Mobility Moment Analysis of Molecular Interactions by Capillary Electrophoresis

Abstract

A new method for the quantitative evaluation of molecular interactions that are observed in electrophoresis is described. One component taking part in the interaction is labeled with a fluorescent dye and is subjected to capillary zone electrophoresis with fluorescence detection in the presence or absence of an unlabeled interacting component. Fluorescence signals are collected at constant time intervals, and the electropherograms are converted to represent the fluorescence signal against mobility. After baseline subtraction, the first statistical moment of fluorescence signals on the mobility axis is calculated. This moment represents the average mobility of a labeled component. The change in the mobility moment in the presence and absence of the unlabeled component is used to evaluate the degree of saturation of the binding site of a labeled molecule with an unlabeled molecule. Mixtures of fluorescence-labeled protein (Fab' fragment of antibody or concanavalin A) and its unlabeled interacting partner (alpha(1)-antitrypsin or succinylated ovalbumin, respectively) at various concentrations were injected into a bare-silica capillary, and zone electrophoresis was carried out. The change in the mobility moment of the fluorescence-labeled molecules was used to determine the dissociation constants of the complexes. The determined constants are comparable to those obtained by a well-established method, that is, an analysis based on the peak height of the complex. Since the mobility moment analysis is not affected by the total intensity of the signals, it should be advantageous in analyses in which multiple capillaries are used, in which the injection volume and the sensitivity of detection might be more difficult to control at constant values. The mobility moment analysis also has advantages for the analysis of heterogeneous samples, since the identification of peaks is not necessarily required.