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Flow‐through partial‐filling affinity capillary electrophoresis can estimate binding constants of neutral ligands to receptors via a competitive assay technique
Author(s) -
Kaddis John,
Mito Erica,
Heintz Joseph,
Plazas Alfredo,
Gomez Frank A.
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200390129
Subject(s) - capillary electrophoresis , ligand (biochemistry) , affinity electrophoresis , chemistry , electrophoresis , carbonic anhydrase , analytical chemistry (journal) , chromatography , binding constant , spark plug , receptor , binding site , thermodynamics , enzyme , affinity chromatography , biochemistry , physics
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.