2.2. EPR Study of the BMPO-Adducts

A modified protocol from our previous study was used [13]. First, ^•BMPO-OOH was prepared by the addition of the saturated KO₂/DMSO solution (10% v/v DMSO/final buffer) into the BMPO (30 mmol L⁻¹ final concentration, 37 °C) diluted in the phosphate buffer. The BMPO/KO₂ sample was mixed for 10 s and then the studied compounds, H₂S_n, SeO_n²⁻ or H₂S_n/SeO_n²⁻ mixtures, were added. The sample was mixed again for 5 s and transferred to a standard cavity aqueous EPR flat cell (WG 808-Q, Wilmad-LabGlass, Vineland, NJ, USA). The first EPR spectrum was recorded 100 s after the addition of the compounds. The sets of the individual EPR spectra of the BMPO spin-adducts were recorded as 15 sequential scans, each 42 s, with a total acquisition time of 11 min. Each experiment was repeated at least twice. EPR spectra of the BMPO spin-adducts were measured on a Bruker EMX spectrometer (Rheinstetten, Germany), X-band ~9.4 GHz, 335.15 mT central field, 8 mT scan range, 20 mW microwave power, 0.1 mT modulation amplitude, 42 s sweep time, 20.48 ms time constant and 20.48 ms conversion time at 37 °C. To compare the relative potency of the compounds to decrease overall trapped radical concentration, a second integral of the total EPR spectra intensity of the BMPO-adducts was evaluated. To obtain the ^•BMPO-OH/^•BMPO-OOH radical proportion, the simulated spectra ratio was calculated using EasySpin program working on MatLab platform [22].