Kuromizu K, Shimokawa Y, Abe O, Izumiya N
Anal Biochem. 1985 Dec;151(2):534-9. doi: 10.1016/0003-2697(85)90216-7.
A new fluorogenic substrate, benzyloxycarbonyl-L-phenylalanine 4-methylcoumaryl-7-ester, has been developed for determination of the esterase activity of alpha-chymotrypsin and related enzymes. Synthesis of the substrate was achieved simply by the carbodiimide condensation of benzyloxycarbonyl-L-phenylalanine and 7-hydroxy-4-methylcoumarin in a 86% yield. The esterase activity was measured by increase of the fluorescence intensity at excitation and emission wavelengths of 325 and 465 nm, respectively. An initial rate of hydrolysis was linear over a 100-fold range of the enzyme concentration. As little as 2 ng of alpha-chymotrypsin could be detected in the standard assay. A typical enzyme assay, stability of the substrate, kinetic parameters, and specific activity have been reported.
已开发出一种新型荧光底物——苄氧羰基-L-苯丙氨酸4-甲基香豆素-7-酯，用于测定α-胰凝乳蛋白酶及相关酶的酯酶活性。该底物的合成只需通过苄氧羰基-L-苯丙氨酸与7-羟基-4-甲基香豆素的碳二亚胺缩合反应即可完成，产率为86%。酯酶活性通过分别在激发波长325 nm和发射波长465 nm处荧光强度的增加来测定。在酶浓度相差100倍的范围内，水解初始速率呈线性关系。在标准测定中，低至2 ng的α-胰凝乳蛋白酶都能被检测到。文中报道了典型的酶测定、底物稳定性、动力学参数和比活性。
