Role of Charge in the Radioprotection of E. Coli by Thiols

Abstract

The role of net charge (Z) of thiols in their ability to radioprotect cells has been investigated in a glutathione (GSH)-deficient strain of E. coli. This strain, 7, is deficient in the enzyme γ-glutamylcysteine synthetase and allows the effects of added low molecular weight thiols to be studied. Using the gas explosion system it is possible to measure the chemical repair of the free-radical precursors of lethal lesions by thiols in intact cells. The first-order chemical repair rate in strain 7 is 280 s⁻¹ in comparison with 1100 s⁻¹ in the wild-type strain 1157. From the measured difference in the intracellular concentration of GSH between the wild-type and the mutant, this gives a second-order repair rate, k_r, of 1·23 ± 0·3 × 10⁵ dm³mol⁻¹s⁻¹. Measurement of intracellular thiol levels after addition of various low molecular weight thiols showed that uptake was rapid, leading to stable thiol levels within 1 min. The ratios of the intracellular to extracellular concentrations (C_in/C_out) were 0·74 for 3-mercaptopropionic acid (Z = −1), 0·56 for 2-mercaptoethanol (Z = 0), 1·47 for cysteamine (Z = +1) and 1·04 for WR1065 (Z = +2). The k_r's for these thiols were 1·3 ± 0·5 × 10⁵ dm³mol⁻¹s⁻¹ for 30-mercaptopropionic acid, 3·3 ± 1·6 × 10⁵ dm³mol⁻¹s⁻¹ for 2-mercaptoethanol, 3·9 ± 1·1 × 10⁵ dm³mol⁻¹s⁻¹ for cysteamine and 2·7 ± 1·1 × 10⁶ dm³mol⁻¹s⁻¹ for WR1065. These are lower and increase less with charge than previously published values for chemical repair in isolated pBR322 DNA, probably because of the association of nucleoproteins and polyamines with the cellular DNA of E. coli. However, the approximate three-fold increase in k_r per unit increase in Z shows that the counter-ion condensation and co-ion depletion are important in determining the effectiveness of charged thiols in the radioprotection of E. coli.