Study of lipid and apolipoprotein binding interactions using vesicle affinity capillary electrophoresis

Vesicle affinity capillary electrophoresis (VCE), a newly developed technique, was designed to assess the effect of physicochemical properties of apolipoprotein (apo) on the binding to lipoproteins, under physiological conditions (phosphate-saline buffer system at pH 7.4 and 37 degreesC), using vesicle as a model. The technique results in similar lipid binding properties of apo CIII (CIII) and its peptides compared to other techniques.(1,2) It also offers a fast and more sensitive tool in determining the lipid affinity of apos in a unique system simulating the dynamic binding properties of apo in vivo. A noncompetitive binding model is used to determine the multiple binding properties of CIII and its peptides to vesicle. The VCE binding constants are dependent on temperature, physicochemical properties of the protein (hydrophobicity and charge), and nature of the vesicle. The vesicles used in the VCE experiments described here have been fully characterized and found to be stable under different temperatures (4 and 37 degreesC) and voltage conditions. Migration behavior of CIII and related peptides is reported in terms of relative mobility in order to correct for variability in viscosity at different vesicle concentrations. The VCE method provides very precise data on the migration time from 0.1 to 3.3% RSD at the highest concentration of vesicle. The model and current data have been used to determine VCE binding constants and protein-to-lipid binding ratios. The model predicts that higher lipid affinity (K-B), protein-lipid binding ratio (n), and lower protein concentration result in a shift of the binding isotherm toward a lower concentration range of vesicle. A higher vesicle mobility, reflecting the size and charge of the vesicle, results in a larger separation window between the migration time of the free protein and the complex. The value of VCE for structure-function studies and drug design for peptides and proteins that are strongly bound to lipids has been illustrated.