Methods for the determination of binding constants by capillary electrophoresis

The qualitative and quantitative aspects of capillary electrophoretic methods used to study drug-protein interactions, viz. the affinity capillary electrophoresis (ACE). Hummel-Dreyer (HD), frontal analysis (FA), vacancy peak (VP) and vacancy affinity capillary electrophoresis (VACE) methods have been investigated. In the ACE and the VACE methods the binding parameters can be calculated from the change in the electrophoretic mobility of the drug on complexation with a protein. In the frontal analysis and the vacancy peak method the free drug concentration is measured with UV detection. In the Hummel-Dreyer method the amount of drug bound is measured with UV detection. For the comparison of these five methods the warfarin-bovine serum albumin (BSA) system was used. Several factors that might influence the determination of association parameters were examined. With the FA, VP, HD and VACE methods the absolute numbers of the different binding sites involved in the complex formation can be determined, a major advantage in drug-binding studies.