在偶联对氨基苯甲脒的旭pak GS凝胶上对胰蛋白酶进行高效亲和色谱分析。

High-performance affinity chromatography of trypsins on Asahipak GS-gel coupled with p-aminobenzamidine.

作者信息

Ito N, Noguchi K, Shimura K, Kasai K

出版信息

J Chromatogr. 1985 Sep 27;333(1):107-14. doi: 10.1016/s0021-9673(01)87328-3.

Abstract

An adsorbent for high-performance affinity chromatography of trypsins was prepared, based on a micro-particulate polyvinyl alcohol gel for high-performance liquid chromatography, Asahipak GS-gel. After the hydroxyl groups had been activated with 1,1'-carbonyldiimidazole, 6-aminohexanoic acid was coupled as a spacer, then p-aminobenzamidine, a specific ligand for trypsin-family enzymes, was immobilized on the spacer. Fluorometric detection of eluted protein and on-line assay of enzyme activity using a fluorogenic substrate, peptidylmethylcoumarylamide, made it possible to attain very high sensitivity. Microgram amounts of bovine trypsin and Streptomyces griseus trypsin could easily be analyzed in a short time (less than 1 h).

摘要

基于用于高效液相色谱的微颗粒聚乙烯醇凝胶（Asahipak GS - 凝胶），制备了一种用于胰蛋白酶高效亲和色谱的吸附剂。在用1,1'-羰基二咪唑活化羟基后，偶联6 - 氨基己酸作为间隔臂，然后将对氨基苯甲脒（一种胰蛋白酶家族酶的特异性配体）固定在间隔臂上。使用荧光底物肽基甲基香豆素酰胺对洗脱蛋白进行荧光检测和酶活性在线测定，使得能够获得非常高的灵敏度。在短时间内（不到1小时）可以轻松分析微克量的牛胰蛋白酶和灰色链霉菌胰蛋白酶。