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.