Capillary electrophoretic reactor for estimation of spontaneous dissociation rate of Trypsin-Aprotinin complex

A capillary electrophoretic reactor was used to analyze the dissociation kinetics of an enzyme-inhibitor complex in a homogeneous solution without immobilization. The complex consisting of trypsin (Try) and aprotinin (Apr) was used as the model. Capillary electrophoresis provided a reaction field for Try-Apr complex to dissociate through the steady removal of free Try and Apr from the Try-Apr zone. By analyzing the dependence of peak height of Try-Apr on separation time, the dissociation rate k_d^H was obtained as 2.73 × 10^-4 s^-1 (298 K) at pH 2.46. The dependence of k_d^H on the proton concentration (pH = 2.09-3.12) revealed a first-order dependence of k_d^H on [H⁺]; k_d^H = k_d + k₁[H⁺], where k_d is the spontaneous dissociation rate and was 5.65 × 10^-5 s^-1, and k₁ is the second-order rate constant and was 5.07 × 10^-2 M^-1 s^-1. From the k_d value, the half-life of the Try-Apr complex at physiological pH was determined as 3.4 h. The presence of the proton-assisted dissociation can be explained by the protonation of -COO^- of the Asp residue in Try, which breaks the salt bridge with the -NH₃⁺ group of Lys in Apr.