PRINCIPLES AND LIMITATIONS OF METHODS AVAILABLE FOR THE DETERMINATIONOF BINDING CONSTANTS WITH AFFINITY CAPILLARY ELECTROPHORESIS

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 affin ity capillary electrophoresis, the vacancy peak and the vacancy affini ty capillary electrophoresis methods. These methods exhibit their own advantages and limitations. In this paper the limitations of these fiv e CZE methods will be explored with the aid of simulated concentration -position profiles of the interacting species. With the frontal analys is, the Hummel and Dreyer and the vacancy peak methods e.g., correct r esults for the binding parameters can only be obtained when the mobili ties of e.g., a protein and the complex are equal. When the mobilities differ, the binding constants obtained with these methods will deviat e 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 n ecessary difference in the mobility between the free protein and the c omplex may lead to a deviation in the free ligand concentration and co nsequently in the binding constant. (C) 1997 Elsevier Science B.V.