title: A study of photochemically-generated protein radical spin adducts on bovine serum albumin: the detection of genuine spin-trapping and artefactual, non-radical addition in the same molecule.
abstract: Photo-oxidation of bovine serum albumin (BSA) by porphyrins produces protein-centred radicals that can be spin trapped by 3, 5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS) and 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO). In the case of DMPO, a thiyl radical from the Cys-34 residue is trapped, whereas with DBNBS signals from both this thiyl and tertiary carbon-centred species are observed. However, specific chemical modification of the Cys-34 residue, in combination with dual-isotope spin-trapping techniques, shows that the signal assigned to the Cys-34 thiyl adduct with DBNBS is a nitroxide artefact resulting from sequential (non-radical) nucleophilic addition and oxidation. In contrast, both the Cys-34 thiyl DMPO adduct and the tertiary carbon-centred DBNBS adducts result from genuine spintrapping. This study shows that such artefacts can be detected-even with anisotropic EPR spectra-through the use of appropriately substituted spin-traps, and that nitroso spin-traps need to be employed with great care in systems containing free thiol groups.
authors.affiliation: a Department of Chemistry , University of York , York , UK.
authors.forename: J A
authors.initials: JA
authors.lastname: Silvester
authors.affiliation: c EPR Center, Department of Radiology , Dartmouth Medical School , Hanover , USA.
authors.forename: X D
authors.initials: XD
authors.lastname: Wei
authors.affiliation: b The Heart Research Institute , Camperdown, Sydney , Australia.
authors.forename: M J
authors.initials: MJ
authors.lastname: Davies
authors.affiliation: a Department of Chemistry , University of York , York , UK.
authors.forename: G S
authors.initials: GS
authors.lastname: Timmins