RESUMO
This work evaluates the use of mobility ratios (M) to estimate binding constants of proteins to ligands using affinity capillary electrophoresis (ACE). This concept is demonstrated using two model systems: vancomycin (Van) from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). A plot of change in M (deltaM) over the concentration of ligand [L] versus deltaM yields a more useful representation of the Scatchard plot in capillary electrophoresis (CE) than traditional plots of the change in mobility delta mu over [L] versus delta mu in a wide set of circumstances, especially when comparing electropherograms obtained in the presence of substantial variations in electroosmotic flow. Altering the voltage and/or capillary length of the CE system produced only small variations in M, but much larger changes in the more standard measures of migration used by the mu form of analysis. The use of M in the Scatchard analysis offers a new approach to estimating binding constants of ligands to proteins using ACE.
