MASKE: macroscopic approach to studying kinetics at equilibrium.

The kinetics of biomolecular interactions at equilibrium is typically studied by "microscopic" methods that monitor concentration fluctuations of molecules in an "observation" volume in which the number of molecules is so small that the equilibrium is statistically impossible. Here, we introduce a "macroscopic" method for studying kinetics of biomolecular interactions at equilibrium which does not rely on monitoring the fluctuation of concentrations. We termed this method MASKE: a "macroscopic approach to studying kinetics at equilibrium". Conceptually, in MASKE, two equilibrium reaction mixtures, "unlabeled" and "labeled", are both prepared with two reactants and their complex; in the labeled mixture, one reactant is labeled for detection. A "macroscopic" amount of the labeled mixture is introduced into a long and narrow reactor filled with the unlabeled mixture, and a differential mobility of the reactant versus the complex is then induced by an external action along the reactor. The kinetics of complex formation and dissociation is then studied from the label-propagation pattern. In this work, we developed the theory of MASKE and experimentally proved it with a capillary as a reactor, a fluorophore as a label, and an electric field as a differential mobility inducer. Two pairs of molecules interacting with significantly different rate constants were used in this proof-of-principle work.