288 Sweeney and Walker2.6. 5. Assay
The assay is based on the hydrolysis of casein. The reaction condi-
tions consist of 1% casein in 0.1MTris-HC1, pH 7.8. Enzyme is added,
and the mixture is incubated at 25°C for 10 min. The reaction is stopped
by adding an equal volume of 10% TCA, and the mixture is allowed
to stand for a further 15 min. The mixture is then filtered or centrifuged;
the absorbance of the supernatant is read at 280 nm, and the enzyme
activity calculated. Using this assay, the enzyme is usually supplied
with a specific activity of 500 U/mg, where 1 U is the amount of
enzyme that caused 0.001 A280 unit of change/min at 25°C, pH 7.8.
Alternatively, an assay based on the hydrolysis of N-CBZ-L-glutamyl
t~-phenyl ester can be used. Using this assay, the enzyme is usually
supplied with a specific activity of 500-1000 U/mg, where 1 U is
defined as the amount of enzyme that will hydrolyze 1 pmol of sub-
strate/min at pH 7.8, at 37°C. (One unit is equivalent to approx 0.004
casein digestion units.)
Also, an assay based on the hydrolysis of carbobenzoxy-Phe-Leu-
Glu-4-nitroanilide, at pH 7.8,410 nm, and 25°C has been described.
The enzyme is supplied with a specific activity of 10-20 U/mg when
this assay is used, where 1 U hydrolyzes 1 ~'nol of substrate/min at pH
7.8 and 25°C.
2.6. 6. Stability
The enzyme is active in the presence of 0.2% SDS, and retains 50%
activity in 4M urea (55). The enzyme has been shown to be equally
resistant to heat denaturation over a wide range of temperatures. Sev-
enty percent of activity remained after heating at 100°C for 3 min (50).
Aqueous solutions are stable to freeze/thawing. The enzyme is stable
at pH 4-10, but precipitates below pH 4. It retains activity in 1M
guanidine hydrochloride and 10% acetonitrile. The enzyme is stable
at 4°C, when stored dry. A solution in redistilled water may be used for
1-2 d at maximum, if stored at 4°C.
2.6. 7. Inhibitors
Being a serine protease, the enzyme is inhibited by DFP (50) (see
Chymotrypsin, Section 2.1.7, for practical details). The enzyme is not
inhibited by EDTA, nor does the addition of a range of divalent metal
ions have any effect on enzyme activity (50). The enzyme is also inhibited
by t~ 2 macroglobulin at concentrations equimolar with the enzyme.