Sasaki Yumiko, Sato Yosuke, Takahashi Toru, Umetsu Mitsuo, Iki Nobuhiko
Graduate School of Environmental Studies, Tohoku University, 6-6-07 Aramaki-Aoba, Aoba, Sendai, 980-8579, Japan.
Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui, 910-8507, Japan.
Anal Biochem. 2019 Nov 15;585:113406. doi: 10.1016/j.ab.2019.113406. Epub 2019 Aug 22.
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.
使用毛细管电泳反应器分析均匀溶液中未固定化的酶 - 抑制剂复合物的解离动力学。以胰蛋白酶（Try）和抑肽酶（Apr）组成的复合物作为模型。毛细管电泳通过从Try - Apr区稳定去除游离的Try和Apr，为Try - Apr复合物提供了一个解离的反应场。通过分析Try - Apr峰高对分离时间的依赖性，在pH 2.46时，解离速率k为2.73×10⁻⁴ s⁻¹（298 K）。k对质子浓度（pH = 2.09 - 3.12）的依赖性表明k对[H⁺]呈一级依赖性；k = k₀ + k₁[H⁺]，其中k₀是自发解离速率，为5.65×10⁻⁵ s⁻¹，k₁是二级速率常数，为5.07×10⁻³ M⁻¹ s⁻¹。根据k值，确定Try - Apr复合物在生理pH下的半衰期为3.4小时。质子辅助解离的存在可以通过Try中Asp残基的 - COO⁻质子化来解释，这打破了与Apr中Lys的 - NH基团的盐桥。
