Peptide Production 293
Oxalate, citrate, and phosphate also inhibit the enzyme, so phosphate
buffers should be avoided. Mercuric chloride and silver nitrate at 5
mM also cause complete inactivation. The enzyme is also inhibited by
o~ 2 macroglobulin and diethyl pyrocarbonate. The enzyme is not inhib-
ited by DFP, TPCK, soyabean trypsin inhibitors, cysteine, or sodium
cyanide (62, 66).
2.10. Trypsin (EC 3.4.21.4)
2.10.1. General Information
Trypsin is a serine protease that is formed by cleavage of the inac-
tive precursor trypsinogen. Activation is achieved by limited proteolytic
cleavage at a single peptide bond, Lys6-Ile7, near the N terminus of the
zymogen. The activation process is catalyzed by a variety of enzymes,
including enterokinase, mold proteases, and trypsin itself. The com-
mercial form of the enzyme is from bovine pancreas (67).
2.10.2. Specificity
Trypsin is a highly specific endopeptidase whose protease activity
is restricted to the positively charged side chains lysine and arginine
(67,68). Cleavage occurs C terminal to these residues. The enzyme
also hydrolyzes ester and amide linkages of synthetic derivatives of
these amino acids. No hydrolysis at X--Pro bonds occurs, and reduced
hydrolysis occurs ifXis preceded or followed by an acidic residue. Some
nonspecific cleavage may occur because of the presence of ~-trypsin in
some commercial preparations. Therefore, results from a tryptic digest
should be interpreted carefully, since the number of peptides may not add
up to the total number of arginine and lysine residues.
2.10.3. Molecular Mass
The bovine enzyme has a mol mass of approx 24,000 Da. The
enzyme has been sequenced, and the amino acid composition has been
reported (67).
2.10.4. pH Optimum
The enzyme has a pH optimum of 7-9 (67).
2.10.5. Assay
The assay is based on hydrolysis of N-benzoyl-L-arginine ethyl ester.
The substrate solution is 46.7 mM Tris-HC1, pH 8.0, 0.9 mM BAEE,
and 19 mM CaC12. Following the addition of enzyme, the change in