Pepsin - Assay

Source:
Porcine Stomach
CAS:
9001-75-6
EC:
3.4.23.1

Pepsin Assay

Method

The Worthington assay is based on the stop-point assay of hemoglobin degradation developed by Anson (1938).

The rate of hydrolysis of denatured hemoglobin is measured. One unit releases 0.001 A280 as TCA soluble hydrolysis products per minute at 37°C under the specified conditions.

Reagents
  • 1.0 N HCl
  • 0.3 N HCl
  • 0.01 N HCl
  • 2.5% w/v Hemoglobin: Prepare by dissolving 2.5 grams Worthington bovine erythrocyte hemoglobin powder (Code: HB) in 100 ml reagent grade water. Blend in a Waring blender at maximum speed for 3-5 minutes. Filter through gauze. Dilute 80 ml of filtrate with 20 ml of 0.3 N HCl.
  • 5% w/v Trichoracetic acid (TCA)
Enzyme

Pepsin activity: Dissolve pepsin at a concentration of 0.5 mg/ml in 0.01 N HCl. Keep chilled. Immediately prior to assay, dilute further in 0.01 N HCl to 10-20 micrograms per ml. Three dilutions are recommended.

Pepsinogen: Dissolve 25 mg pepsinogen in approximately 40 ml reagent grade water. Adjust the pH to 8.0 with 0.01 N NaOH and allow 10 minutes to inactivate any contaminating pepsin activity. Lower the pH to 2.0 with HCl and dilute to a final volume of 50 ml with reagent grade water. For assay dilute further to 10-20 micrograms per ml with 0.01 N HCl.

Procedure

Into each of six numbered test tubes pipette 2.5 ml hemoglobin substrate. Place in a 37°C water bath to equilibrate. Tubes 1-3 are blanks. Into each, pipette 5 ml of TCA followed by 0.5 ml of respective enzyme dilution. Remove from bath after 5 minutes and filter. Read A280 of clear filtrate.

Tubes 4-6 are for test. At timed intervals, add 0.5 ml of respective enzyme dilution to each and incubate at 37°C for exactly 10 minutes, stop the reaction by adding 5 ml of 5% TCA at timed intervals. Remove from bath after 5 minutes and filter. The filtrates should be clear. Record filtrate absorbance at 280 nm and subtract A280 of appropriate blank.

Calculation

Pepsin Products

Description
Activity
Code
Cat. #
Size
Price
Description
Pepsin A
Source:
Porcine Stomach
Two times crystallized from dilute alcohol. A lyophilized powder.
Store at 2-8°C.
Activity
≥2,500 units per mg dry weight
Code
PM
LS003319
1 gm
$100.00
LS003317
10 gm
$700.00
LS003322
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 500000 x 1 gm. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.
Description
Pepsin A
Source:
Porcine Stomach
Salt-precipitated protein. A dialyzed, lyophilized powder. 
Store at 2-8°C.
Activity
≥3,000 units per mg protein
Code
PM2
LS003324
1 gm
$70.00
LS003326
10 gm
$500.00
LS003328
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 200 x 1 gm. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.