboratory of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zürich, Switzerland.
Nauser
Thomas
T
Koppenol
Willem H
WH
D005609:Free Radicals / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry*; D019803:Glutathione Disulfide / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D011675:Pulse Radiolysis; D013056:Spectrophotometry, Ultraviolet
D005609:Free Radicals; D006859:Hydrogen; D005978:Glutathione; D019803:Glutathione Disulfide