Peptide Production 287
presence of 5 mM CaC12 appears to stabilize the enzyme somewhat
with residual activity still remaining at 60°C (46). The enzyme is
stable at 4°C, stored dry. A solution in redistilled water may be used
for 1 wk at maximum if stored at 4°C (44). Aqueous solutions of the
enzyme are stable up to 1 mo at-20°C. Freeze/thawing of enzyme
solutions is not recommended. The enzyme is stable in 0.01% SDS,
0.1M guanidine hydrochloride, and 10% (v/v) acetonitrile (44).
2.5. 7. Inhibitors
Being a metalloenzyme, the enzyme is inhibited by chelating agents,
such as EDTA, and 1,10-phenanthrolene (46,47). Activity can be restored
by the addition of zinc or cobalt ions, but not by adding calcium or
magnesium ions (46,47). The enzyme is not inhibited by iodoacetic acid,
PCMB, dithiothreitol, PMSF, or soyabean trypsin inhibitor (46,47).
- Endoproteinase Glu-C (EC 3.4.21.9)
2.6.1. General Information
The enzyme is purified from the culture filtrates of Staphylococcus
aureus strain V8 (50-52). The enzyme has also been named staphylo-
coccal protease and V8 protease.
2.6.2. Specificity
The enzyme cleaves C terminal to Asp and Glu residues in protein
substrates when used in phosphate buffer, pH 7.8. However, when
used in ammonium buffers (ammonium bicarbonate, pH 7.8, or ammo-
nium acetate, pH 4.0), cleavage is restricted to Glu residues only.
--Glu--X-- or Asx--X bonds are not cleaved ifX is Pro or S-carboxy-
methylcysteine (53). IfX is a bulky hydrophobic residue, cleavage is
slow. In addition to the expected cleavages at Asp and Glu, a number
of other workers have noted a number of nonspecific cleavages, often
of Ser--X bonds (54-56).
2.6.3. Molecular Mass
The enzyme is a serine protease (see Section 2.1.1.) with a mol mass
of 12,000 Da (50). The amino acid composition has been published (50).
2.6.4. pH Optimum
The protease is active in the pH range 3.5-9, and exhibits maximum
proteolytic activity at pH 4.0 and 7.8 with hemoglobin as substrate (55).