Procedure for Enzymatic Assay of Chymotrypsin (α‑Chymotrypsin, EC


This procedure is for products with a specification for Chymotrypsin activity. It is not to be used to assay Insoluble Chymotrypsin such as Catalog No. C9134. The procedure is a continuous spectrophotometric rate determination (A256, Light path = 1 cm) based on the following reaction:

BTEE + H2O     Chymotrypsin    > N-Benzoyl-L-tyrosine + ethanol

BTEE – N-Benzoyl-L-tyrosine ethyl ester

Unit Definition – One unit of chymotrypsin will hydrolyze 1.0 µmole of BTEE per minute at pH 7.8 at 25 °C.


Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents and Equipment Required

Trizma® Base (Catalog No. T1503)

N‑Benzoyl-L‑Tyrosine Ethyl Ester (Catalog No. B6125)

Methanol (Catalog No. M1775)

Calcium chloride, dihydrate (Catalog No. C3881)

Hydrochloric acid solution (Catalog No. 318949)

Preparation Instructions

Use ultrapure water (≥18 MΩxcm resistivity at 25 °C) for the preparation of reagents.

Buffer (80 mM Tris HCl Buffer, pH 7.8 at 25 °C) – Prepare a 9.69 mg/ml solution in ultrapure water using Trizma® Base (Catalog No. T1503). Adjust the pH of this solution to 7.8 at 25 °C.

BTEE Solution (1.18 mM N‑Benzoyl-L‑Tyrosine Ethyl Ester) – Weigh 37 mg of N‑Benzoyl-L‑Tyrosine Ethyl Ester (Catalog No. B6125) into a 100 ml Class A volumetric flask. Add 63.4 ml of Methanol (Catalog No. M1775) and mix by swirling. Bring the final volume of the solution to 100 ml using ultrapure water. Invert the flask several times to ensure complete mixing.

CaCl2 Solution (2 M Calcium Chloride) – Prepare a 294 mg/ml solution in ultrapure water using Calcium chloride, dihydrate (Catalog No. C3881).

HCl Solution (1 mM Hydrochloric Acid) – Prepare a solution by diluting 0.10 ml of 1.0 M Hydrochloric acid solution (Catalog No. 318949) to 100 ml with ultrapure water in a 100 ml Class A volumetric flask. Mix by inversion and place on ice.

Enzyme Solution (Chymotrypsin) – Immediately before use, prepare a solution containing 2‑5 chymotrypsin units per milliliter in cold (2‑8 °C) HCl Solution.


In a 3.00 ml reaction mix, the final concentrations are 38 mM Tris, 0.55 mM N-Benzoyl-L-Tyrosine Ethyl Ester, 30% (v/v) Methanol, 53 mM Calcium Chloride, 0.03 mM Hydrochloric Acid, and 0.2‑0.5 units of Chymotrypsin.

1. Pipette the following reagents into suitable quartz cuvettes:

Reagent Blank (ml) Test (ml)
Buffer 1.42 1.42
BTEE Solution 1.40 1.40
CaCl2 Solution 0.08 0.08

2. Mix by inversion and equilibrate to 25 °C using a suitably thermostatted spectrophotometer.

3. Add the following to the cuvettes:

Reagent Blank (ml) Test (ml)
HCl Solution 0.10
Enzyme Solution 0.10

4. Immediately mix by inversion and record the increase in A256 for 3‑5 minutes.

5. Obtain the ΔA256/minute for both the blank and test reactions using the maximum linear rate over a one minute interval using at least 4 points.




Units/ml enzyme = (ΔA256/minute Test – ΔA256/minute Blank) x (3) x (df)
(0.964) x (0.10)

3 = volume (ml) of reaction mix
df = dilution factor
0.964 = millimolar extinction coefficient of BTEE at 256 nm
0.10 = volume (ml) of test sample used in assay


Units/mg solid = units/ml enzyme
mg solid/ml enzyme




  1. Wirnt, R., and Bergmeyer, H.U., eds., Chymotrypsin, in Methods of Enzymatic Analysis, Academic Press (NY, New York: 1974) 1009-1012.


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