EPR Study of KO<sub>2</sub> as a Source of Superoxide and <sup>•</sup>BMPO-OH/OOH Radical That Cleaves Plasmid DNA and Detects Radical Interaction with H<sub>2</sub>S and Se-Derivatives
Anton Misak,
Vlasta Brezova,
Miroslav Chovanec,
Karol Luspai,
Muhammad Jawad Nasim,
Marian Grman,
Lenka Tomasova,
Claus Jacob,
Karol Ondrias
Affiliations
Anton Misak
Biomedical Research Center, Department of Molecular Physiology, Institute of Clinical and Translational Research, Slovak Academy of Sciences, Dúbravská Cesta 9, 84505 Bratislava, Slovakia
Vlasta Brezova
Institute of Physical Chemistry and Chemical Physics, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinského 9, 81237 Bratislava, Slovakia
Miroslav Chovanec
Biomedical Research Center, Department of Genetics, Cancer Research Institute, Slovak Academy of Sciences, Dúbravská Cesta 9, 84505 Bratislava, Slovakia
Karol Luspai
Institute of Physical Chemistry and Chemical Physics, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinského 9, 81237 Bratislava, Slovakia
Muhammad Jawad Nasim
Division of Bioorganic Chemistry, School of Pharmacy, University of Saarland, D-66123 Saarbruecken, Germany
Marian Grman
Biomedical Research Center, Department of Molecular Physiology, Institute of Clinical and Translational Research, Slovak Academy of Sciences, Dúbravská Cesta 9, 84505 Bratislava, Slovakia
Lenka Tomasova
Biomedical Research Center, Department of Molecular Physiology, Institute of Clinical and Translational Research, Slovak Academy of Sciences, Dúbravská Cesta 9, 84505 Bratislava, Slovakia
Claus Jacob
Division of Bioorganic Chemistry, School of Pharmacy, University of Saarland, D-66123 Saarbruecken, Germany
Karol Ondrias
Biomedical Research Center, Department of Molecular Physiology, Institute of Clinical and Translational Research, Slovak Academy of Sciences, Dúbravská Cesta 9, 84505 Bratislava, Slovakia
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.
