title: Using nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) for simultaneous determination of concentration and equilibrium constant.
abstract: 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.
authors: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University , Toronto, Ontario M3J 1P3, Canada.
authors: Mirzo
authors: M
authors: Kanoatov
authors: Victor A
authors: VA
authors: Galievsky
authors: Svetlana M
authors: SM
authors: Krylova
authors: Leonid T
authors: LT
authors: Cherney
authors: Hanna K
authors: HK
authors: Jankowski
authors: Sergey N
authors: SN
authors: Krylov
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D003198:Computer Simulation; D004268:DNA-Binding Proteins / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry / Q000378:metabolism*
chemical_list: C119202:AT-rich DNA-binding protein 25; D052157:Aptamers, Nucleotide; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli