Partial-filling affinity capillary electrophoresis and quartz crystal microbalance with adsorption energy distribution calculations in the study of biomolecular interactions with apolipoprotein E as interaction partner.

ABSTRACT

Adsorption energy distribution (AED) calculations were successfully applied to partial-filling affinity capillary electrophoresis (PF-ACE) to facilitate more detailed studies of biomolecular interactions. PF-ACE with AED calculations was employed to study the interactions between two isoforms of apolipoprotein E (apoE) and dermatan sulfate (DS), and a quartz crystal microbalance (QCM) was used in combination with AED calculations to examine the interactions of the 15-amino-acid peptide fragment of apoE with DS. The heterogeneity of the interactions was elucidated. Microscale thermophoresis was used to validate the results. The interactions studied are of interest because, in vivo, apolipoprotein E localizes on DS-containing regions in the extracellular matrix of human vascular subendothelium. Two-site binding was demonstrated for the isoform apoE3 and DS, but only one-site binding for apoE2-DS. Comparable affinity constants were obtained for the apoE2-DS, apoE3-D3, and 15-amino-acid peptide of apoE-DS using the three techniques. The results show that combining AED calculations with modern biosensing techniques can open up another dimension in studies on the heterogeneity and affinity constants of biological molecules.