Multiple-injection affinity capillary electrophoresis to estimate binding constants of receptors to ligands

Abstract

Multiple-injection affinity capillary electrophoresis (MIACE) is used to determine binding constants (Kb) between receptors and ligands using as model systems vancomycin and teicoplanin from Streptomyces orientalis and Actinoplanes teichomyceticus, respectively, and their binding to D-Ala-D-Ala peptides and carbonic anhydrase B (CAB. EC 4.2.1.1) and the binding of the latter to arylsulfonamides. A sample plug containing a non-interacting standard is first injected followed by multiple plugs of sample containing the receptor and then a final injection of sample containing a second standard. Between each injection of sample, a small plug of buffer is injected which contains an increasing concentration of ligand to effect separation between the multiple injections of sample. Electrophoresis is then carried out in an increasing concentration of ligand in the running buffer. Continued electrophoresis results in a shift in the migration time of the receptor in the sample plugs upon binding to their respective ligand. Analysis of the change in the relative migration time ratio (RMTR) or electrophoretic mobility (mu) of the resultant receptor-ligand complex relative to the non-interacting standards, as a function of the concentration of ligand yields a value for Kb. The MIACE technique is a modification in the ACE method that allows for the estimation of binding affinities between biological interactions on a timescale faster than that found for standard ACE. In addition sample volume requirements for the technique are reduced compared to traditional ACE assays. These findings demonstrate the advantage of using MIACE to estimate binding parameters between receptors and ligands.

Chromatographic and Electrophoretic Methods for the Determination of Binding Constants for Dye-Protein Complexes

“…This work is an extension of the earlier methods of multiple-injection ACE [75] and of partial-filling ACE. [76] As illustrated in Fig.…”

Recent applications of capillary electrophoresis–mass spectrometry (CE–MS): CE performing functions beyond separation

“…By comparing the relative migration time ratio of the receptor to the noninteracting internal standard analytes, as a function of the concentration of ligand, the binding constant was deduced. In another example, the binding constants between receptors and ligands was determined using multiple-injection affinity CE [176]. Complexes attached with different number of ligands were clearly separated during electrophoresis, and the binding constant of these complexes were determined.…”

Recent developments in CE and CEC of peptides

“…In the technique of multiple-injection CAE (MICAE) [122,123], separate plugs of sample containing noninteracting standards, peptide I, buffer, and peptide II, are injected into the capillary and electrophoresed. Peptides I and II migrate through the capillary at similar electrophoretic velocities and remain as distinct zones due to the buffer plug between peptides.…”