290 Sweeney and Walker
2.8. Pepsin (EC 3.4.23.1)
2.8.1. General Information
The term "pepsin" refers to a group of gastric proteinases that are
active at acid pH values (pH 1-5). These enzymes are formed by
partial proteolysis of the inactive zymogens, the pepsinogens (58).
Pepsin is very suitable for investigating arrangement of disulfide bonds
in proteins owing to minimization of disulfide interchange reactions
because of low pH. The commercially available form of the enzyme
is Pepsin A from porcine gastric mucosa (59).
2.8.2. Specificity
Pepsin has broad specificity with a preference for peptides contain-
ing linkages with aromatic or dicarboxylic L-amino acids. It preferen-
tially cleaves C terminal to Phe and Leu and to a lesser extent Glu
linkages. The enzyme does not cleave at Val, Ala, or Gly.
2.8.3. Molecular Mass
The enzyme has a mol mass of 35,000 Da. The amino acid sequence
and composition have been determined (58,59).
2.8.4. pH Optimum
Pepsin is optimally active at pH 2-4 (58).
2.8.5. Assay
The assay is based on hydrolysis Of denatured hemoglobin. Five
milliliters of hemoglobin solution (20 mg/mL in 60 mM HC1) and 0.1
mL enzyme in HCI (10 mM) are combined and incubated for 10 min
at 37°C. Ten milliliters of TCA (5% [w/v]) are then added, and the
mixture centrifuged at 8000g or filtered. Five milliliters of filtrate are
combined with l0 ~L of 0.5M NaOH and 3 mL of Folin and Ciocalteau's
reagent; after l0 min, the absorbance is read at 578 nm. The quantity
of tyrosine released can be determined from a standard curve. Using
this assay, the enzyme is supplied with a specific activity of 1 Anson
unit/gram, where 1 Anson unit is the amount of enzyme that hydro-
lyzes hemoglobin, such that hydrolysis products formed/min give the
same absorbance with Folin's reagent as 1 mmol of tyrosine.
Alternatively, an assay based on the hydrolysis of acetyl-L-
phenylalanyl-3,5-diiodo-L-tyrosine can be used. Using this assay, the
enzyme is supplied with a specific activity of approx 150 U/g, where