Farmer W H, Yuan Z Y
Syntex Research, Mississauga, Ontario, Canada.
Anal Biochem. 1991 Sep 2;197(2):347-52. doi: 10.1016/0003-2697(91)90403-g.
A continuous caseinolytic activity assay has been developed and characterized with trypsin, a serine protease, and transin, a metalloproteinase. Beta-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 microM for trypsin and Km = 13.2 +/- 1.9 microM 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.
已开发出一种连续酪蛋白溶解活性测定法，并对丝氨酸蛋白酶胰蛋白酶和金属蛋白酶转移素进行了特性鉴定。在该测定中，用N-(7-二甲基氨基-4-甲基香豆素基)-马来酰亚胺（DACM）和异硫氰酸荧光素（FITC）标记的β-酪蛋白用作底物。底物蛋白水解的作用是减少从DACM到FITC的分子间能量转移。然后通过荧光增加来监测酪蛋白溶解活性。两种蛋白酶的活性均符合米氏动力学，胰蛋白酶的Km = 1.6±0.2 microM，转移素的Km = 13.2±1.9 microM。蛋白酶浓度低至10 ng/mL即可使用。已测定了两种酶的酪蛋白溶解活性对pH的依赖性。
