A CONTINUOUS FLUORESCENT ASSAY FOR MEASURING PROTEASE ACTIVITY USING NATURAL PROTEIN SUBSTRATE

被引：33

作者：

FARMER, WH ^{[1
]}

YUAN, ZG ^{[1
]}

机构：

[1] SYNTEX RES,2100 SYNTEX COURT,MISSISSAUGA L5N 3X4,ONTARIO,CANADA

来源：

ANALYTICAL BIOCHEMISTRY | 1991年 / 197卷 / 02期

关键词：

D O I：

10.1016/0003-2697(91)90403-G

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A continuous caseinolytic activity assay has been developed and characterized with trypsin, a serine protease, and transin, a metalloproteinase. β-casein labeled with both N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and fluorescein isothiocyanate (FITC) is used as the substrate in this assay. The effect of proteolysis of the substrate is a reduction of the intermolecular energy transfer from DACM to FITC. The caseinolytic activity is then monitored by the fluorescence increase. The activities of both proteases obey Michaelis-Menten kinetics with Km = 1.6 ± 0.2 μm for trypsin and Km = 13.2 ± 1.9 μm for transin. Protease concentrations as low as 10 ng/mL can be utilized. The pH dependence of the caseinolytic activity has been determined for both enzymes. © 1991.

引用

页码：347 / 352

页数：6

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