具有新型荧光标记物的胰凝乳蛋白酶荧光底物。

Fluorogenic substrates for chymotrypsin with new fluorescent markers.

作者信息

Kokotos G, Tzougraki C

出版信息

Int J Pept Protein Res. 1986 Aug;28(2):186-91. doi: 10.1111/j.1399-3011.1986.tb03246.x.

DOI:10.1111/j.1399-3011.1986.tb03246.x

Abstract

Several substrates for chymotrypsin Glt-Phe-NHRx have been synthesized, where RxNH2 are the compounds 3-aminocoumarin (2), 6-aminocoumarin (3), 3-acetamido-6-aminocoumarin (4), 3-acetamido-8-aminocoumarin (5), 7-amino-4-methyl-2-quinolinone (AMQ), (6). The fluorescence properties of the new substrates and those of the corresponding free amines were examined. The compound 7-glutarylphenylalaninamido-4-methyl-2-quinolinone (Glt-Phe-AMQ), (15), provided a new suitable substrate for chymotrypsin determination. The enzymatic release of the fluorophore AMQ was measured at lambda ex = 360 nm and lambda em = 435 nm. The Km of 15 was 0.5 mM and its kcat/Km ratio was 47 M-1 s-1. By using this substrate, the detection limit of chymotrypsin was 10 ng/ml.

摘要

已合成了几种用于胰凝乳蛋白酶Glt-Phe-NHRx的底物，其中RxNH2为化合物3-氨基香豆素（2）、6-氨基香豆素（3）、3-乙酰氨基-6-氨基香豆素（4）、3-乙酰氨基-8-氨基香豆素（5）、7-氨基-4-甲基-2-喹啉酮（AMQ）（6）。研究了新底物及相应游离胺的荧光性质。化合物7-戊二酰基苯丙氨酰胺基-4-甲基-2-喹啉酮（Glt-Phe-AMQ）（15）为胰凝乳蛋白酶测定提供了一种新的合适底物。在激发波长λex = 360 nm和发射波长λem = 435 nm处测量荧光团AMQ的酶促释放。15的米氏常数为0.5 mM，其催化常数与米氏常数之比为47 M-1 s-1。使用该底物时，胰凝乳蛋白酶的检测限为10 ng/ml。