Pepsin Assay

A pepsin assay based upon decreasing fluorescence as a fluorescent dye is released from a protein substrate has been described by Spencer et al. (1975). See also Sachdev and Fruton (1975). Robinson and White (1970) report on a spectrophotometric assay at 320 nm using p-nitrophenyl sulfite as a substrate and Stein et al. (1971) describe one using phenyl sulfite at 270 nm. Doleschel and Auerswald (1973) report on a gel radial diffusion assay. The Worthington assay is based on that of 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 glass wool. 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 5-20 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 5-20 micrograms per ml with 0.01 N HCl.

Procedure

Into each of six numbered test tubes pipette 5.0 ml hemoglobin substrate. Place in a 37°C water bath to equilibrate. Tubes 1-3 are blanks. Into each, pipette 10 ml of TCA followed by 1 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 1 ml of respective enzyme dilution to each and incubate at 37°C for exactly 10 minutes, stop the reaction by adding 10 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.