Direct Evidence for apo B-100-Mediated Copper Reduction: Studies with Purified apo B-100 and Detection of Tryptophanyl Radicals

• Published in: Archives of biochemistry and biophysics 2000-12, Vol.384 (2), p.335-340
• Main Authors: Batthyány, Carlos, Santos, Célio X.C., Botti, Horacio, Cerveñansky, Carlos, Radi, Rafael, Augusto, Ohara, Rubbo, Homero
• Format: Article
• Language: English
• Subjects: apo B-100; Apolipoprotein B-100; Apolipoproteins B - isolation & purification; Apolipoproteins B - metabolism; Chromatography, High Pressure Liquid; copper; Copper - metabolism; Electron Spin Resonance Spectroscopy; free radicals; Free Radicals - metabolism; Humans; Kinetics; LDL oxidation; lipid oxidation; Lipoproteins, LDL - isolation & purification; Lipoproteins, LDL - metabolism; nitric oxide; Nitroso Compounds - metabolism; Oxidation-Reduction; protein oxidation; Spin Trapping; Tryptophan - metabolism; tryptophan-centered radical
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.2000.2102

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 α-tocopherol-dependent and independent phases). While at early time points, the LDL copper reducing activity was higher due to the presence of α-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.