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Activity Assay for Endothia parasitica Protease Using Trypsinogen as Substrate¹

Department of Food Science and Technology, University of California, Davis 95616

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 30.0 C using 3.0 mg/ml of bovine trypsinogen and 1.25 g/ml of purified Endothia parasitica protease in .04 M sodium formate –.3 M KCl buffer. At intervals of 1, 2, 3, 5, and 7 minutes the amounts of trypsin formed were determined on .05 ml aliquots with 1.95 ml of 1% casein in .2 M Tris –.01 M CaCl₂ buffer, pH 8.0 and 30 C for 10 min. The trichloroacetic acid-soluble products were determined at 280 nm. 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, V_max, 2,950 ± 177 micromoles trypsinogen activated per minute per micromole Endothia parasitica protease, and K_m was 8.32 ± .30 x 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 30 C and 600 times more sensitive than a milk clotting assay at pH 5.1 and 30 C.

¹ Supported in part by the National Institutes of Health (AM-13165)