Study of Lipid and Apolipoprotein Binding Interactions Using Vesicle Affinity Capillary Electrophoresis

Abstract

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 degrees C), using vesicle as a model. The technique results in similar lipid binding properties of apo CIII (CIII) and its peptides compared to other techniques. 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 degrees C) 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.

Capillary electrophoresis of proteins 2003–2005

“…CZE was found useful for investigation of the Maillard reaction of glucosamine with fibrinogen and HSA [294], formation of avidin-biotine complexes [295], adsorption of small ions on proteins [296], composition of metallo-blactamase from Aeromonas hydrophila [297], structural and conformational variants of human b 2 -microglobulin [298], its folding, unfolding, and aggregation [299], apolipoprotein binding interactions [130], interaction between b-lactoglobulin and pectin [300], distribution of Se in nut proteins [301], folding of carbonic anhydrase [302], coexisting protein conformations [303], interaction of human immunodeficiency virus (HIV) inhibitors with trans-activator of transcription protein [304], and behavior of the MS2 virus and a corresponding antibody [149].…”

Recent applications of affinity interactions in capillary electrophoresis

“…Otherwise, pure lipids must be analyzed as micellar preparations, as vesicles/liposomes, in the presence of detergents (enabling the formation of mixed micelles), or by using nonaqueous CE. The lipid binding of apolipoproteins was studied using an ACE approach with negatively charged phospholipid vesicles added to the physiological electrophoresis buffer [45]. Unilamellar vesicles resembling cell membranes were found more suitable for binding studies than multilamellar vesicles and liposomes.…”

Electrophoresis of a non-conducting Newtonian drop of low electrical potential normal to a plane

“…Relevant results on non-rigid entities, although of both fundamental and practical significance, are very limited. The application of capillary electrophoresis in characterization and separation of vesicles and multi-components liquid separation, for instance, all involve this problem (Breyer et al, 2003;Ashok and Joykrishna, 2005). The analysis of the electrophoresis of non-rigid particles is more complicated than that of rigid ones because both the electrokinetic equations inside a particle and those outside a particle need to be considered simultaneously.…”