Principles and limitations of methods available for the determination of binding constants with affinity capillary electrophoresis

Abstract

At present there are five capillary zone electrophoresis (CZE) methods available for the measurement of binding (association) parameters viz. the frontal analysis method, the Hummel and Dreyer method, the affinity capillary electrophoresis, the vacancy peak and the vacancy affinity capillary electrophoresis methods. These methods exhibit their own advantages and limitations. In this paper the limitations of these five CZE methods will be explored with the aid of simulated concentration–position profiles of the interacting species. With the frontal analysis, the Hummel and Dreyer and the vacancy peak methods e.g., correct results for the binding parameters can only be obtained when the mobilities of e.g., a protein and the complex are equal. When the mobilities differ, the binding constants obtained with these methods will deviate systematically. The affinity capillary electrophoresis method on the other hand can only be performed when the mobility of the protein is not equal to the mobility of the complex. It is shown that this very necessary difference in the mobility between the free protein and the complex may lead to a deviation in the free ligand concentration and consequently in the binding constant.