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In the biomedical research of molecular bases of both normal and pathological biological processes, it is currently necessary not only to detect, identify, and quantify individual compounds, but also to study their interactions with endo- and exogenous compounds. Obviously, for these purposes it is crucial to develop new advanced high‑performance analytical methods providing high sensitivity, high selectivity, and high throughput. These challenging requirements are well met by capillary electromigration (CE) methods. They have developed in the last three and half decades into high‑performance separation techniques suitable for the analysis of a wide spectrum of both low- and high‑molecular mass bioactive compounds.
In the biomedical research of molecular bases of both normal and pathological biological processes, it is currently necessary not only to detect, identify, and quantify individual compounds, but also to study their interactions with endo- and exogenous compounds. Obviously, for these purposes it is crucial to develop new advanced highâperformance analytical methods providing high sensitivity, high selectivity, and high throughput. These challenging requirements are well met by capillary electromigration (CE) methods. They have developed in the last three and half decades into highâperformance separation techniques suitable for the analysis of a wide spectrum of both low- and highâmolecular mass bioactive compounds (1).
Among the variety of modern CE techniques, affinity capillary electrophoresis (ACE) represents a special separation mode (2,3). It is based on the monitoring of migration times and on the measurement of effective electrophoretic mobilities of the interacting species. It is used not only for selective analysis of particular types of compounds (bio)specifically interacting with the affinity ligands present in the separation medium, but also for the identification and quantification of these (bio)specific interactions. Various ACE modes are available that can be used for the investigation of thermodynamically weak or strong bindings and kinetically fast or slow interactions both in homogeneous liquid phase and on the heterogeneous solid–liquid interface. ACE allows the determination of both binding (stability, association) constants and stoichiometries of the biomolecular complexes (3,4).
ACE possesses the advantages of CE methods, that is, high separation efficiency, ultra-small sample volume with high mass sensitivity, short analysis time, low consumption of reagents and solvents, and the capability to separate a wide range of biologically active compounds, such as amino acids, peptides, proteins, nucleobases, nucleosides, nucleotides, nucleic acids, saccharides, steroids, flavonoids, and other (bio)molecules.
For investigation of biomolecular interactions, the following ACE methods are available: The first mode is nonequilibrium ACE of equilibrated mixtures of analyte and ligand at different analyte–ligand ratios in the background electrolyte (BGE) free of ligand and analyte can be used for strong or slowly dissociating complexes. From the peak areas of the analyte, ligand, and analyte–ligand complex, their equilibrium concentrations and the stability constant of the complex can be determined.
The second mode is the dynamic equilibrium ACE of an analyte in the BGE containing a free ligand at several distinct concentrations. Electrophoretic migration of the analyte is retarded as a result of the formation of the analyte–ligand complex. The advantage of this mode, the “so-called” mobility shift assay, is that the analyte need not be perfectly pure (the admixtures can be separated during the ACE experiment) and concentration of the analyte need not be exactly known since estimation of the stability constant is based on measurement of analyte effective mobility.
Partial filling ACE (PF-ACE) is a special ACE mode in which only a part of the capillary is filled with ligand solution in the BGE. This technique has several advantages over classical ACE. Instead of adding the ligand at several concentrations to the BGE in the whole capillary and in one or both electrode vessels, the use of a short ligand zone means that the consumption of the valuable ligand is very low. The binding constants can be calculated from a slope of linear dependence of analyte migration time changes on the substance amount of ligand in the ligand zone in the capillary.
In frontal analysis ACE (FA-ACE), a long zone of equilibrated mixture of analyte–ligand complex at different analyte–ligand ratios is introduced into the capillary, and in the applied electric field the complex dissociates and the zones of free analyte, analyte–ligand complex, and free ligand are formed. From the heights of the analyte or ligand zones on the electropherograms, their equilibrium concentrations, binding constants, and stoichiometry of the complexes can be determined. These parameters can also be determined by a special mode of FA-ACE, continuous FA-ACE, in which the analyte–ligand complex at different analyte–ligand ratios is electrokinetically introduced in the capillary.
In ACE with immobilized ligand, the ligand is covalently or by physical sorption attached to the inner capillary wall and the analyte is electrophoretically or electroosmotically transported through this affinity open-tubular column. The strength of the analyte–ligand interaction is evaluated from the reduced electrophoretic mobility of the analyte as a result of its interaction with the ligand.
Václav KašiÄka, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic.