Koritsas V M, Atkinson H J
Centre for Plant Biochemistry and Biotechnology, University of Leeds, United Kingdom.
Anal Biochem. 1995 May 1;227(1):22-6. doi: 10.1006/abio.1995.1247.
A rapid, cheap, and sensitive method has been developed for determining proteolytic activity of different classes of endoproteinases. The method is based on a solid-phase assay employing as substrate biotinylated gelatin adsorbed onto microtiter plates. Enzymatic activity is measured by incubating proteinase with the immobilized biotin-protein. Any remaining, undigested substrate bound to the microtiter plate is assayed with streptavidin-alkaline phosphatase. It was established that papain, pepsin, thermolysin, and trypsin all hydrolyzed the biotinylated substrate to varying degrees. Furthermore, the activity of these proteinases was blocked by their respective inhibitors. The assay presented is quick, highly reproducible, inexpensive, and useful for detecting all classes of endoproteolytic enzymes. By using different biotinylated proteins or peptides as substrates, and employing specific buffers and inhibitors, this assay may be utilized for detecting other and more specific endoproteinases.
已开发出一种快速、廉价且灵敏的方法来测定不同类别的内切蛋白酶的蛋白水解活性。该方法基于一种固相测定法，使用吸附在微量滴定板上的生物素化明胶作为底物。通过将蛋白酶与固定化的生物素化蛋白一起孵育来测量酶活性。用链霉亲和素-碱性磷酸酶测定与微量滴定板结合的任何剩余未消化底物。已确定木瓜蛋白酶、胃蛋白酶、嗜热菌蛋白酶和胰蛋白酶都不同程度地水解了生物素化底物。此外，这些蛋白酶的活性被它们各自的抑制剂所阻断。所介绍的测定方法快速、高度可重复、廉价，并且可用于检测所有类别的内切蛋白酶。通过使用不同的生物素化蛋白质或肽作为底物，并采用特定的缓冲液和抑制剂，该测定法可用于检测其他更特异的内切蛋白酶。
