EPR Study of KO(2) as a Source of Superoxide and (•)BMPO-OH/OOH Radical That Cleaves Plasmid DNA and Detects Radical Interaction with H(2)S and Se-Derivatives

Superoxide radical anion (O(2)(•−)) and its derivatives regulate numerous physiological and pathological processes, which are extensively studied. The aim of our work was to utilize KO(2) as a source of O(2)(•−) 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 KO(2) formed relatively stable (•)BMPO-OOH and (•)BMPO-OH radicals and H(2)O(2), 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. H(2)S 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 KO(2) as a source of O(2)(•−)/H(2)O(2) 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.