Procedure for Enzymatic Assay of α-Chymotrypsin (EC


This procedure may be used for the determination of α‑Chymotrypsin activity using N-Benzoyl-L-tyrosine ethyl ester (BTEE) as the substrate. It is not to be used to assay α‑Chymotrypsin agarose (Catalog Number C9134). The procedure is a continuous spectrophotometric rate determination (A256, Light path = 1 cm) based on the following reaction:

BTEE – N-Benzoyl-L-tyrosine ethyl ester

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

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. H9892)

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

Preparation Instructions

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

Buffer (80 mM Trizma-base Buffer, pH 7.8 at 25 °C) – Prepare a 12.5-fold dilution of stock 1.0 M Trizma Base buffer, prepared using Catalog Number T1503, with ultrapure water. Adjust the pH to 7.8 at 25 °C using 1 M HCl or 1 M NaOH.

BTEE Solution (1.18 mM N‑Benzoyl-L‑Tyrosine Ethyl Ester) – To prepare 100 ml:

  • Weigh 37.0±0.5 mg of N‑Benzoyl-L‑Tyrosine Ethyl Ester (Catalog Number B6125) into a 100 ml Class A volumetric flask.
  • Add 63.4 ml of Methanol (Catalog Number M1775) and mix by swirling.
  • Bring the final volume of the solution to 100 ml using ultrapure water.
  • Cover flask opening and invert several times to ensure complete mixing.
    Note: Volume prepared can be adjusted if needed.

CaCl2 Solution (2 M Calcium Chloride) – Prepare a 294 mg/ml solution using Calcium chloride, dihydrate (Catalog Number C3881) in ultrapure water.

HCl Solution (1 mM Hydrochloric Acid) – Prepare a 1,000-fold dilution of 1 M Hydrochloric acid solution (Catalog Number H9892), with ultrapure water in a 100 ml Class A volumetric flask. Cover flask opening and invert several times to ensure complete mixing. Place solution on ice.

Enzyme Solution (α‑Chymotrypsin) – Immediately before use, prepare a solution containing 2‑5 α‑chymotrypsin units/ml 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 α‑Chymotrypsin.

1. Pipette the following reagents into suitable quartz cuvettes:

Reagent Test 1 (ml) Test 2 (ml) Test 3 (ml) Blank (ml)
Buffer 1.42 1.42 1.42 1.42
BTEE Solution 1.40 1.40 1.40 1.40
CaCl2 Solution 0.08 0.08 0.08 0.08

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

3. Pipette the following reagents into the cuvettes:

Reagent Test 1 (ml) Test 2 (ml) Test 3 (ml) Blank (ml)
HCl Solution 0.10
Enzyme Solution 0.05 0.07 0.10

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

5. Determine 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.





TV = total volume (ml) of reaction mix in cuvette
df = dilution factor
0.964 = millimolar extinction coefficient of BTEE at 256 nm
VTest = volume (ml) of Enzyme Solution used in assay








  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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