Rate and equilibrium constants of weak non-covalent molecular interactions are extremely difficult to measure. Here, we introduced a homogeneous approach called equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM) to determine k_(on), k_(off), and K_(d) of weak (K_(d) > 1 (mu)M) and fast kinetics (relaxation time, tau < 0.1 s) in quasi-equilibrium for multiple unlabeled ligands simultaneously in one microreactor. Conceptually, an equilibrium mixture (EM) of a ligand (L), target (T), and a complex (C) is prepared. The mixture is introduced into the beginning of a capillary reactor with aspect ratio >1000 filled with T. Afterward, differential mobility of L, T, and C along the reactor is induced by an electric field. The combination of differential mobility of reactants and their interactions leads to a change of the EM peak shape. This change is a function of rate constants, so the rate and equilibrium constants can be directly determined from the analysis of the EM peak shape (width and symmetry) and propagation pattern along the reactor. We proved experimentally the use of ECEEM for multiplex determination of kinetic parameters describing weak (3 mM > K_(d) > 80 (mu)M) and fast (0.25 s >= tau >= 0.9 ms) noncovalent interactions between four small molecule drugs (ibuprofen, S-flurbiprofen, salicylic acid and phenylbutazone) and alpha- and beta-cyclodextrins. The affinity of the drugs was significantly higher for beta-cyclodextrin than alpha-cyclodextrin and mostly determined by the rate constant of complex formation.
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