•BMPO-OOH Spin-Adduct as a Model for Study of Decomposition of Organic Hydroperoxides and the Effects of Sulfide/Selenite Derivatives. An EPR Spin-Trapping Approach

Abstract

Lipid hydroperoxides play an important role in various pathophysiological processes. Therefore, a simple model for organic hydroperoxides could be helpful to monitor the biologic effects of endogenous and exogenous compounds. The electron paramagnetic resonance (EPR) spin-trapping technique is a useful method to study superoxide (O2•−) and hydroxyl radicals. The aim of our work was to use EPR with the spin trap 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO), which, by trapping O2•− produces relatively stable •BMPO-OOH spin-adduct, a valuable model for organic hydroperoxides. We used this experimental setup to investigate the effects of selected sulfur/selenium compounds on •BMPO-OOH and to evaluate the antioxidant potential of these compounds. Second, using the simulation of time-dependent individual BMPO adducts in the experimental EPR spectra, the ratio of •BMPO-OH/•BMPO-OOH—which is proportional to the transformation/decomposition of •BMPO-OOH—was evaluated. The order of potency of the studied compounds to alter •BMPO-OOH concentration estimated from the time-dependent •BMPO-OH/•BMPO-OOH ratio was as follows: Na2S4 > Na2S4/SeO32− > H2S/SeO32− > Na2S2 ~Na2S2/SeO32− ~H2S > SeO32− ~SeO42− ~control. In conclusion, the presented approach of the EPR measurement of the time-dependent ratio of •BMPO-OH/•BMPO-OOH could be useful to study the impact of compounds to influence the transformation of •BMPO-OOH.

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

“…The shapes of the EPR spectra of the BMPO adducts depended on the pH ( Figure 1 ) and changed over time, indicating a dynamic superposition of signals corresponding to the generation of individual BMPO adducts. Therefore, we analyzed the EPR spectra by simulation, as in [ 6 ]. Two conformers each of • BMPO-OOH and • BMPO-OH adducts were inserted into the spin Hamiltonian calculations of the experimental spectra measured in the pH range of 2–8.…”

“…The detailed procedure for the sample preparation for EPR measurement is described in the Supplementary Materials . The sample was transferred to a standard cavity aqueous EPR flat cell (WG 808-Q, Wilmad-LabGlass, Vineland, NJ, USA), and the EPR spectra were measured as described previously [ 6 ]. The first EPR spectrum was recorded 100 ± 15 s after the addition of the BMPO solution to powdered KO 2 .…”

“…The sets of individual EPR spectra of the BMPO spin adducts were recorded as 15 or 30 sequential scans of 42 s each, with a total acquisition time of 11 or 22 min, respectively. The EPR spectra of the BMPO spin adducts were measured on a Bruker EMX spectrometer (Rheinstetten, Germany) essentially as in our previous study [ 6 ]. All of the EPR spectra were recorded at 37 °C.…”

“…A pDNA cleavage assay with the use of the pBR322 plasmid (N3033 L, New England BioLabs, Inc., Ipswich, MA, USA) was performed as reported previously, with some modifications [ 6 ]. All of the final samples contained the studied compounds and 0.2 μg of pDNA in 20 µL of the sodium phosphate buffer (25 mmol L −1 sodium phosphate, 50 μmol L −1 DTPA; pH 6.5, 7.4, 8.0, or 8.5).…”

“…For experiments involving radical O 2 •− reactions, O 2 •− can be generated using various methods [ 3 ], e.g., enzymatically by xanthine/xanthine oxidase [ 4 , 5 ], which is a system too complex for some types of O 2 •− radical models. It can also be generated by ionizing radiation or by dissolving superoxide salts in aprotic solvents, e.g., KO 2 in anhydrous DMSO; however, DMSO may not be desirable in some biological experiments [ 3 , 6 ]. On the other hand, KO 2 dissolved in an aqueous solution is a simple source of O 2 • − , but its lifetime is in the range of milliseconds [ 7 ].…”