Analytical Chemistry · 1994 · 172 citations · 15 references
PharmacyPharmaceutical AnalysisAnalytical ChemistryElectroanalytical MethodsMolecular RecognitionBinding AffinityLigand BindingProtein ElectrophoresisProtein-ligand BindingProtein-ligand InteractionsProtein-ligand RecognitionBinding Affinity MeasurementsBinding ConstantsBiomolecular Binding AffinityCapillary ElectrophoresisElectroosmotic FlowLigand Binding AffinityLigand-binding AssaysProtein-ligand Binding Assays
This paper describes the estimation of binding constants (Kb) between carbonic anhydrase B (CAB, EC 4.2.1.1, from bovine erythrocytes) and charged benzenesulfonamides by affinity capillary electrophoresis (ACE) under conditions in which the migration time is affected by changes in electroosmotic flow and by nonspecific interactions accompanying changes in the concentration of ligand. Comparisons of values of migration times of the protein of interest, and of "noninteracting" marker proteins, with those of a neutral internal standard provide the basis for corrections for variable electroosmotic flow; these corrections make possible the estimation of Kb and its uncertainty even in the presence of substantial variations in electroosmotic flow.
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