Yoshimoto Y, Shibukawa A, Sasagawa H, Nitta S, Nakagawa T
Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
J Pharm Biomed Anal. 1995 Apr;13(4-5):483-8. doi: 10.1016/0731-7085(95)01337-k.
Michaelis constant of enzymatic reaction was evaluated by affinity capillary electrophoresis using beta-galactosidase as a model enzyme and o- and p-isomers of nitrophenyl-beta-galactoside as substrates. The enzyme was immobilized on the inner surface of a fused-silica capillary by the covalent bonding through a bridging group, and the substrates were introduced into the capillary. The reaction products migrated electrophoretically toward the detection side (anodic side), while the unreacted substrates moved toward the injection side (cathodic side) on a slow electroosmotic flow generated by the weak negative charge of the immobilized enzyme. The initial velocity of the enzymatic reaction was estimated from the peak height of the product, and the Michaelis constant was calculated according to Lineweaver-Burk equation. The results (Km, 2.34 mM for o-isomer and 1.09 mM for p-isomer) were reproducible (RSD < 11.8%, n = 5). Although the estimated Michaelis constants were larger than the reported values measured in homogeneous solution, the ratio of the Michaelis constants of o-/p-isomers was in good agreement with the literature value. The present method required as low as a few microgram amount of enzyme and nanogram amount of substrate which is far smaller than those required in a conventional affinity HPLC.
以β-半乳糖苷酶为模型酶，邻硝基苯基-β-半乳糖苷和对硝基苯基-β-半乳糖苷的异构体为底物，通过亲和毛细管电泳评估酶促反应的米氏常数。酶通过桥连基团共价键固定在熔融石英毛细管的内表面，底物被引入毛细管。反应产物在固定化酶的弱负电荷产生的缓慢电渗流作用下，电泳迁移向检测端（阳极端），而未反应的底物则向进样端（阴极端）移动。根据产物的峰高估算酶促反应的初始速度，并根据Lineweaver-Burk方程计算米氏常数。结果（邻异构体的Km为2.34 mM，对异构体的Km为1.09 mM）具有可重复性（相对标准偏差<11.8%，n = 5）。尽管估算的米氏常数大于在均相溶液中测得的报道值，但邻/对异构体米氏常数的比值与文献值吻合良好。本方法所需酶量低至几微克，底物量低至纳克，远低于传统亲和高效液相色谱法的需求量。
