title: Flow-through partial-filling affinity capillary electrophoresis can estimate binding constants of neutral ligands to receptors via a competitive assay technique.
abstract: This work evaluates the use of a competitive binding assay using flow-through partial-filling affinity capillary electrophoresis (FTPFACE) to estimate binding constants of neutral ligands to a receptor. We demonstrate this technique using, as a model system, carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides. In this technique, the capillary is first partially filled with a negatively charged ligand, a sample containing CAB and two noninteracting standards, and a neutral ligand, then electrophoresed. Upon application of a voltage the sample plug migrates into the plug of negatively charged ligand (L(-)) resulting in the formation of a CAB-L(-) complex. Continued electrophoresis results in mixing between the neutral ligand (L(0)) and the CAB-L(-) complex. L(0) successfully competes out L(-) to form the new CAB-L(0) complex. Analysis of the change in the relative migration time ratio (RMTR) of CAB relative to the noninteracting standards, as a function of neutral ligand concentration, yields a value for the binding constant. These values are in agreement with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented.
authors.affiliation: Department of Chemistry and Biochemistry, California State University, Los Angeles, CA 90032-8202, USA. fgomez2@calstatela.edu
authors.forename: John
authors.initials: J
authors.lastname: Kaddis
authors.forename: Erica
authors.initials: E
authors.lastname: Mito
authors.forename: Joseph
authors.initials: J
authors.lastname: Heintz
authors.forename: Alfredo
authors.initials: A
authors.lastname: Plazas
authors.forename: Frank A
authors.initials: FA
authors.lastname: Gomez
mesh_terms: D001667:Binding, Competitive; D024401:Carbonic Anhydrase I / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D055672:Static Electricity; D013449:Sulfonamides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008024:Ligands; D013449:Sulfonamides; D024401:Carbonic Anhydrase I