Pronase 273incubated at 40°C. After 10 min, 2 mL of protein precipitation agent
are added (1.63 g trichloroacetic acid, 0.82 g sodium acetate, 0.57 mL
glacial acetic acid made up to 100 mL with water). The mixture is
incubated for a further 10 min at 40°C, and then centrifuged for 5 min
at 3000 rpm. Five milliliters of 0.4M sodium carbonate solution and 1
mL Folin and Ciocalteau's reagent (diluted 1:5 just prior to use) are
added to 1 mL of supernatant. The mixture is then incubated for a
further 10 min at 40°C, and the absorbance at 660 nm then measured.
One PUK (Proteolytic Units of Kaken; "Kaken" is a Japanese com-
pany) unit is defined as the amount of enzyme that produces an absor-
bance of 1.0 at 660 nm. Using this assay, the enzyme is usually supplied
with a specific activity of about 70,000 PUK/g.
Alternatively, the enzyme is supplied with a specific activity of
about 7000 PU/g where one PU (proteolytic unit) is the enzyme activ-
ity that liberates Folin-positive amino acids and peptides correspond-
ing to 1 ~nol of tyrosine within 1 min under the aforementioned assay
conditions. One PUK corresponds to 50 PU. Because of its broad
specificity, the enzyme is capable ofhydrolyzing many peptide, amide,
and ester bonds, including the majority of the specific substrates of
most proteolytic enzymes. Pronase also hydrolyzes peptides involv-
ing D amino acid residues (24).
2.6. Stability
The calcium ion dependence for the stability of some of the compo-
nents (mainly exopeptidases) was one of the earliest observations made
of Pronase (2). Pronase is therefore normally used in the presence of
5-20 mM calcium. The addition of excess EDTA results in the irrever-
sible loss of 70% of proteolytic activity (18). Two peptidase components
are inactivated by EDTA, but activity is restored by the addition of Co z÷
or Ca 2+. One of these components, the leucine aminopeptidase, is heat
stable up to 70°C. All other components of Pronase lose 90% of their
activity at this temperature (5). The leucine aminopeptidase is not inac-
tivated by 9M urea, but is labile on dialysis against distilled water (2).
Some of the other components of Pronase are also reported to be stable
in 8M urea (2), and one of the serine proteases retains activity in 6M
guanidinium chloride (24). Pronase retains activity in 1% SDS (w/v)
and 1% Triton (w/v) (15). The enzyme is stable at 4°C for at least 6 mo
and is usually stored as a stock solution of 5-20 mg/mL in water at-20°C.