286 Sweeney and Walker2.5. Endoproteinase Asp-N
2.5.1. General Information
This enzyme has been isolated from the culture filtrates of Pseudomo-
nasfragic (46,47). The enzyme is a neutral protease and is a metallo-
enzyme having an essential zinc atom at the active site (46,47).
2.5.2. Specificity
The enzyme cleaves protein substrate on the N-terminal side of
aspartic acid residues or cysteic acid residues (48). Cleavage occurred
for all such residues in oxidized ribonuclease, but when myoglobin
was used, a similar specificity was observed, except that only four out
of six aspartyl bonds present were hydrolyzed (48). However, occa-
sional additional cleavage of Glu residues has been reported (49).
2.5.3. Molecular Mass
The mol mass of the enzyme is about 50,000 Da (46,47). The amino
acid analysis of the enzyme has been published (47).
2.5.4. pH Optimum
The enzyme has a pH optimum of about pH 7.0, with activity drop-
ping off rapidly above pH 9.0 and below pH 6.0 (46).
2.5.5. Assay
The assay is based on the hydrolysis of azocasein. A 2% azocasein
substrate in 50 mM phosphate buffer, pH 7.0, is used. Substrate (250 lxL)
is incubated at 25°C and 150 l.tL of enzyme then added. After incubation
for 20 min, the reaction is terminated by addition of 1.2 mL of 10% (w/
v) TCA, and the mixture allowed to stand for a further 15 min to allow
complete precipitation of the remaining azocasein. The mixture is
centrifuged (8000g) or filtered, and 1-2 mL of the supernatant are then
combined with 1.4 mLof 1.0MNaOH. The absorbance is read at 440 nm,
and the concentration of enzyme calculated. One unit of enzyme acti-
vity is defined as the amount of enzyme that produces an absorbance
change of 1.0 in a 1-cm path length cuvet under the above conditions.
2.5.6. Stability
The enzyme is fully active in 2M urea (48). It is stable between pH
7.0 and 9.5, but is rapidly inactivated outside these values (46). The
enzyme is stable at temperatures up to 40°C in the absence of Ca 2+. The