Chymotrypsinogen A Assay

Method: Chymotrypsinogen is assayed first for intrinsic activity, then activated and re-assayed. The activity will be determined in the chymotrypsin assay as described above.

Reagents

• 1 N H₂SO₄
• 0.5 M Sodium potassium phosphate buffer, pH 7.6
• Trypsin solution: Dissolve Worthington trypsin (Code: TRL), at a concentration of 1 mg/ml in 0.001 N HCl. Prepare immediately before use.

Procedure

Intrinsic chymotrypsin activity: Dissolve enzyme at a concentration of 10 mg/ml in 0.001 N HCl. Assay for chymotrypsin as described above. Calculate units/mg protein.

Chymotrypsinogen activation: Dissolve 100 mg enzyme in 5 ml reagent grade water. Adjust pH to 3.0 with 1 N H₂SO₄. Add 2.5 ml of 0.5 M phosphate buffer pH 7.6 and readjust pH to 7.6 if necessary. Add 1 ml (1 mg) trypsin and dilute to a final volume of 10 ml with reagent grade water. A 1:500 dilution will probably be required. Assay immediately using the chymotrypsin assay as described above. Repeat the assay at 10 minute intervals until the activity begins to decline. As activation continues, dilution may increase. Calculate units/mg chymotrypsin activity.