On-column ligand synthesis coupled to partial-filling affinity capillary electrophoresis to estimate binding constants of ligands to a receptor.
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.
Zhang
Y
Y
Department of Chemistry and Biochemistry, California State University, Los Angeles, 90032-8202, USA.
Kodama
C
C
Zurita
C
C
Gomez
F A
FA
D019075:Electrophoresis, Capillary / Q000379:methods*; D008024:Ligands; D011485:Protein Binding; D011955:Receptors, Drug / Q000378:metabolism*; D014640:Vancomycin / Q000378:metabolism*
D008024:Ligands; D011955:Receptors, Drug; D014640:Vancomycin