Measurement of Copper-Binding Sites on Low Density Lipoprotein

Abstract

Abstract-Copper is often used to oxidize low density lipoprotein (LDL) in experiments in vitro and is a candidate for oxidizing LDL in atherosclerotic lesions. The binding of copper ions to LDL is usually thought to be a prerequisite for LDL oxidation by copper, although estimates of LDL copper binding vary widely. We have developed and validated an equilibrium dialysis assay in a MOPS-buffered system to measure copper binding to LDL and have found 38.6Ϯ0.7 (meanϮSEM, nϭ25) copper binding sites on LDL. The binding was saturated at a copper concentration of 10 mol/L at LDL concentrations of up to 1 mg protein/mL. Copper-binding capacity increased progressively and markedly when LDL was oxidized to increasing extents. Chemical modification of histidyl and lysyl residues on apolipoprotein B-100 reduced the number of binding sites by 56% and 23%, respectively. As an example of the potential of this method to assess the effects of antioxidants on copper binding to LDL, we have shown that the flavonoids myricetin, quercetin, and catechin (but not epicatechin, kaempferol, or morin), at concentrations equimolar to the copper present (10 mol/L), significantly decreased copper binding to LDL by 82%, 56%, and 20%, respectively.

Prooxidant and antioxidant properties of human serum ultrafiltrates toward LDL: important role of uric acid

“…The reasons for this are unknown, but may possibly relate to our finding that LDL has about 40 binding sites for copper per particle (38) and at 50 g of LDL protein/ml (about 0.1 M LDL) there would be insufficient copper at 1 M to bind to all the sites. Alternatively, the ratio of Cu 2ϩ to Cu ϩ may vary with the concentration of copper and this may affect the antioxidant/prooxidant activities observed.…”

Relationship Between Protein Oxidation Markers and Oxidative Stress Biomarkers

“…Ascorbic acid protects apolipoprotein E of oxidation (Jedidi et al 2003) LDL oxidation was mediated by water gamma radiolysis (*)OH initiate oxidation leading to apob carbonylation in presence of aminoguanidine (Roland et al 2001) LDL and Cu(2+) Flavonoids myricetin, quercetin, and catechin decreased copper binding to LDL (Patterson et al 2003) LDL, human serum ultrafiltrate of Mr below 500 and hydroperoxide or Cu(2+) Low Cu(2+) inhibit tocopherol induced oxidation in LDL, promote breakdown of lipid hydroperoxides into radicals (Makedou et al 2009) Copper-induced Low-density lipoprotein (LDL) oxidizability Progeny with a positive family history for hyperlipidemia have increased LDL susceptibility to oxidation (Hockerstedt et al 2004) Copper-induced oxidation of purified HDL LCAT causes estradiol esterification and thus provide antioxidant protection to HDL (Moreno & Fuster 2004) LDL oxidation susceptibility to Cu(2+) Apolipoprotein E polymorphism partially explain differences in individual responses to diet (Popa et al 2009) HDL ability to inhibit copperinduced oxidation of lowdensity lipoprotein (LDL)…”

“…Copper ions bind to LDL being the copper-binding capacity progressively and markedly higher when LDL is increasingly oxidized. It was assessed that the flavonoids myricetin, quercetin, and catechin (but not epicatechin, kaempferol, or morin), at concentrations equimolar to the copper present significantly decreased copper binding to LDL (Roland, Patterson, & Leake 2001). Later, the same group proposed uric acid as both antioxidant and prooxidant for LDL.…”

The Antiatherogenic Effect of Allicin: Possible Mode of Action

“…Oxidation of LDL by Cu 2+ is dependent on its binding to LDL [7] . Application of electron spin resonance (ESR) is based on a decreased Cu 2+ ESR signal as a result of its binding and immobilization on a polymer surface [40] .…”

“…This protein is made up of 4,536 amino acids, 25 of which are cysteinyl residues. Nine of them exist as free thiols, located in the hydrophobic regions of LDL, and the remainder form disulfi de bonds [7] .…”