Using Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM) for Simultaneous Determination of Concentration and Equilibrium Constant

• Published in: Analytical chemistry (Washington) Vol. 87; no. 5; pp. 3099 - 3106
• Main Authors: Kanoatov, Mirzo, Galievsky, Victor A, Krylova, Svetlana M, Cherney, Leonid T, Jankowski, Hanna K, Krylov, Sergey N
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03.03.2015
• Subjects: Aptamers, Nucleotide - chemistry; Aptamers, Nucleotide - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binders; Binding sites; Capillarity; Capillary electrophoresis; Chemical equilibrium; Computer Simulation; Dilution; DNA-Binding Proteins - metabolism; Electrophoresis; Electrophoresis, Capillary - instrumentation; Electrophoresis, Capillary - methods; Equilibrium; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Kinetics; Mathematical analysis; Mathematical models; Molecules; MutS DNA Mismatch-Binding Protein - chemistry; MutS DNA Mismatch-Binding Protein - metabolism
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.5b00171

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a versatile tool for studying affinity binding. Here we describe a NECEEM-based approach for simultaneous determination of both the equilibrium constant, K d, and the unknown concentration of a binder that we call a target, T. In essence, NECEEM is used to measure the unbound equilibrium fraction, R, for the binder with a known concentration that we call a ligand, L. The first set of experiments is performed at varying concentrations of T, prepared by serial dilution of the stock solution, but at a constant concentration of L, which is as low as its reliable quantitation allows. The value of R is plotted as a function of the dilution coefficient, and dilution corresponding to R = 0.5 is determined. This dilution of T is used in the second set of experiments in which the concentration of T is fixed but the concentration of L is varied. The experimental dependence of R on the concentration of L is fitted with a function describing their theoretical dependence. Both K d and the concentration of T are used as fitting parameters, and their sought values are determined as the ones that generate the best fit. We have fully validated this approach in silico by using computer-simulated NECEEM electropherograms and then applied it to experimental determination of the unknown concentration of MutS protein and K d of its interactions with a DNA aptamer. The general approach described here is applicable not only to NECEEM but also to any other method that can determine a fraction of unbound molecules at equilibrium.