Journal of Chromatography A, 897 (2000) 339–347 www.elsevier.com / locate / chroma Multiple-step ligand injection affinity capillary electrophoresis for determining binding constants of ligands to receptors * Ying Zhang, Frank A. Gomez Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8202, USA Received 10 January 2000; received in revised form 31 July 2000; accepted 9 August 2000 Abstract This work demonstrates the use of multiple-step ligand injection affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique a sample plug of receptor and non-interacting standards is injected by pressure and electrophoresed in a buffer containing a given concentration of ligand. The sequence is repeated for all concentrations of ligand generating a single electropherogram containing a series of individual sample plugs superimposed on environments of buffer containing increasing concentrations of ligand. Analysis of the change in the relative migration time ratio, RMTR, relative to the non-interacting standards, as a function of the concentration of the ligand, yields a value for the binding constant. A competitive assay using the technique is also demonstrated using neutral ligands for CAB. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method are presented.  2000 Elsevier Science B.V. All rights reserved. Keywords: Multiple-step ligand injection; Binding constants; Receptor–ligand interactions; Carbonic anhydrase; Vancomycin; Proteins 1. Introduction and precursors that have been achieved using com- binatorial approaches to drug development. The Accurate and expeditious analysis of receptor– increased output of potential drugs compared to ligand interactions is critical to rational drug design traditional techniques of drug design has made and development. The importance of the former goes expeditious and facile analysis of potential drugs a without saying. Clinical trials are based on reliable must in any new analytical technique. and reproducible chemical analyses. The latter is Affinity capillary electrophoresis (ACE) is a ver- problematic given the dramatic advances in chemical satile technique for studying biomolecular non-co- syntheses and the great number of potential drugs valent interactions and has been shown to be an effective means of determining binding and dissocia- tion constants of formed complexes [1–38]. For *Corresponding author. Tel.: 11-323-3432-368; fax: 11-323- example, Kiessig et al. has used ACE to examine the 3436-490. E-mail address: fgomez2@calstatela.edu (F.A. Gomez). interaction of the enzyme cyclophilin with the 0021-9673 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0021-9673(00)00853-0