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

Anton Misak; Vlasta Brezova; Miroslav Chovanec; Karol Luspai; Muhammad Jawad Nasim; Marian Grman; Lenka Tomasova; Claus Jacob; Karol Ondrias

doi:10.3390/antiox10081286

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

Antioxidants (Basel). 2021 Aug 13;10(8):1286. doi: 10.3390/antiox10081286.

Authors

Anton Misak¹, Vlasta Brezova², Miroslav Chovanec³, Karol Luspai², Muhammad Jawad Nasim⁴, Marian Grman¹, Lenka Tomasova¹, Claus Jacob⁴, Karol Ondrias¹

Affiliations

¹ Biomedical Research Center, Department of Molecular Physiology, Institute of Clinical and Translational Research, Slovak Academy of Sciences, Dúbravská Cesta 9, 84505 Bratislava, Slovakia.
² Institute of Physical Chemistry and Chemical Physics, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinského 9, 81237 Bratislava, Slovakia.
³ Biomedical Research Center, Department of Genetics, Cancer Research Institute, Slovak Academy of Sciences, Dúbravská Cesta 9, 84505 Bratislava, Slovakia.
⁴ Division of Bioorganic Chemistry, School of Pharmacy, University of Saarland, D-66123 Saarbruecken, Germany.

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.

Keywords: EPR spectra simulation; KO2; antioxidants; cleavage DNA; hydrogen sulfide; radical; selenium-derivatives; superoxide; •BMPO-OOH spin adduct.

Abstract

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