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328 Heegaard et al.
2. Introduce pre-incubated samples into the capillary by applying pressure (0.5 psi)
for 99 s (injection volume ∼121 nL) (see Note 10).
3. Perform electrophoretic separation of drug standard solutions and HAS-containing
samples using a voltage of +15 kV in the normal polarity mode and a detection
wavelength of 200 nm (see Note 11).
4. Construct a calibration curve by plotting plateau peak heights as a function of drug
concentration of the standard solutions and determine the free drug concentration
from the plateau heights by aid of the calibration curve.
5. Determine binding parameters by suitable data analysis (see Note 12).
Electropherograms obtained by CE-FA using experimental setups very
similar to the one outlined above for warfarin-HSA binding are depicted in
Fig. 7. The rectangular plateau peaks of the standard solution and the consecutive
plateaus of the ligand-protein solution are characteristic of CE-FA. HSA
and warfarin are both negatively charged with the apparent mobility of warfarin
being slightly smaller than that of HAS, which leads to incomplete separation
and the free warfarin plateau passing the detector after HSA. A positively
charged ligand would be detected as a plateau before HAS, and complete
separation would be obtained because of the large difference in mobility. Note
that Fig. 7A was prepared for illustration of the CE-FA principle. With the long
analysis time and very broad plateaus, the method would be of little practical
interest. Figure 7B represents a more normal CE-FA experiment.
7. Conclusions
To the extent that proteins are recovered during conditions that are relevant
for their native or in vivo function, there is a great deal to be learnt about their
function from ACE experiments. Close attention to peak shapes and analyte
recovery, reproducible temperature conditions, inclusion of non-interacting
markers and proper coverage of binding isotherms will make useful characterization
of protein interactions possible also in cases where only few other
methods succeed.
8. Notes
1. The term ACE is normally used to cover both the mobility shift and the pre-eq
formats. A number of alternative names for mobility shift ACE methodology have
appeared; ACE, classical ACE (17), dynamic complexation CE (DCCE) (94) and
mobility change analysis (95). Pre-eq CZE has been termed CZE (96), equilibriummixture
analysis (95), CE mobility shift assay (CEMSA), pre-incubation ACE
(PI-CE) (10) and a variant hereof non-equilibrium CE of equilibrium mixtures
(NECEEM) (82,83). The recommended acronym for CE in the FA mode is CE-FA
as the abbreviation FACE (97) has been used for fluorescence anisotropy CE.