ESR measurement of rapid penetration of DMPO and DEPMPO spin traps through lipid bilayer membranes

The passive permeation rates of DMPO and DEPMPO spin traps and their hydroxyl radical adducts through liposomal membranes were measured using ESR spectroscopy. For the spin traps, we measured the time-dependent change in the signal intensity of the OH-adduct, which is formed by a reaction between the penetrated spin trap and hydroxyl radicals produced by the UV-radiolysis of H2O2 inside the liposomes. The hydroxyl radicals produced outside the liposomes were quenched with polyethylene glycol. For the OH-adduct, pre-formed adduct was mixed with liposomes and the time-dependent change of the ESR signal was measured in the presence of a line-broadening reagent outside the liposomes to make the signal outside the liposomes invisible. Both the spin traps and their OH-adducts diffused across the lipid membranes rapidly and reached equilibrium within tens of seconds. These findings suggest that if used for the detection of free radicals inside cells, these spin traps should be well distributed in cells and even in organelles.