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 was obtained as 2.73 × 10 s (298 K) at pH 2.46. The dependence of k on the proton concentration (pH = 2.09-3.12) revealed a first-order dependence of k on [H]; k = k + k[H], where k is the spontaneous dissociation rate and was 5.65 × 10 s, and k is the second-order rate constant and was 5.07 × 10 M s. From the k 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 -NH group of Lys in Apr.