Chymotrypsin Assay

Method: The reaction velocity is determined according to Hummel (1959) by measuring an increase in absorbance at 256 nm resulting from the hydrolysis of benzoyl-L-tyrosine ethyl ester. One unit hydrolyzes one micromole of benzoyl-L-tyrosine ethyl ester (BTEE) per minute at pH 7.8 and 25°C under the specified conditions.

Reagents

• 0.08 M Tris⋅HCl buffer, pH 7.8 containing 0.1 M calcium chloride
• 0.00107 M Benzoyl-L-tyrosine ethyl ester (BTEE) in 50% w/w methanol (63 ml absolute methanol added to 50 ml reagent grade water)
• 0.001 N HCl

Enzyme

Dissolve enzyme at one mg/ml in 0.001 N HCl. Dilute in 0.001 N HCl to 10-30 μg/ml for assay.

Procedure

Adjust the spectrophotometer to 256 nm and 25°C.

Pipette into cuvettes as follows:

0.08 M Tris⋅HCl buffer, pH 7.8 with 0.1 M CaCl2	1.5 ml
0.00107 M BTEE	1.4 ml

Incubate in spectrophotometer at 25°C for 4-5 minutes to achieve temperature equilibrium and record blank rate, if any. Add 0.1 ml of appropriately diluted enzyme and record increase in absorbance at 256 nm for 4-5 minutes. Calculate ΔA₂₅₆/min from the initial linear portion of the curve.

Calculation