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

The above unit can be expressed as micromoles of tyrosine equivalents released per minute by multiplying by 16/1250 where 16 represents the final filtrate volume and 1250 is the extinction coefficient of tyrosine.