Flow-through partial-filling affinity capillary electrophoresis for the estimation of binding constants of ligands to receptors

A new approach for estimating binding constants of ligands to receptors, fl ow-through partial-filling affinity capillary electrophoresis (FTPFACE), is introduced for studying the interaction of carbonic anhydrase B (CAB, EC 4 .2.1.1) with arylsulfonamides and vancomycin from Streptomyces orientalis w ith D-Ala-D-Ala peptides. In this technique the capillary is first partiall y-filled with ligand followed by a sample of receptor and non-interacting s tandards. Upon application of a voltage the receptor and standards flow int o the ligand plug where equilibrium is achieved between the receptor and li gand. Continued electrophoresis results in the receptor and standards flowi ng through the domain of the ligand plug. Analysis of the change in the rel ative migration time ratio of the receptor, relative to the non-interacting standards, as a function of the concentration of ligand, yields a value fo r the binding constant. These values are comparable to those estimated usin g other binding and ACE techniques. Data demonstrating the quantitative pot ential of this method is presented.