Trypsin, chymotrypsin, and total proteolytic activity of pancreas, pancreatic juice, and intestinal contents from the bovine

Abstract

A procedure was developed for assaying total proteolytic activity, or esterase activities of trypsin and chymotrypsin in bovine pancreas, pancreatic juice, and intestinal contents. Trypsinogen and chymotrypsinogen were released from pancreatic tissue by homogenization in 0.15 M NaCl containing 0.1% Triton X-100. The homogenate was centrifuged and diluted to contain 0.3 to 1% tissue. Pancreatic juice was diluted in saline to 0.25 to 0.75 mg protein/ml. Maximum enzyme activity and stability were obtained by activating with a crude enteropeptidase solution containing 0.05 M CaCl 2 at 5°C for 2 to 6 hr for chymotrypsinogen and 5 to 7 days for trypsinogen. The normal curvilinear response of enzyme concentration and casein digestion was transformed to a linear process by plotting absorbancy (A) + A 3 2 , using a tyrosine standard at 280 mμ. Rate of BTEE hydrolysis by activated pancreatic juice was 2.2 times greater in 4.7% methanol than in 26%, v/v (BTEE dissolved in 10 or 56% methanol). Interfering substances in intestinal contents which produced an apparent esterase activity, in the absence of enzyme substrates, were attributed to increased turbidity as a result of calcium complexing with certain dietary constituents or bile salts, or both. Addition of CaCl 2, (NH 4) 2SO 4, and adjustment of the intestinal contents to pH 8 removed most of the interfering substances from the supernatant, and also augmented trypsin and chymotrypsin activity in most samples.