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Affinity Capillary Electrophoresis 319
(81–83). As indicated in Table 1, a number of approaches exist. The most
widely used modes for the determination of binding constants are mobility shift
ACE, pre-eq CZE and CE-FA. The principles of these methods will be outlined
here. Partial-filling ACE and FACCE may be considered as specialized variants
of mobility shift ACE and CE-FA, respectively. The workflow for conducting
affinity experiments using these two methods has previously been described
in the Methods in Molecular Biology series (84,85). After a brief description
of mobility shift ACE, pre-eq CZE and CE-FA, a few general comments
on how to approach interaction studies using ACE are provided. Practical
examples on how to conduct mobility shift ACE and CE-FA are presented in
Subheading 6.2.
The fundamental parameter of all CE experiments is the electrophoretic
mobility, . The value of is determined by
=
q eff
6r
(1)
where is the viscosity of the background electrolyte; q eff and r are the
effective charge and radius of the analyte, respectively (86). After introduction
of a molecule into an electrical field, a steady state is attained in which the
ionic attraction is balanced by the frictional drag acting on the molecule. The
charge-to-size ratio of Eq. 1 represents this balance between forces, which
makes a charged molecule (analyte or ligand) migrate with constant velocity
in an electrical field of constant magnitude. The interaction of an analyte with
another molecule present in the electrophoresis medium is likely to alter the
charge-to-size ratio of the analyte. This will make the analyte migrate with
a different velocity in the presence of interacting species. In other words,
the analyte–ligand complex formed most often has an electrophoretic mobility
different from that of the free analyte. This complexation-induced change in
mobility is the basis of ACE. The high efficiency of CE makes it possible to
detect even subtle differences in and consequently makes CE a strong tool
for interaction analysis.
Mobility shift ACE is especially well suited for low-to-medium affinity
interactions. A prerequisite for mobility shift ACE is that the dynamics of the
binding equilibrium is fast, i.e. that the association and dissociation processes
are rapid. If a 1:1 binding stoichiometry for the interaction between the analyte
A and the ligand L is assumed, the corresponding binding equilibrium and
stability constant for the interaction will be given by Eqs. 2 and 3, respectively.
A + L = AL (2)
K = AL
AL
(3)