Enzymatic determination of pepsin
Introduction
This method can be used for the determination of pepsin activity using haemoglobin as a substrate. It is a spectrophotometric rate termination assay.
Unit definition: One unit of pepsin at 37°C, pH2.0, dissolved in trichloroacetic acid (TCA) produces 0.001 ΔA280 per minute of product using haemoglobin as substrate. (Final volume =16 mL, optical range =1cm.)
Reagents and equipment required
1.0M Hydrochloric Acid
Bovine haemoglobin (Product No. H195714)
6.1N [~100% (w/v)] Trichloroacetic acid solution (Product No. A291718)
Cautions
Please refer to the Product Chemical Safety Technical Sheet for information on hazards and safe handling practices.
Preparation instructions
Use ultrapure water (≥18 MΩxcm resistivity at 25 °C) to prepare the reagent.
10 mM HCl (hydrochloric acid)-1.0M hydrochloric acid diluted 100 times with ultrapure water.
2.5% (w/v) Haemoglobin stock solution - Prepare a 25 mg/mL solution using bovine blood haemoglobin in ultrapure water. Stir vigorously, creating a vortex, for at least 10 minutes at 37°C, then filter through a polypropylene column using a coarse filter (90-130 mm).
Substrate [2.0% (w/v) haemoglobin solution]-80 mL of filtered 2.5% (w/v) haemoglobin stock solution was transferred to a suitable vessel. The pH of the solution was adjusted to 2.0 using 5M HCl at 37°C. The final volume was added to 100 mL with ultrapure water.
TCA solution [5% (w/v) TCA] - dilute 6.1N [~100% (w/v)] TCA solution 20 times with ultrapure water.
Enzyme solution (pepsin) - Prepare a stock solution of 1 mg/mL with cold (2-8°C) 10 mM HCl. If insoluble material is present, place the stock solution on ice until dissolved. If pepsin (Product No. P128678) has dissolved, or if insoluble material is still present, after 1 hour, further dilute the 1 mg/mL stock solution to 0.01-0.05 mg/mL with cold 10 mM HCl solution.
Operating procedures
1. Transfer the substrate into a suitable glass vial.
Reagents | Blank(ml) | Specimen1(ml) | Specimen2(ml) | Specimen3(ml) |
Substrates | 5.00 | 5.00 | 5.00 | 5.00 |
2. Place the vial in a constant temperature water bath and equilibrate to 37°C for approximately 10 minutes, then add.
Reagents | Blank(ml) | Specimen1(ml) | Specimen2(ml) | Specimen3(ml) |
Enzyme solutions | – | 1.00 | 1.00 | 1.00 |
3. Vortex mix and incubate at 37ºC for a full 10 minutes, then add.
Reagents | Blank(ml) | Specimen1(ml) | Specimen2(ml) | Specimen3(ml) |
Trichloroacetic acid solution | 10.0 | 10.0 | 10.0 | 10.0 |
Enzyme solutions | 1.00 | – | – | – |
4. Vortex the mixture and incubate for a further 5 minutes at 37°C.
5. Filter the blank and specimen mixture through a 0.45µm injection filter. Using a spectrophotometer, record the A280 of each filtered solution relative to air in each vial.
Results
Calculation
Units/ml Enzyme = (A280 Specimen - A280 Blank) x (df)
(10) x (1.0) x (.001)
Where：
df = dilution factor
10 = incubation time of the assay in minutes
1.0 = volume of enzyme solution added (ml)
0.001 =ΔA280 per unit of pepsin (unit definition)