Pepsin Assay

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

Method: 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