Peptide Production 281Chymotrypsin is a basic protein, and hence, it sticks easily to glass.
Therefore, dilute solutions of the enzyme should be avoided (2).
2.1.7. Inhibitors
The most commonly used serine protease inhibitors are DFP and
PMSF (Section 2.1.1.). PMSF is preferred, since it is much less toxic
than DFP. PMSF is prepared as a stock solution (1M) in a dry solvent,
such as propan-2-o/or methanol (stable for months at 4°C). It is added
to the enzyme solution with vigorous stirring to a final concentration
of 1 mM. It has a half-life of about 1 h in aqueous solution. DFP (highly
toxic !) is prepared as a 500 mM stock solution in propan-2-ol and added
to a final concentration of 0.1 mM. It also has a half-life of 1 h in
aqueous solution. Other low-mol-mass inhibitors include chymostatin
(mol mass 605 Da), a microbial amino acid aldehyde, active at 10-100
gM (11), and tosyl phenylalanyl chloromethyl ketone (TPCK) (12),
active at 10-100 gM. TPCK reacts with histidine 57 involved in the
catalytic triad shown in Fig. 1. High-mol-mass inhibitors include
aprotinin, o~ 1 antitrypsin, and o~ macroglobulin, and they are all effective
at equimolar concentrations with chymotrypsin. The enzyme is also
completely inhibited in the presence of 10 mM Cu 2÷ and Hg 2÷, and
partially inhibited by 1 mM Zn 2÷. Chymotrypsin that has been com-
pletely inhibited by Cu a÷ can be reactivated by the addition of Ca a÷ or
Versene (13).2.2. Clostripain (EC 3.4.22.8)
2.2.1. General Information
Clostripain (clostridiopeptidase B) is a sulfhydryl protease isolated
from culture filtrates of Clostridium histolyticum (14,15). Sulfhydryl
proteases are all characterized by a thiol group at the active site that is
essential for proteolytic activity.
2.2.2. Specificity
The enzyme has amidase and esterase activity as well as protease
activity. The enzyme primarily cleaves proteins at the C-terminal side
of arginine residues, i.e.,--Arg--X--bonds, although --Lys--X--
bonds are also cleaved, but at a much slower rate (14). Thus, under
conditions of controlled hydrolysis, the enzyme may be used to cause
cleavage effectively at arginine residues only (16,17).