Radical damage to proteins studied by EPR spin-trapping techniques

We report the use of the novel spin trap DEPMPO in conjunction with EPR spe ctroscopy to provide evidence for free-radical mediated oxidation of some p roteins in aqueous solution, with systems including metal ions, peroxides o r both. In addition we have used a novel approach involving detailed spectr al simulation (to reproduce anisotropic features of phosphorus, hydrogen an d nitrogen splittings) to confirm structural assignments. Oxidation of bovine serum albumin (BSA) with either Ce4+ or IrCl62- leads t o the predominant formation of spin-trapped oxygen-centred free-radicals, a ttributed to tyrosyl-based adducts, a reaction also observed for sulfur-fre e analogues (lysozyme and tyrosine-containing models). Similar spectra were obtained from reactions involving oxidation with HRP/H2O2 and methaemoglob in/H2O2 couples, through intermolecular oxidation of the proteins via oxo-i ron intermediates. Oxidation of BSA with peroxynitrite leads to an anisotropic spectrum which is assigned to the trapping of a sulfur-centred radical. This assignment ha s been confirmed via denaturation of the spin-adduct (and simulation of the resulting less immobilized spectrum). Reaction of the hydroxyl radical wit h BSA and lysozyme, in Fenton-type reactions, leads to the detection of ani sotropic spectra derived from the spin-trapping of carbon-centred radicals, whose assignment is discussed.