Generation and Reactions of the Disulphide Radical Anion Derived from Metallothionein: A Pulse Radiolytic Study

Abstract

OH-radicals were generated by pulse radiolysis of aqueous solutions of rabbit (Zn,Cd)-metallothionein (MT). They react with MT mainly by forming a thiyl radical with a rate constant of 1·7 × 10¹² dm³ mol⁻¹ s⁻¹. The thiyl radical reacts rapidly but reversibly with a thiolate function to form RSSR^.−: RS. + RS⁻ RSSR^.−. The kinetics of the formation and decay of this radical anion have been studied pulse radiolytically by monitoring the evolution of the optical absorption of RSSR^.− at 450 nm. This process is mostly intermolecular, i.e. bimolecular in MT. In the absence of O₂, RSSR^.− decays bimolecularly: RSSR^.− + RS. RSSR + RS⁻. In the presence of O₂, RS. may be scavenged by O₂ and thus the yield of RSSR^.− decreases: RS. + O₂ RSOO.. Under these conditions RSSR^.− decays by first-order kinetics: RSSR^.− + O₂ RSSR + O^.−₂. The rate constants of these reactions have been determined at room temperature: k₄ = 1·8 × 10⁹ dm³ mol⁻¹ s⁻¹, k₅ = 7 × 10⁴ s⁻¹, k₁₀ = 9·2 × 10⁸ dm³ mol⁻¹ s⁻¹, and k₁₈ about 3 × 10⁷ dm³ mol⁻¹ s⁻¹. From the dependence of the maximal absorbance at 450 nm on the thiolate concentration in the absence of oxygen, ϵ(RSSR^.−) = 9 × 10³ dm³ mol⁻¹ cm⁻¹ and the stability constant (K_4/5) of 2·3 × 10⁴ dm³ ml⁻¹ was determined. K_4/5 is in good agreement with that determined kinetically, k₄/k₅ = 2·6 × 10⁴ dm³ mol⁻¹. The stability constant K_15/16 of the thiylperoxyl radical, RSOO., was determined to be 5·5 × 10³ dm³ mol⁻¹.