Enzymatic Assay of Pepsin (3.4.23.1)

Description

This procedure may be used for determination of Pepsin activity using hemoglobin as the substrate. It is a spectrophotometric stop rate determination.

Unit Definition: One unit of Pepsin will produce a ΔA₂₈₀ of 0.001 per minute at pH 2.0 at 37 °C measured as trichloroacetic acid (TCA)-soluble products using hemoglobin as the substrate. (Final volume = 16 ml, light path = 1 cm.)

Reagents and Equipment Required

1.0 M Hydrochloric Acid

Hemoglobin from bovine blood (Catalog Number H2625)

6.1 N [~100% (w/v)] Trichloroacetic acid solution (Catalog Number T0699)

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.

10 mM HCl (Hydrochloric Acid) – Dilute 1.0 M Hydrochloric Acid 100‑fold with ultrapure water.

2.5% (w/v) Hemoglobin Stock Solution – Prepare a 25 mg/ml solution in ultrapure water using Hemoglobin from bovine blood (Catalog Number H2625). Stir vigorously, creating a vortex, for a minimum of 10 minutes at 37 °C. Then filter through a polypropylene column with a coarse filter (90–130 mm).

Substrate [2.0% (w/v) Hemoglobin solution] – Transfer 80 ml of the filtered 2.5% (w/v) Hemoglobin Stock Solution into a suitable container. Adjust the pH of this solution to 2.0 at 37 °C using 5 M HCl. Bring the final volume to 100 ml with ultrapure water.

TCA Solution [5% (w/v) Trichloroacetic Acid] – Dilute 6.1 N [~100% (w/v)] Trichloroacetic acid solution (Catalog Number T0699) 20-fold with ultrapure water.

Enzyme Solution (Pepsin) – Prepare a 1 mg/mL stock solution in cold (2–8 °C) 10 mM HCl. If insoluble material is present, allow the stock solution to sit on ice until dissolved. When the Pepsin has dissolved or, if insoluble material is still present, after 1 hour, dilute the 1 mg/mL stock solution further to 0.01–0.05 mg/ml Enzyme Solution in cold 10 mM HCl.

Procedure

1. Pipette the Substrate into suitable glass vials.

Reagent	Blank (ml)	Test 1 (ml)	Test 2 (ml)	Test 3 (ml)
Substrate	5.00	5.00	5.00	5.00

2. Place vials in a thermostatted water bath and equilibrate to 37 °C for ~10 minutes, then add:

Reagent	Blank (ml)	Test 1 (ml)	Test 2 (ml)	Test 3 (ml)
Enzyme Solution	–	1.00	1.00	1.00

3. Mix by swirling and incubate at 37 °C for exactly 10 minutes, then add:

Reagent	Blank (ml)	Test 1 (ml)	Test 2 (ml)	Test 3 (ml)
TCA Solution	10.0	10.0	10.0	10.0
Enzyme Solution	1.00	–	–	–

4. Mix by swirling and incubate at 37 °C for an additional 5 minutes.

5. Filter the Blank and Test mixtures through a 0.45 µm syringe filter. Using a spectrophotometer, record the A₂₈₀ versus air for each filtered solution for each vial.

Results

Calculations

Units/ml enzyme = (A280 Test – A280 Blank) x (df) (10) x (1.0) x (.001)

where:

df = Dilution factor
10 = Assay incubation time in minutes
1.0 = Volume of Enzyme Solution (ml) added
0.001 = ΔA₂₈₀ per unit of Pepsin (unit definition)

Reference

1. Anson ML. 1938. THE ESTIMATION OF PEPSIN, TRYPSIN, PAPAIN, AND CATHEPSIN WITH HEMOGLOBIN. 22(1):79-89. https://doi.org/10.1085/jgp.22.1.79