284 Sweeney and Walker
2.3.6. Stability
The enzyme is active in 0.1% SDS. The lyophilized enzyme is stable
indefinitely at-20°C and for at least 6 mo at 4°C. The enzyme rapidly
autolyzes if stored at room temperature near its pH optimum of 8.8.
However, in solution at pH 5.0, proteolytic activity is slight, and solu-
tions at this pH can be used at room temperature with minimal autoly-
sis. The addition of 0.1 mM Ca 2+ (or 0.1 mM Mg 2+ to a lesser extent)
stabilizes the native form of the enzyme against thermal denaturation,
even if a destabilizing ion (e.g., Cu 2÷) is present (32). Elastase retains
activity in 2M urea. Note that the enzyme sticks to glass, and therefore,
plasticware should be used where possible. The addition of Triton X-
100 to incubation solutions at 0.05% helps minimize this adhesion (31).
2.3.7. Inhibitors
Since it is a serine protease, elastase is inhibited by DFP and PMSF
(see Section 2.1.7. for practical details). However, it is not inhibited by
TPCK (see Section 2.1.7.). It is inhibited by elastinal, a low-mol-mass
(mol mass 513 Da) amino acid aldehyde at 10-100 laM, and also by ot 2
macroglobulin (at equimolar concentrations with elastase) and by ct 1
antitrypsin. Various sulfonyl fluorides and p-dinitrophenyl diethyl
phosphate also inhibit the enzyme (33). The activity of elastase has
been shown to be appreciably affected by salts. Sodium chloride (50-
70 mM) gave 50% inhibition (31), and similar effects were observed
with potassium chloride, ammonium sulfate, and sodium cyanide (31).
Copper sulfate (10 mM) gave 50% inhibition, but millimolar concen-
trations of zinc, manganese, cobalt, magnesium, or calcium had no
effect (34,35). The enzyme is unaffected by EDTA (35-37).
2.4. Endoproteinase Arg-C (EC 3.4.21.40)
2.4.1. General Information
The enzyme is prepared from mouse submaxillary glands (38,39).
It is also known as submaxillary protease.
2.4.2. Specificity
This enzyme cleaves specifically C terminal to arginine residues,
although cleavage at some residues does not always go to completion
(40). Other workers have found occasional bonds that are resistant to
cleavage. For example, when the immunoglobulin ~ chain was used as