Procedure for Enzymatic Assay of Trypsin (EC


This procedure is for determining Trypsin activity using Nα-Benzoyl-L-arginine ethyl ester (BAEE) as the substrate. The procedure is a continuous spectrophotometric rate determination (A253, Light path = 1 cm) based on the following reaction:

BAEE + H2O     Trypsin    > Nα-Benzoyl-L-arginine + ethanol

BAEE – Nα-Benzoyl-L-arginine ethyl ester

Unit Definition – One BAEE unit of trypsin activity will produce a ΔA253 of 0.001 per minute with BAEE as substrate at pH 7.6 at 25 °C in a reaction volume of 3.20 ml.


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

Reagents and Equipment Required

Sodium phosphate, monobasic (Catalog No. S0751)

Nα-Benzoyl-L-arginine ethyl ester (BAEE, Catalog No. B4500)

1 M NaOH solution

1 M Hydrochloric acid (Catalog No. 318949)

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 (67 mM Sodium Phosphate Buffer, pH 7.6 at 25 °C) – Prepare a 8.04 mg/ml solution using sodium phosphate, monobasic (Catalog No. S0751) in ultrapure water. Adjust to pH 7.6 at 25 °C with 1 M NaOH solution.

Substrate Solution (0.25 mM Nα-Benzoyl-L-arginine ethyl ester) – Prepare a 0.086 mg/ml solution using Nα-Benzoyl-L-arginine ethyl ester (BAEE, Catalog No. B4500) in Buffer.

HCl Solution (1 mM Hydrochloric Acid) – Prepare a 1,000-fold dilution of 1 M Hydrochloric acid solution (Catalog No. 318949) in ultrapure water.

Enzyme Solution (Trypsin) – Immediately before use, prepare a solution containing 425‑575 units/ml of Trypsin in cold (2‑8 °C) HCl Solution.


In a 3.20 ml reaction mix, the final concentrations are 62.8 mM sodium phosphate, 0.23 mM Nα‑Benzoyl- L-arginine ethyl ester, 0.031‑0.063 mM hydrochloric acid, 42.5‑115.0 units of trypsin.

1. Pipette the following reagents into suitable quartz cuvettes:

Reagent Blank (ml) Test (ml)
Substrate Solution 3.00 3.00
HCl Solution 0.200 0.125

Note: Perform Test in triplicate.

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

Reagent Blank (ml) Test (ml)
Enzyme Solution 0.075

Note: Final volume in each cuvette must be 3.2 ml per unit definition.

3. Immediately mix by inversion and record the increase in A253 for 5 minutes. Using a 1 minute time period and a minimum of 4 data points, obtain the ΔA253/minute using the maximum linear rate for both the Blank and Tests.




df = dilution factor
0.001 = The change in A253/minute based on unit definition
0.075 = volume (ml) of test sample used in assay
Note: The total volume in the cuvette is not used in the calculation since the unit definition is based on 3.2 ml.


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




  1. Bergmeyer, H.U. et al., in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.) Volume I, 2nd ed., Academic Press, Inc., (New York, NY:1974) 515-516.



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