Solubility: Soluble in distilled water or dilute buffer
Stability: -20° C; -4° F
Activity: 3000 U/mg
Protein: 95%
Unit Definition
The amount of enzyme which renders TCA soluble 0.001 E280 nm per minute at 37°C, using a denatured hemoglobin substrate.
Assay Method
Pepsin cleaves peptides from hemoglobin which are soluble in trichloroacetic acid (TCA). The tyrosine and tryptophan content of these TCA-soluble peptides is determined by the measurement of the extinction at 280 nm.
Applications
Pepsin (3.4.23.1) is the principal proteolytic enzyme which occurs in the gastric juices of all mammals. Pepsinogen, its precursor, is secreted by the stomach mucosa. Activation of pepsinogen to pepsin is facilitated by the hydrogen ion concentration of the gastric juice, and it is accomplished autocatalytically by pepsin.
Pepsin is an endopeptidase, which preferentially hydrolyzes those peptide linkages which involve the amino group contributed by the aromatic amino acids phenylalanine, tyrosine and tryptophan. Although pepsin digests proteins mainly into polypeptides of varying length, some shorter peptides and even some free amino acids, notably tyrosine and phenylalanine, may be released. The optimum pH for pepsin activity varies from 1.5-2.0 depending on the substrate. Pepsin from porcine stomach mucosa has been studied most extensively and has a molecular weight of 35,000.
Reagents
1.0 N HCl.
0.3 N HCl.
0.01 N HCl.
2.0% (w/v) Hemoglobin.
5% (w/v) Trichloroacetic acid (TCA).
Pepsin (enzyme) solution. Dissolve to a concentration of 0.5 mg/ml in 0.01 N HCl. Just prior to assay dilute further in 0.01 N HCl to a concentration of 5-20 micrograms per ml.
Procedure
Set spectrophotometer (equipped with strip chart recorder and temperature control) at 280 nm and 37°C. Prepare water bath at 37°C.
Into each of 6 numbered test tubes pipette 5.0 ml hemoglobin substrate. Place all tubes in water bath at 37°C to equilibrate. Use tubes 1-3 as blanks; pipette 10 ml TCA, followed by 1 ml of diluted enzyme, into each of the blank tubes.
Remove tubes 1-3 from water bath and clarify. Read E280 nm of clear filtrate.
Tubes 4-6, remaining in water bath, are for the enzyme test. Add 1 ml of the diluted enzyme to each of these tubes at timed intervals for exactly 10 min.
Stop the reaction by adding 10 ml of 5% TCA at timed intervals.
Remove tubes 4-6 from water bath after 5 min. and clarify (filtrates should be clear). Read E280 nm of filtrate and subtract E280 nm of the appropriate blank.
