Ferrous ion-induced strand breaks in the DNA plasmid pBR322 are not mediated by hydrogen peroxide

Ferrous ion-induced generation of single and multiple strand breaks in the DNA plasmid pBR322 induces the formation of two new plasmid forms with altered electrophoretic mobility. The yield of these plasmid forms, the circular relaxed and the linear forms, depended on the applied Fe2+ concentration. This property was independent of the presence of hydrogen peroxide in the incubation mixture indicating the lack of Fenton chemistry to explain the DNA degradation. The removal of dioxygen or the presence of superoxide dismutase diminished partially the yield of ferrous ion-induced DNA plasmid degradation, while catalase was without any effect. Autoxidation of divalent iron as followed by the formation of a coloured iron-phenanthroline complex was enhanced in a concentration-dependent manner by phosphate and bicarbonate and very efficiently using a mixture of 0.15 M NaCl, 1.2 mM phosphate, 23.8 mM bicarbonate, pH 7.4, that concentrations correspond closely to the intracellular values of buffer components. Thus, the formation of a yet unknown reactive species from Fe2+, and dioxygen, that is complexed to buffer components especially phosphate and its contribution in DNA plasmid degradation is more likely than the often cited formation of hydroxyl radicals in result of the Fenton reaction from Fe2+ and hydrogen peroxide.