Partial filling affinity capillary electrophoresis including adsorption energy distribution calculations--towards reliable and feasible biomolecular interaction studies.

In this work, a method to study and analyze the interaction data in free solution by exploiting partial filling affinity capillary electrophoresis (PF-ACE) followed by adsorption energy distribution calculations (AED) prior model fit to adsorption isotherms will be demonstrated. PF-ACE-AED approach allowed the possibility to distinguish weak and strong interactions of the binding processes between the most common apolipoprotein E protein isoforms (apoE2, apoE3, apoE4) of high density lipoprotein (HDL) and apoE-containing HDL2 with major glycosaminoglycan (GAG) chain of proteoglycans (PGs), chondroitin-6-sulfate (C6S). The AED analysis clearly revealed the heterogeneity of the binding processes. The major difference was that they were heterogeneous with two different adsorption sites for apoE2 and apoE4 isoforms, whereas interestingly for apoE3 and apoE-containing HDL2, the binding was homogeneous (one site) adsorption process. Moreover, our results allowed the evaluation of differences in the binding process strengths giving the following order with C6S: apoE-containing HDL2 > apoE2 > apoE4 > apoE3. In addition, the affinity constant values determined could be compared with those obtained in our previous studies for the interactions between apoE isoforms and another important GAG chain of PGs - dermatan sulfate (DS). The success of the combination of AED calculations prior to non-linear adsorption isotherm model fit with PF-ACE when the concentration range was extended, confirmed the power of the system in the clarification of the heterogeneity of biological processes studied.