Procedure for Enzymatic Assay of Trypsin (EC 3.4.21.4)

Description

This procedure is for determining Trypsin activity using N_α-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 = N_α-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)

N_α-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 N_α-Benzoyl-L-arginine ethyl ester) – Prepare a 0.086 mg/mL solution using N_α-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 N_α‑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.

Results

Calculations

1.

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

1. Bergmeyer H. 1974. Methods of Enzymatic Analysis. 2. New York: Academic Press, Inc..