Substrates were dissolved in dimethyl sulphoxide
(DMSO) to give stock solutions of 4 mM. The assay
was performed in Eppendorf tubes and the reaction
mixture contained 800 ml of chromogenic substrate and
50 ml of suitably diluted protease solution in Tris–HCl
buffer. After 30 min of incubation at 37°C, the reaction
was stopped by the addition of 200 ml of 2 N acetic acid
and the amount of p-nitroaniline released was assayed
at 405 nm on a spectrophotometer (Perkin–Elmer,
Ubelingen, FRG). The molar absorbance of p-nitroaniline
at 405 nm was equal to 9260 l mol−1 cm−1
.
Enzyme activities were expressed in arbitrary units
defined as the amount of enzyme that liberated 1 nmol
of p-nitroaniline in 1 min under the assay conditions.