272 Sweeney and Walker
sion of substrates (5). Pronase has also been used to study membrane
topology and protein membrane translocation in the same manner as
described for proteinase K in Chapter 14 (14,15), and has been used
to disperse mast cells prior to purification and characterization of
these cells (16).
- Enzyme Data
2.1. Specificity
Because it is a mixture of exo- and endoproteases, pronase has a
broad specificity, cleaving nearly all peptide bonds (17).
2.2. Purification
The purification of individual proteolytic components of pronase
has been reported by a number of authors (18-21). In 1988, the puri-
fication and characterization of an additional amino acid-specific
endopeptidase from Pronase were described by Yoshida et al. (22).
2.3. Molecular Mass
Molecular masses of 15,000-27,000 Da have been reported for com-
ponents of pronase. These weights have usually been determined by
gel filtration (18,20-22).
2.4. pH Optimum
Pronase has optimal activity at pH 7-8. However, individual com-
ponents are reported to retain activity over a much wider pH range
(20-23). The neutral components are stable in the pH range 5.0-9.0,
in the presence of calcium, and have optimal activity at pH 7.0-8.0.
The alkaline components are stable in the pH range 3.0-9.0, in the
presence of calcium, and have optimal activity at pH 9.0-10.0 (4). The
aminopeptidase and carboxypeptidase components are stable at pH
5.0-8.0 in the presence of calcium (23).
2.5. Assay
The assay is normally based on the hydrolysis of casein, which is
followed spectrophotometrically by reaction with Folin and
Ciocalteau's reagent. One milliliter of enzyme solution is suitably
diluted in 0.067M phosphate buffer, pH 7.4. A 2% casein solution,
adjusted to pH 7.4 with NaOH, is also prepared. One milliliter of the
2% casein solution is added to the enzyme solution, and the mixture