Journal of Chromatography B, 715 (1998) 203–210 Use of mobility ratios to estimate binding constants of ligands to proteins in affinity capillary electrophoresis * Jane Kawaoka, Frank A. Gomez Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8202, USA Abstract 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 (DM ) over the concentration of ligand [L] versus DM yields a more useful representation of the Scatchard plot in capillary electrophoresis (CE) than traditional plots of the change in mobility Dm over [L] versus Dm 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 m 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.  1998 Elsevier Science B.V. All rights reserved. Keywords: Mobility ratios; Binding constants; Ligands; Proteins; Vancomycin; Carbonic anhydrase 1. Introduction ing binding and dissociation constants of formed complexes [3–30]. For example, Mammen et al. [27] Capillary electrophoresis (CE) is a versatile mi- have shown that ACE can be used to determine the croanalytical technique that has gained much atten- two dissociation constants of the complex between tion, particularly from those working with biological- an antibody and charged N-dinitrophenyl ligands. ly active molecules. CE differentiates charged Kwak and Gomez [26] have demonstrated the use of species on the basis of mobility under the influence ACE to study the binding of adamantane carboxylic of an applied electric field gradient. The characteri- acids to cyclodextrins using indirect detection. Final- zation of specific interactions is the focus of much ly, Chu et al. [6] have used ACE to determine biochemical research [1]. There are a number of binding stoichiometries of protein–ligand interac- methods to measure binding parameters for noncov- tions. alent interactions [2]. Affinity capillary electropho- Analysis via ACE requires the measurement of the resis (ACE) is a new technique that has been shown electrophoretic mobility, m, of a species which is to be an efficient and accurate tool in studying directly related to the net charge and inversely biomolecular noncovalent interactions and determin- related to its hydrodynamic drag. In this form of analysis changes in the electrophoretic mobility m P * Corresponding author. of a protein (P) on complexation with a ligand (L) 0378-4347 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII: S0378-4347(98)00161-3