title: Direct evidence for apo B-100-mediated copper reduction: studies with purified apo B-100 and detection of tryptophanyl radicals.
abstract: Copper binding to apolipoprotein B-100 (apo B-100) and its reduction by endogenous components of low-density lipoprotein (LDL) represent critical steps in copper-mediated LDL oxidation, where cuprous ion (Cu(I)) generated from cupric ion (Cu(II)) reduction is the real trigger for lipid peroxidation. Although the copper-reducing capacity of the lipid components of LDL has been studied extensively, we developed a model to specifically analyze the potential copper reducing activity of its protein moiety (apo B-100). Apo B-100 was isolated after solubilization and extraction from size exclusion-HPLC purified LDL. We obtained, for the first time, direct evidence for apo B-100-mediated copper reduction in a process that involves protein-derived radical formation. Kinetics of copper reduction by isolated apo B-100 was different from that of LDL, mainly because apo B-100 showed a single phase-exponential kinetic, instead of the already described biphasic kinetics for LDL (namely alpha-tocopherol-dependent and independent phases). While at early time points, the LDL copper reducing activity was higher due to the presence of alpha-tocopherol, at longer time points kinetics of copper reduction was similar in both LDL and apo B-100 samples. Electron paramagnetic resonance studies of either LDL or apo B-100 incubated with Cu(II), in the presence of the spin trap 2-methyl-2-nitroso propane (MNP), indicated the formation of protein-tryptophanyl radicals. Our results supports that apo B-100 plays a critical role in copper-dependent LDL oxidation, due to its lipid-independent-copper reductive ability.
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Uruguay.
authors.forename: C
authors.initials: C
authors.lastname: Batthyány
authors.forename: C X
authors.initials: CX
authors.lastname: Santos
authors.forename: H
authors.initials: H
authors.lastname: Botti
authors.forename: C
authors.initials: C
authors.lastname: Cerveñansky
authors.forename: R
authors.initials: R
authors.lastname: Radi
authors.forename: O
authors.initials: O
authors.lastname: Augusto
authors.forename: H
authors.initials: H
authors.lastname: Rubbo
mesh_terms: D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000302:isolation & purification / Q000378:metabolism; D009603:Nitroso Compounds / Q000378:metabolism; D010084:Oxidation-Reduction; D019097:Spin Trapping; D014364:Tryptophan / Q000378:metabolism
chemical_list: D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D005609:Free Radicals; D008077:Lipoproteins, LDL; D009603:Nitroso Compounds; D003300:Copper; D014364:Tryptophan; C034526:tert-nitrosobutane