Methods

Protein concentration of trypsinogen and trypsin was determined by measuring the absorbance at 280 rnp in a Beckman DU spectrophotometer. The extinction coefficient E:‘&= 13.9 was used for trypsinogen and 14.4 for trypsin (5). Trypsin activity was determined by a slight modification of the procedure of Schwert and Takenaka(12), with the rate of increase in absorbance at 253 rnp occurring during the hydrolysis of benzoyl-L-arginine ethyl ester as a measure of enzyme activity. A Beckman model DU spectrophotometer equipped with a photomultiplier and a circulating water jacket to maintain the cell chamber at 25” was used to follow the reaction. An aliquot of 0.025 ml of the solution to be assayed was added to a 1.0 ml solution of 0.0005 M BAE in 0.1 M Tris buffer, pH 7.0, with 0.02 M CaClz contained in a 1.5-ml cuvette. The cuvette was inverted a number of times with the use of Parafilm as a cover and readings were taken every 15 or 30 seconds for 3 minutes. A unit of trypsin activity per ml of the incubation mixture is defined as that activity contained in an aliquot of 0.025 ml of the incubation mixture which caused an increase in absorbancy of 0.001 per minute when assayed under the above conditions. With the use of this procedure with standard trypsin solutions it was found that an aliquot of 0.025 ml of a solution containing 0.001 mg per ml of crystalline trypsin caused an increase in