Using Nonequilibrium Capillary Electrophoresis of Equilibrium
Mixtures (NECEEM) for Simultaneous Determination of Concentration
and Equilibrium Constant
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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, <i>K</i><sub>d</sub>, and
the unknown concentration of a binder that we call a target, T. In
essence, NECEEM is used to measure the unbound equilibrium fraction, <i>R</i>, 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 <i>R</i> is plotted as
a function of the dilution coefficient, and dilution corresponding
to <i>R</i> = 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 <i>R</i> on the concentration of L is fitted with a function
describing their theoretical dependence. Both <i>K</i><sub>d</sub> 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 <i>in silico</i> by using computer-simulated NECEEM electropherograms and then applied
it to experimental determination of the unknown concentration of MutS
protein and <i>K</i><sub>d</sub> 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
