Epr study of ko2 as a source of superoxide and•bmpo-oh/ooh radical that cleaves plasmid dna and detects radical interaction with h2s and se-derivatives

Abstract

Superoxide radical anion (O2•−) and its derivatives regulate numerous physiological and pathological processes, which are extensively studied. The aim of our work was to utilize KO2 as a source of O2•− and the electron paramagnetic resonance (EPR) spin trapping 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) technique for the preparation of•BMPO-OOH and/or•BMPO-OH radicals in water solution without DMSO. The method distinguishes the interactions of various compounds with•BMPO-OOH and/or•BMPO-OH radicals over time. Here, we show that the addition of a buffered BMPO-HCl mixture to powdered KO2 formed relatively stable•BMPO-OOH and•BMPO-OH radicals and H2O2, where the•BMPO-OOH/OH ratio depended on the pH. At a final pH of ~6.5–8.0, the concentration of•BMPO-OOH radicals was ≥20 times higher than that of•BMPO-OH, whereas at pH 9.0–10.0, the•BMPO-OH radicals prevailed. The•BMPO-OOH/OH radicals effectively cleaved the plasmid DNA. H2S decreased the concentration of•BMPO-OOH/OH radicals, whereas the selenium derivatives 1-methyl-4-(3-(phenylselanyl) propyl) piperazine and 1-methyl-4-(4-(phenylselanyl) butyl) piperazine increased the proportion of•BMPO-OH over the•BMPO-OOH radicals. In conclusion, the presented approach of using KO2 as a source of O2•−/H2O2 and EPR spin trap BMPO for the preparation of•BMPO-OOH/OH radicals in a physiological solution could be useful to study the biological effects of radicals and their interactions with compounds.