Study On Catalytic Properties Of Protease Using SDS-PAGE-the Establishment Of A New Method

Protease is one of hydrolytic enzymes which there are abundance in vivo, protease plays an important role in metabolism. The substrate of Protease should be protein, but the substrate of the reaction which used to test and evaluate the activity of the protease were not protein in reports. The un-protein substrate made some effect on the accuracy of the results. Filter from protein substrates and bovine serum albumin(BSA) was chosen as a substrate of protease-catalyzed reaction finally in the study. The activity of trypsin, papain and acid protease were be tested by SDS-PAGE and results showed that SDS-PAGE was an effective new method for evaluating proteinase properties such as the activity. Moreover, SDS-PAGE analysis pattern can monitor the process of reaction between protease with protein, it is conducive to the understanding of properties of the protease.Using SDS-PAGE to optimize the enzymatic reaction of trypsin, papain and acid protease at different temperatures and pH values, and draws the most suitable hydrolysis conditions for these protease and conclusions consistent with the existing reports: 1. The optimum temperature of trypsin is 40 °C, the optimum pH is pH8.5-pH8.7; 2. The optimum temperature of papain is 60 °C, the optimum pH is pH8.0; 3. The optimum temperature of acid protease is 60 °C, the optimum pH is pH2.6-pH3.6.At the same time, SDS-PAGE also be applied to study the mechanism of trypsin inhibitors which can prevent BSA from being hydrolyzed by trypsin. According to pattern of SDS-PAGE, molecular weight of trypsin and inhibitors were determined, the process of the reaction also be analyzed. Based on the results and analysis presumed that trypsin inhibitors can bind to the active site of trypsin and that can prevent substrates bind to trypsin, so the hydrolysis of the substrate be prevented.