View - ResearchGate

318 Heegaard et al.
f
M
s
5.0
0.01
s
3.6
A200 nm
s
s
1.1
0.0
0.00
9 10 11 12 13 14 15
Time (min.)
Fig. 5. Mobility shift ACE for studying binding activity of CE-separated
2 -microglobulin ( 2 m) conformers. Congo red dye was added to the electrophoresis
buffer in separations of conformationally heterogeneous 2 m. The sample was 0.17
mg/ml 2 m and 0.05 mg/ml peptide marker (M) in 33% (v/v) trifluoroethanol and
injections took place for 4 s. CE was performed at 17 kV. Under these conditions
the 2 m analyte separates into two conformer peaks (see f and s), corresponding to
a native (see f) and a more unfolded (s) conformation. Congo red was added to the
running buffer from a 0.144 mM stock solution in electrophoresis buffer to the final
concentrations (M) given in the figure. The s-peak is much more sensitive to the
presence of Congo red than the f-peak (Adapted with permission from (112)).
rates and activation enthalpy and energy are accessible by CE (77–79) at
the same time as the binding activities of the individual conformers may be
estimated (45). Binding assays such as these are executed exactly as any other
CE-based binding assays but exploit the high-resolution capabilities of the
technique (see Fig. 5).
6. Quantitative Protein-Binding Parameters
6.1. Theory and Strategy
The binding strength is an important parameter in the functional evaluation
of a protein and its interactions with established and putative ligands. While
ACE may be used for identification of ligands (c.f. 4 above), it may also be used
quantitatively, i.e. for the determination of binding stoichiometries (80) and
binding constants. In special cases, also determination of the association rate
and dissociation rate constants relating to the equilibrium constant is possible