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

• Main Authors: Anton Misak, Vlasta Brezova, Miroslav Chovanec, Karol Luspai, Muhammad Jawad Nasim, Marian Grman, Lenka Tomasova, Claus Jacob, Karol Ondrias
• Format: Article
• Language: English
• Published: MDPI AG 2021-08-01
• Series: Antioxidants
• Subjects: KO₂; antioxidants; EPR spectra simulation; ^•BMPO-OOH spin adduct; superoxide; radical
• Online Access: https://www.mdpi.com/2076-3921/10/8/1286
• ISSN: 2076-3921

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-<i>tert</i>-butoxycarbonyl-5-methyl-1-pyrroline <i>N</i>-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.