Rates for Repair of pBR 322 DNA Radicals by Thiols as Measured by the Gas Explosion Technique: Evidence that Counter-ion Condensation and Co-ion Depletion are Significant at Physiological Ionic Strength

Summary

Rates of repair of pBR 322 plasmid DNA radicals by thiols of varying net charge (Z) at pH 7 and physiological ionic strength were measured using the oxygen explosion technique. The extent of conversion of supercoiled to relaxed circular plasmid was measured by HPLC as a function of the time of oxygen exposure before or after irradiation, the time-courses being fitted by a pseudo-first-order kinetic expression with k₁ = k₂[RSH]. Values of k₂ (M⁻¹ s⁻¹) were: 2·1 × 10⁵ (GSH, Z = −1), 1·4 × 10⁶ (2-mercaptoethanol, Z = 0), 1·2 × 10⁷ (cysteamine, Z = +1), 6·6 × 10⁷ (WR-1065 or N-(2-mercaptoethyl)-1,3-diaminopropane, Z = +2). The approximately 6-fold increase in rate with each unit increase in Z is attributed to concentration of cationic thiols near DNA as a consequence of counter-ion condensation and reduced levels of anionic thiols near DNA owing to co-ion depletion. The results are quantitatively consistent with chemical repair as a significant mechanism for radioprotection of cells by neutral and cationic thiols under aerobic conditions, but indicate that repair by GSH will compete effectively with oxygen only at low oxygen tension.