Procedure for Enzymatic Assay of Trypsin (EC 3.4.21.4)

Description

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


where:
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.

Precautions

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.

Procedure

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:

Note: Perform Test in triplicate.

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

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.

Results

Calculations

1.

calculation

where:
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.

2.

Units/mg solid = Units/ mL enzyme
mg solid/mL enzyme
Materials
Loading
1.
Bergmeyer H. 1974. Methods of Enzymatic Analysis. 2. New York: Academic Press, Inc..

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