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Affinity Capillary Electrophoresis 323
HSA (d) and warfarin (e). The concentration of free warfarin is proportional
to the height of the free ligand plateau (region (a) in Fig. 7A). In zone (b)
where both warfarin and HSA is present the equilibrium is preserved. Thus,
the amount of warfarin leaving this zone and entering zone (a) is equal to the
free warfarin concentration in the original pre-incubated sample. CE-FA is well
established for studying interactions between low-molecular weight ligands and
macromolecules where the mobility of the macromolecule is equal to that of
the complex (9). However, theory indicates that the free concentrations are
overestimated when these mobility requirements are not fulfilled, and this may
be the case for some low-affinity protein–protein interactions (92). For systems
characterized by slow binding kinetics where re-equilibration does not occur
during the separation CE-FA performs as pre-eq CZE, and the mobilities of
the complex relative to the free species is not an issue. Quantitation and data
analysis are usually conducted as described for pre-eq CZE except that plateau
heights are used rather than peak areas.
The first step in the characterization of an interaction system is to demonstrate
binding. This is most easily accomplished using the mobility shift ACE format
by conducting electrophoresis with and without the putative ligand added to
the electrophoresis buffer. The sample should contain the analyte and a noninteracting
marker molecule to correct for changes in the EEO flow. The
existence of interactions will be revealed as a change in analyte mobility.
The selection of the interacting species to be added to the electrophoresis
buffer should be based on properties such as size, charge, UV-absorption
properties and availability. Provided that the interaction kinetics is rapid and
a 1:1 stoichiometry can be expected, mobility shift ACE may be used for
further characterization of the system. If higher order stoichiometries are likely,
one of the pre-incubation approaches should be considered if quantitative data
are desired. In that case, the FA approach should be used initially as it is
conducive to the study of interactions characterized by both fast- and slowdissociation
kinetics. In case of slow kinetics, however, the use of pre-eq CZE
may be advantageous as compared with CE-FA because resolution is much
◭
Fig. 6. to the detector. Electrophoresis took place at 8.5 kV in 0.1 M phosphate,
pH 8.13, with additions of double-stranded 32mer biotin-DNA (dsDNA) at the concentrations
given in the figure. Detection at 200 nm. (B) Data from binding experiments
such as those presented in (A) plotted as outlined in Subheading 6.2.1. Data points
represent the mean and the standard deviation of triplicate experiments. The curve
represents a non-linear curve fit using a one-site binding hyperbola (GraphPadPrism).
R 2 = 0.99. The equation for the curve yields a K d for the Mab–dsDNA interaction of
0.10 M (Adapted with permission from (113)).