Peptide Production 297
then taken, a final addition of enzyme made, and incubation continued
for a further 8 h, when a final sample is taken. Samples are either
immediately frozen for later analysis by reverse-phase HPLC (see vol.
1 of this series, Chapter 5) or dried directly onto a cellulose TLC plate
(which effectively stops the reaction), and when fully loaded, the plate
is run in an appropriate solvent, such as butanol/pyridine/acetic acid/
water (15:12:3:10) and peptides identified by spraying with ninhydrin
or fluorescamine (see vol. 1). Whichever method is used, the time of
digestion that gives an unchanging pattern of peptides is used for the
large-scale digestion.
- Termination of the Digestion
The simplest method of termination is freezing followed by lyophiliza-
tion. Such a procedure will retain biological activity of limited proteolysis
products where necessary. Where total digestion has taken place, samples
may be easily (and more quickly) rotary evaporated to dryness, since
one is not attempting to retain any tertiary structures in the proteolysis
products. Alternatively, the pH digest may be altered (normally by the
addition of acid) to a pH value at which the enzyme is no longer active,
or the sample boiled rapidly to denature the enzyme. The reaction can
also be stopped by the addition of a specific inhibitor for the enzyme.
In general, serine proteases are inhibited by the addition of PMSF to
1 mM. (PMSF is prepared as a stable 1M stock solution in propan-2-
ol and added to the reaction with vigorous mixing. PMSF cannot be
prepared as an aqueous stock solution, since it is rapidly hydrolyzed
in water with a half-life of about 1 h. Metalloenzymes can be inhibited
by the addition of disodium EDTA to a concentration of 10 mM (from
a 1M stock solution), and sulfhydryl proteases inhibited by the addi-
tion of iodoacetic acid to a concentration just in excess of that of the
sulfhydryl reagent added to activate the enzyme.
- Termination of the Digestion
3.6. Typical Digestion Conditions
The conditions given below are suitable for producing total diges-
tion, e.g., suitable for peptide mapping.
3.6.1. Chymotrypsin
Dissolve the protein substrate at a concentration up to 10 mg/mL in
0.1M ammonium bicarbonate (pH 8.0). Add enzyme solution to give
a ratio of 2% (w/w), and incubate for 4 h at 37°C (10).