Activity Assay for Endothia parasitica Protease Using Trypsinogen as Substrate

Abstract

A sensitive activity assay method for Endothia parasitica protease based on activation of trypsinogen has been developed. Activation of trypsinogen was performed at pH 4.0 and 30.0C using 3.0 mg/ml of bovine trypsinogen and 1.25 μ/ml of purified Endothia parasitica protease in .04M sodium formate −.3M KCl buffer. At intervals of 1, 2, 3, 5, and 7 minutes the amounts of trypsin formed were determined on .05ml aliquots with 1.95ml of 1% casein in .2M Tris −.01M CaCl2 buffer, pH 8.0 and 30C for 10min. The trichloroacetic acid-soluble products were determined at 280nm. Enzyme activity was determined from the initial rate of trypsinogen activation. The pH optimum for activation of trypsinogen by Endothia parasitica protease was 3.5 to 3.6, Vmax, 2,950±177 micromoles trypsinogen activated per minute per micromole Endothia parasitica protease, and Km was 8.32±.30×10−5 M. Sensitivity of the trypsinogen activation assay was 11,400 times that of direct determination of Endothia parasitica protease activity on casein at pH 3.0 and 30C and 600 times more sensitive than a milk clotting assay at pH 5.1 and 30C.