Chavira R, Burnett T J, Hageman J H
Anal Biochem. 1984 Feb;136(2):446-50. doi: 10.1016/0003-2697(84)90242-2.
Azocoll, an insoluble, ground collagen to which a bright-red azodye is attached has been 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 has been offered. We have found that assays of both crude extracts of Bacillus subtilis and commercial preparations of subtilisin BPN' gave linear rates of hydrolysis of azocoll as a function of protease concentration; however, both gave increasing rates of hydrolysis of azocoll as a function of time. In attempting to improve and standardize proteolytic assays using azocoll we have found: (a) the absorption maximum of solubilized azocoll at pH 7.8 is 516 nm and is not significantly altered at acid pH; (b) assays which are perfectly linear as a function of time can be obtained by using azocoll that has been vigorously prewashed with buffer; (c) the soluble filtrate removed by prewashing can regenerate the nonlinear time courses previously observed; and (d) the rate of hydrolysis of azocoll can be varied by a factor of 3 by varying the rates of agitation of the assay tubes. In summary, to obtain reproducible, linear assays it was essential to prewash commercial azocoll and agitate reaction tubes vigorously.
偶氮胶原是一种附着有鲜红色偶氮染料的不溶性磨碎胶原，已被广泛用于蛋白水解酶的测定。早期研究表明，偶氮胶原的水解随蛋白酶浓度呈线性进展，但随时间呈指数增加。对于后一种行为尚未给出解释。我们发现，枯草芽孢杆菌粗提物和枯草杆菌蛋白酶BPN'商业制剂的测定中，偶氮胶原的水解速率均随蛋白酶浓度呈线性；然而，两者的偶氮胶原水解速率均随时间增加。在尝试改进和标准化使用偶氮胶原的蛋白水解测定时，我们发现：(a) 溶解的偶氮胶原在pH 7.8时的最大吸收波长为516 nm，在酸性pH下无明显变化；(b) 通过使用用缓冲液剧烈预洗过的偶氮胶原，可以获得随时间呈完美线性的测定；(c) 预洗除去的可溶性滤液可以重现先前观察到的非线性时间进程；(d) 通过改变测定管的搅拌速率，偶氮胶原的水解速率可以变化3倍。总之，为了获得可重复的线性测定，必须对商业偶氮胶原进行预洗并剧烈搅拌反应管。
