Optimization of conditions for flow-through partial-filling affinity capillary electrophoresis to estimate binding constants of ligands to receptors

This work details the determination of the minimal injection time of ligand required in flow-through partial-filling affinity capillary electrophoresis (FTPFACE) to estimate binding constants of ligands to receptors. Two model systems are examined in this study: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, and vancomycin from Streptomyces orientalis and d-Ala-d-Ala peptides. Using CAB, a minimal injection time of 0.07 min at high pressure was determined that provided for the accurate and reproducible measurement of binding constants. In the FTPFACE technique, the capillary is first partially filled with a zone of ligand followed by a sample plug containing receptor and non-interacting standards. Upon application of a voltage the receptor and standards flow into the zone of ligand where a dynamic equilibrium is achieved between receptor and ligand. Continued electrophoresis results in the receptor and standards flowing through the domain of the ligand plug prior to detection. Analysis of the change in the relative migration time ratio (RMTR) of the receptor, relative to the non-interacting standards, as a function of the concentration of ligand, yields a value for the binding constant. In the present study, variable injection times of 4-carboxybenzenesulfonamide (CBSA) were examined to determine the minimal injection time needed to establish an equilibrium between CAB and ligand. A mathematical relationship was derived that correlated injection time and ligand concentration to the change in RMTR and comparisons made between the experimental and calculated values. Binding constants were obtained for a series of arylsulfonamide ligands and d-Ala-d-Ala terminus peptides to CAB and Van, respectively. The results support the use of FTPFACE to estimate affinity constants under variable experimental conditions.