Whittaker R G, Manthey M K, Le Brocque D S, Hayes P J
CSIRO Division of Biomolecular Engineering, Sydney Laboratory, North Ryde, New South Wales, Australia.
Anal Biochem. 1994 Aug 1;220(2):238-43. doi: 10.1006/abio.1994.1333.
The action of serine (and cysteine) proteases on peptide esters proceeds, as a generalization, orders of magnitude faster than the corresponding enzymatic hydrolysis of peptide bonds or peptide amides. Esterolysis liberates an alcohol while generating a free carboxyl group on the peptide; the proton produced can be detected by the use of an appropriate indicator. The action of trypsin on benzyloxycarbonylalanylarginine methyl ester was used as a model for the development of a simple microtiter plate assay procedure that takes advantage of the speed of these reactions and the ease of detection afforded by the color change of the indicator. A family of ester substrates of the form benzyloxycarbonylalanyl-X-methyl ester, in which X is one of the 20 common amino acids, was synthesized to allow the determination of the primary specificity profiles of serine proteases. Using a 96-well microtiter plate the specificity profiles of four enzymes with all 20 substrates can be carried out in approximately 4 h per enzyme, including setting up and data processing. The primary substrate preferences of trypsin, chymotrypsin, thrombin, pancreatic elastase, alpha-lytic protease, subtilisin, and proteinase K were determined to demonstrate the method and were found to be in good general agreement with reported specificities established by more conventional means.
一般来说，丝氨酸（和半胱氨酸）蛋白酶对肽酯的作用比相应的肽键或肽酰胺的酶促水解快几个数量级。酯解反应释放出一种醇，同时在肽上生成一个游离羧基；产生的质子可用适当的指示剂检测。胰蛋白酶对苄氧羰基丙氨酰精氨酸甲酯的作用被用作开发一种简单的微量滴定板测定方法的模型，该方法利用了这些反应的速度以及指示剂颜色变化带来的易于检测的特性。合成了一系列苄氧羰基丙氨酰-X-甲酯形式的酯底物，其中X是20种常见氨基酸之一，用于确定丝氨酸蛋白酶的主要特异性谱。使用96孔微量滴定板，每种酶与所有20种底物的特异性谱测定大约每酶需要4小时，包括设置和数据处理。测定了胰蛋白酶、胰凝乳蛋白酶、凝血酶、胰腺弹性蛋白酶、α-裂解蛋白酶、枯草杆菌蛋白酶和蛋白酶K的主要底物偏好，以证明该方法，发现其与通过更传统方法确定的报道特异性总体上吻合良好。
