Affinity Capillary Electrophoresis to Examine Receptor–Ligand Interactions

Abstract

Afffinity capillary electrophoresis (ACE) is a new analytical technique that has been shown to be an efficient and accurate tool in studying biomolecular noncovalent interactions and determining binding and dissociation constants of formed complexes. ACE uses as its basis the change in migration time of a receptor upon binding to a ligand found in the electrophoresis buffer. Subsequent Scatchard analysis using noninteracting markers realizes a binding constant. Herein, ACE and three modifications in the technique, partial-filling ACE (PFACE), flow through PFACE (FTPFACE), and multiple-step ligand injection ACE (MSLIACE) are used to probe the binding of ristocetin A (Rist A) and vancomycin (Van) from Streptomyces orientalis to D-Ala-D-Ala terminus peptides and carbonic anhydrase B (CAB, E.C.4.2.1.1) to arylsulfonamides.

Use of CE for the determination of binding constants

“…PFACE and its modification were studied extensively by Gomez and co‐workers 20–23, 25, 52–64. These techniques are illustrated clearly by this group's work and in a previous review 3.…”

“…Multiple injection methods coupled with ACE, PFACE and FTPFACE were described previously 22, 24, 57, 62–64, 76, 77. In multiple‐injection ACE 76, a sample plug containing a noninteracting standard is injected, followed, by multiple plugs of sample containing the substrate and then a final injection of sample containing a second standard.…”

“…Then the voltage is applied and the samples undergo electrophoresis with buffers containing ligands with increasing concentrations. Multiple injection formats coupled with other ACE variations are operated in a similar manner 22, 24, 57, 62–64, 76, 77. Using this approach, analysis times are greatly decreased 22, 24, 57, 62–64, 76, 77 and the effects of fluctuations in the EOF and voltage are minimized.…”

Application of partial‐filling capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in a therapeutic antibody

“…The partial‐filling technique has been used to ascertain the affinity characteristics between receptors and ligands 24–33. In this technique, the capillary is partly filled with the affinity ligand dissolved in running buffer before injection of the sample.…”

Flow‐through partial‐filling affinity capillary electrophoresis using a crossreactive antibody for enantiomeric separations

“…[13,14]. These methodologies are capable of revealing the information on the binding constants and number of binding sites for a solute-protein system, as well as the thermodynamic parameters, rate constants, interaction forces, and binding site structure for the protein and solute [15].…”