On-column ligand synthesis coupled to partial-filling affinity capillary electrophoresis to estimate binding constants of ligands to a receptor

Abstract

This paper describes a two-step procedure whereby on-column ligand synthesis and partial-filling affinity capillary electrophoresis (PFACE) are sequentially coupled to each other to determine the binding constants of 9-fluorenylmethoxy carbonyl (Fmoc)-amino acid– d-Ala– d-Ala species to vancomycin (Van) from Streptomyces orientalis. In this technique four separate plugs of sample are injected onto the capillary column and electrophoresed. The initial sample plug contains a d-Ala– d-Ala terminus peptide and two non-interacting standards. Plugs two and three contain solutions of Fmoc-amino acid– N-hydroxysuccinimide (NHS) ester and running buffer, respectively. The fourth sample plug contains an increasing concentration of Van partially-filled onto the capillary column. Upon electrophoresis the initial d-Ala– d-Ala peptide reacts with the Fmoc-amino acid NHS ester yielding the Fmoc-amino acid d-Ala– d-Ala peptide. Continued electrophoresis results in the overlap of the plugs of Van and Fmoc-amino acid– d-Ala– d-Ala peptide and non-interacting markers. Analysis of the change in the relative migration time ratio of the Fmoc-amino acid– d-Ala– d-Ala peptide relative to the non-interacting standards, as a function of the concentration of Van, yields a value for the binding constant. These values agree well with those estimated using other binding and ACE techniques.