ASSAYING PROTEINASES WITH AZOCOLL
ASSAYING PROTEINASES WITH AZOCOLL
复制标题
DOI：
10.1016/0003-2697(84)90242-2
复制
发表时间：
1984-01-01
影响因子：
2.9
通讯作者：
HAGEMAN, JH
中科院分区：
文献类型：
--
作者：
CHAVIRA, R;BURNETT, TJ;HAGEMAN, JH
Azocoll是一种不溶性的研磨胶原蛋白，其上附着一种亮红色偶氮染料，广泛用于蛋白水解酶的测定。早期的研究表明，偶氮可的水解作为蛋白酶浓度的函数线性地进行，但作为时间的函数以指数增加的方式进行。没有对后一种行为作出解释。枯草芽孢杆菌的粗提物和枯草杆菌蛋白酶BPN的商业制剂的测定给出了作为蛋白酶浓度的函数的偶氮可的水解速率的线性;两者都给出了作为时间的函数的偶氮可的水解速率的增加。在试图改进和标准化使用azocoll的蛋白水解测定中，发现溶解的azocoll在pH7.8的最大吸收为516 nm，并且在酸性pH下没有显著改变;通过使用用缓冲液剧烈预洗涤的azocoll可以获得作为时间函数的完全线性的测定;通过预洗涤除去的可溶性滤液可以再生先前观察到的非线性时间过程;并且通过改变测定管的搅拌速率，可以将唑可的水解速率改变3倍。为了获得可重现的线性测定，必须剧烈预洗商业azocoll和Nitrile反应管。
Azocoll, an insoluble, ground collagen to which a bright-red azodye is attached is widely used for the assay of proteolytic enzymes. Earlier studies showed that hydrolysis of azocoll progressed linearly as a function of proteinase concentration but in an exponentially increasing manner as a function of time. No explanation for the latter behavior was offered. Assays of crude extracts of Bacillus subtilis and commercial preparations of subtilisin BPN'' gave linear rates of hydrolysis of azocoll as a function of protease concentration; both gave increasing rates of hydrolysis of azocoll as a function of time. In attempting to improve and standardize proteolytic assays using azocoll, it was found that the absorption maximum of solubilized azocoll at pH 7.8 is 516 nm and is not significantly altered at acidic pH; assays which are perfectly linear as a function of time can be obtained by using azocoll that was vigorously prewashed with buffer; the soluble filtrate removed by prewashing can regenerate the nonlinear time courses previously observed; and the rate of hydrolysis of azocoll can be varied by a factor of 3 by varying the rates of agitation of the assay tubes. To obtain reproducible, linear assays it was essential to prewash commercial azocoll and agitate reaction tubes vigorously.