316 Winder and Walker
- Denaturing Agents
Eighty percent of activity remains after incubation with 6M urea at
25°C for 1 h (3). The enzyme also retains its activity for extended
periods in 1% SDS (]6). Its stability to denaturing agents makes CPY
very suitable for studying proteins that have inaccessible or poorly
accessible C-termini under normal (nondenaturing) conditions (16).
- Denaturing Agents
2.8. Inhibitors
The enzyme is a serine protease, and is therefore inhibited by DFP
(17) and PMSF (7), but is not inhibited by soy bean and lima bean
trypsin inhibitors. It is inhibited by p-hydroxymercuribenzoate, prob-
ably by reaction with a single thiol group thought to be located near or
at the substrate binding site (3,8). Enzyme activity is affected by metal
ions: Cu 2+, Ag ÷, and Hg 2+ result in complete loss of activity at 10-aM,
1 mM Cu +, Mg 2÷, Ca 2+, Ba 2+, Cr 2÷, Mn 2+, Fe 2+, Fe 3+, Co 2+, or Ni 2+
results in loss of more than 50% activity (7). Certain organic solvents,
such as DMF and ethanol, apparently show competitive inhibition (5).
EDTA and o-phenanthroline have no effect on enzyme activity (10).
2.9. Additional Comments
Commercial preparations may contain free amino acids owing to
autolysis, which should be removed before use. Repeated freeze-thaw-
ing of solutions of the enzyme or prolonged storage at room tempera-
ture can also lead to autolysis and the liberation of free amino acids.
For ammonium sulfate suspensions, centrifuge and wash the pellet in
saturated ammonium sulfate before dissolving in buffer. Alternatively,
the dissolved enzyme can be dialyzed against the pyridine acetate
buffer used for digestion (see Section 3.).
- Experimental Procedure
3.1. Determination of C-Terminal Sequences
in Peptides and Proteins (16)
Dissolve 20 nmol of the protein to be studied in 200 gL of digestion
buffer (0.1M pyridine acetate, 0.1 mM norleucine, pH 5.6, containing
1% SDS). The norleucine is used as an internal standard for amino acid
analysis to allow for compensation of any handling losses or sampling
errors. Heat the solution to 60°C for 20 min to denature the protein.
After cooling, remove a 25-gL aliquot as the zero time sample. Add 2