Plug−Plug Kinetic Capillary Electrophoresis:  Method for Direct Determination of Rate Constants of Complex Formation and Dissociation

We present a method for direct determination of rate constants of complex formation, k on, and dissociation, k off. The method is termed plug−plug kinetic capillary electrophoresis (ppKCE). To explain the concept of the method, we consider the formation of a noncovalent complex C between molecules A and B; A is assumed to migrate slower in electrophoresis than B. In ppKCE, a short plug of A is injected into a capillary, followed by a short plug of B. When a high voltage is applied, the electrophoretic zone of B moves through that of A, allowing for the formation of C. When the zones of A and B are separated, C starts dissociating. The features of the resulting electropherogram are defined by both binding and dissociation. We developed a unique mathematical approach that allows finding k on and k off from a single electropherogram without nonlinear regression analysis. The approach uses algebraic functions with the only input parameters from electropherograms being areas and migration times of electrophoretic peaks. In this work, we explain theoretical bases of ppKCE and prove the principle of the method by finding k on and k off for a protein−ligand complex. The unique capability of the method to directly determine both k on and k off along with its simplicity make ppKCE highly attractive to a broad community of molecular scientists.