Capillary electrophoretic reactor for estimation of spontaneous dissociation rate of Trypsin?Aprotinin complex
Capillary electrophoretic reactor for estimation of spontaneous dissociation rate of Trypsin?Aprotinin complex
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用于估计胰蛋白酶？抑肽酶复合物自发解离速率的毛细管电泳反应器
DOI：
10.1016/j.ab.2019.113406
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发表时间：
2019
影响因子：
2.9
通讯作者：
Iki Nobuhiko
中科院分区：
文献类型：
--
作者：
Sasaki Yumiko;Sato Yosuke;Takahashi Toru;Umetsu Mitsuo;Iki Nobuhiko
用毛细管电泳反应器分析了酶抑制剂复合物在均相溶液中不固定的解离动力学。以胰蛋白酶（Try）和抑酶蛋白（Apr）组成的复合物为模型。毛细管电泳通过稳定地从Try - Apr区去除游离的Try和Apr，为Try - Apr络合物的解离提供了反应场。通过分析其峰高与分离时间的关系，得到在pH为2.46时的解离率为2.73 × 10−4s−1（298 K）。kdhon [H+]与质子浓度（pH = 2.09-3.12）呈一级依赖关系；kdH=kd+k1[H+]，其中为自发解离速率，为5.65 × 10−5s−1，k1为二级速率常数，为5.07 × 10−2M−1s−1。由kdvalue可知，Try-Apr复合物在生理pH下的半衰期为3.4 h。质子辅助解离的存在可以解释为，在4月，Asp残基的- co -质子化，与赖氨酸的-NH3 +基团断开了盐桥。
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 ratekdHwas obtained as 2.73 × 10−4s−1(298 K) at pH 2.46. The dependence ofkdHon the proton concentration (pH = 2.09–3.12) revealed a first-order dependence ofkdHon [H+];kdH=kd+k1[H+], wherekdis the spontaneous dissociation rate and was 5.65 × 10−5s−1, andk1is the second-order rate constant and was 5.07 × 10−2M−1s−1. From thekdvalue, 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.