Use of voltage gradient partial-filling affinity capillary electrophoresis to estimate binding constants of ligands to receptors.
Voltage gradient partial-filling affinity capillary electrophoresis (VGPFACE) is used to determine binding constants between carbonic anhydrase B (CAB, E.C.4.2.1.1) and arylsulfonamides, and vancomycin (Van) from Streptomyces orientalis and teicoplanin (Teic) from Actinoplanes teicomyceticus and D-Ala-D-Ala terminus peptides. Two variations of VGPFACE are described herein. In the first technique, the capillary is partially filled with ligand at increasing concentrations followed by a sample containing receptor and two noninteracting standards and electrophoresed in buffer using a voltage gradient that increases from 0 to 25 kV over the duration of the experiment. Upon continued electrophoresis, zones of solution overlap, and equilibrium is established between the ligand and receptor, causing a shift in the migration time of the receptor with respect to the noninteracting standards. This change in migration time is utilized for estimating a binding constant (K(b)). In the second technique, voltage gradient partial-filling multiple-injection ACE (VGPFMIACE), a multiple-injection sequence is used whereby the capillary is partially filled with ligand at increasing concentrations, a noninteracting standard, three or four separate plugs of receptor each separated by small plugs of buffer, and a plug containing a second noninteracting standard; this is then electrophoresed in buffer with a similar voltage gradient. Upon continued electrophoresis, a similar equilibrium is established and a value for K(b) is obtained for the interaction. The VGPFACE technique expands the functionality and potential of ACE as an analytical tool to examine various receptor-ligand interactions.
Ramirez
Alejandra
A
Department of Chemistry and Biochemistry, California State University, Los Angeles, CA 90032-8202, USA.
Gomez
Frank A
FA
D001667:Binding, Competitive; D024401:Carbonic Anhydrase I / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D006020:Glycopeptides / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D009038:Motion; D009842:Oligopeptides / Q000737:chemistry; D011485:Protein Binding; D015203:Reproducibility of Results; D055672:Static Electricity
D006020:Glycopeptides; D008024:Ligands; D009842:Oligopeptides; D024401:Carbonic Anhydrase I