Determination of Binding Constants of Ligands to Proteins by Affinity Capillary Electrophoresis: Compensation for Electroosmotic Flow

Abstract

This paper describes the estimation of binding constants (Kb) between carbonic anhydrase B (CAB, EC 4.2.1.1, from bovine erythrocytes) and charged benzenesulfonamides by affinity capillary electrophoresis (ACE) under conditions in which the migration time is affected by changes in electroosmotic flow and by nonspecific interactions accompanying changes in the concentration of ligand. Comparisons of values of migration times of the protein of interest, and of "noninteracting" marker proteins, with those of a neutral internal standard provide the basis for corrections for variable electroosmotic flow; these corrections make possible the estimation of Kb and its uncertainty even in the presence of substantial variations in electroosmotic flow.