Figure 5.

Treatment of hhMb with regent HOCl inhibits the formation of protein-centered radicals as detected by spin trapping. Solutions of hhMb (final concentration 500 µM) were exposed to an increasing dose of reagent HOCl (final oxidant:protein mol ratio indicated in the representative spectra shown in panels (A) Control; (B) hhMb pretreated with HOCl 5 mol/mol protein; (C) hhMb pretreated with HOCl 10 mol/mol protein and (D) absence of hhMb). All hhMb solutions contained 100 µM the chelator diethylenetriaminepentaacetic acid (DTPA). Aliquots of reaction mixture were then treated with hydrogen peroxide (final ratio H₂O₂:hhMb ∼5 mol/mol) or phosphate buffer (as a vehicle control) in the presence of 5 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as described in the Methods section. The reaction mixture was then rapidly transferred to a flat cell and radical generation assessed using EPR spectroscopy with the following parameters: Microwave power 20 mW, modulation amplitude 0.1 mT, modulation frequency of 100 kHz and a sweep time of 84 s. EPR spectra were obtained as a cumulative average of five consecutive scans. No signal was obtained in the absence of DMPO or H₂O₂ (data not shown). Data are representative of n = 3 independent experiments using freshly prepared protein radicals.