DETERMINATION OF BINDING CONSTANTS BETWEEN TEICOPLANIN AND D-ALA-D-ALA TERMINUS PEPTIDES BY AFFINITY CAPILLARY ELECTROPHORESIS

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Abstract
Binding constants between the glycopeptide antibiotic, Teicoplanin (Teic), and D-Ala-D-Ala terminus peptides were determined by affinity capillary electrophoresis (ACE) and by on-column ligand synthesis coupled to ACE. In the first technique, a plug of Teic and two non-interacting standards are injected and electrophoresed. Analysis of the change in the relative migration time ratio (RMTR) of Teic, relative to the non-interacting standards, as a function of the concentration of D-Ala-D-Ala peptide, yields a value for the binding constant. In the second technique, 9-fluorenylmethoxycarbonyl (Fmoc)-amino acid-D-Ala-D-Ala species are first synthesized on-column. 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 buffer, respectively. 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 Teic in the running buffer and the plug of Fmoc-amino acid-D-Ala-D-Ala peptide, and non-interacting markers. Subsequent analysis of the change in RMTR of the peptide relative to the non-interacting standards, as a function of the concentration of Teic, yields a value for the binding constant. Binding constants were determined for five ligands and Teic by ACE and by on-column ligand synthesis coupled to ACE. The findings described here demonstrate the advantage of using ACE and on-column ligand synthesis techniques coupled to ACE for estimating binding parameters between antibiotics and ligands.