292 Sweeney and Walker2.9.4. pH Optimum
Thermolysin has optimal activity in the pH range 7.0-8.0, but can
be used up to pH 9.0 if required.
2.9.5. Assay
The assay is based on the hydrolysis of casein. One milliliter of
borate buffer (0.1M, pH 7.2), 1 mL of substrate solution (2 g casein
dissolved in water and l0 mL of 1 M NaOH, pH adjusted to 7.2, and
solution made up to 100 mL), and 0.02 mL of enzyme solution are
incubated for l0 min at 37°C; 2.0 mL of TCA (0.1M) and 0.2 mL CaC1 a
(2 mM) are then added; the mixture is incubated for a further l0 min
at 37°C, and then centrifuged or filtered. Filtrate (1.5 mL) is combined
with 5 mL of Na2CO 3 (0.4M) and 1 mL of Folin and Ciocalteau's
reagent (commercial reagent diluted 1:3). This mixture is then incu-
bated for 20 min at 37°C. The absorbance is read at 578 nm, and using
standard solutions, the concentration of the enzyme is calculated. The
enzyme is usually supplied with a specific activity of 4 U/mg, where
1 U is defined as the amount of enzyme that liberates 1 ~unol Folin-
positive amino acids and peptides (calculated as tyrosine)/min at pH
7.2 and 37°C.
2.9.6. Stability
The enzyme is thermostable, and at 80°C has a half-life of 1 h (62).
There is no loss of activity at 60°C over 1 h. A zinc atomat the active
site is essential for activity, but the enzyme also has an absolute require-
ment for calcium ions for stability, and this should therefore be included
in digestion buffers, although most preparations contain sufficient
Ca 2+ to be active without the addition of extra Ca 2+. The enzyme is
stable in 8M urea, and also in 20% methanol or ethanol. However,
these conditions enhance thermal denaturation and digestion should
then be carried out at 20°C (62). Aqueous solutions of the enzyme can
be stored frozen for weeks without loss of activity (62). The enzyme
is also active in 1% SDS, and 0.1M NaC1. The lyophilized enzyme is
stable at 4°C for many months.
2.9. 7. Inhibitors
Because of the requirement for a zinc atom at the active site, chelat-
ing agents, such as EDTA and l, l0 phenanthroline, are strong inhibi-
tors (62). Complete inhibition occurs in 5 mM EDTA at 40°C in 3 min.