Flow-through partial-filling affinity capillary electrophoresis can estimate binding constants of neutral ligands to receptors via a competitive assay technique.
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.
Kaddis
John
J
Department of Chemistry and Biochemistry, California State University, Los Angeles, CA 90032-8202, USA. fgomez2@calstatela.edu
Mito
Erica
E
Heintz
Joseph
J
Plazas
Alfredo
A
Gomez
Frank A
FA
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*
D008024:Ligands; D013449:Sulfonamides; D024401:Carbonic Anhydrase I