Use of affinity capillary electrophoresis to determine kinetic and equilibrium constants for binding of arylsulfonamides to bovine carbonic anhydrase.

Affinity capillary electrophoresis (ACE) provides a new approach to studying protein-ligand interactions. The basis for ACE is the change in the electrophoretic mobility of the protein when it forms a complex with its ligand. This binding interaction can be quantified directly for charged ligands or indirectly for neutral ligands in competition with a previously characterized charged ligand. Determination of kinetic and equilibrium constants using ACE relies only on the changes in the migration time and shape (but not the area) of the peak due to protein. Simulation of the protein mobility under conditions of ACE suggests that the experimentally obtained electropherograms can be explained in terms of few variables: on and off rates (and thus, binding constant), concentration of the ligand(s), and relative mobilities of the protein and its complex(es).