Peptide Production 285
substrate, cleavage occurred at most arginine residues, but one Arg--
Val bond and one Arg--Arg bond were resistant to cleavage (41). The
specificity of the enzyme appears to broaden during long incubations,
so it is best to use the enzyme at high ratios for short periods, e.g., 1:50
dilution for 2 h at 37°C (40).
2.4.3. Molecular Mass
The enzyme is a serine protease and has a mol mass of about 28,000
Da (42). The amino acid composition is also known (43).
2.4.4. pH Optimum
The enzyme has a pH optimum between pH 7.5 and 8.5 (42).
2.4.5. Assay
The assay is based on the hydrolysis of Na-p-tosyl-L-arginine methyl
ester (TAME). The substrate solution is 0.8M TAME in 0.1M phos-
phate, pH 8.0. The reaction is started by the addition of 0.1 mL of
enzyme (suitably diluted in redistilled water) to 2.9 mL of substrate
solution. The change in absorbance with time at 247 nm is monitored,
and enzyme activity calculated. Using this assay, the enzyme is sup-
plied with a specific activity of 100-250 U/mg where 1 U is defined
as the amount of enzyme that hydrolyzes 1 pmol of TAME/min.
2.4.6. Stability
The enzyme retains 90% activity after 1 h at 25°C in 0.1% SDS. It
is unstable at low pH, and the enzyme is denatured in 4M urea. Solu-
tions of the enzyme can be stored at-20°C without loss of activity. The
enzyme loses specificity during long incubations; digestions proceed
more rapidly if the substrate is first denatured. The purified form of the
enzyme (enzyme A) undergoes autolysis on storage at pH 8.0 over-
night, resulting in the formation of enzyme D, formed by the cleavage
of a fragment of mol mass 3000-5000 Da from enzyme A (44,45).
Commercially available enzyme is a mixture of enzymes A and D.
2.4. 7. Inhibitors
The enzyme is inhibited by low pH (<2), DFP, TLCK (43) (see
Section 2.1.7. for practical details), and a 2 macroglobulin when used
in equimolar ratios with the enzyme. The enzyme has also been shown
to be inhibited at 1 mM concentrations by Hg 2+ (100% inhibition),
Cu 2+ (80%), Zn 2+ (65%), Cd 2+ (50%), and Fe 2+ (50%) (42).