Measurement of copper-binding sites on low density lipoprotein

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 38.6+/-0.7 (mean+/-SEM, n=25) copper binding sites on LDL. The binding was saturated at a copper concentration of 10 micromol/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 micromol/L), significantly decreased copper binding to LDL by 82%, 56%, and 20%, respectively.