Electron paramagnetic resonance (EPR) spectroscopy assessment of inter-conversions of nitroxides and hydroxylamines.
(a) Effect of Ascorbate, Ascorbate oxidase and Cyt c/H2O2 on the EPR signal of XJB 5-131. A: XJB-5-131 alone in 35 μL of PBS plus 35 μL of DMSO. B: A plus 250 μM Ascorbate, 1 min. C: A plus 250 μM Ascorbate, 5 min. D: A plus 250 μM Ascorbate, 5 min plus ascorbate oxidase (1U, 15 min). E: D plus10 μM Cyt c. F: D plus10 μM Cyt c/200 μM H2O2. (b) Effect of ascorbate, ascorbate oxidase, and NADH on the EPR signal of XJB-5-131 in brain homogenates. A: 35 μL of Brain Homogenate (350 μg protein) in PBS plus 7 μM XJB-5-131 plus 35μL of DMSO. B: A plus Ascorbate Oxidase (2U), 30 min. C: B plus NADH (200 μM), 30 min. D: C plus K3Fe(CN)6 (100 μM). (c) Effect of pH on the EPR signal of XJB-5-131-OH. 50 μL of 7 μM XJB-5-131-OH in DMSO were mixed with 50 μL of 50 μM HEPES pH 7.45 (open bars) or with 50 μL of 50 μM HEPES pH 8.25 (closed bars). Error bars, standard deviation.