296 Sweeney and Walkerproteolytic fragments from the enzyme being used. Bacterial growth
and protein cleavage by released enzymes can also be a problem in
extended hydrolysis. In all cases, therefore, it is wise to carry out a
preliminary small-scale trial digestion to determine the minimum diges-
tion time necessary to generate a stable peptide pattern. For limited
proteolysis experiments, much lower enzyme-to-substrate ratios (0.1%
or less), lower temperatures (10-20°C), and shorter times (15-30 min)
may all be used in conjunction to reduce the amount of proteolysis that
occurs. Again, it is necessary to carry out preliminary small-scale trial
digests to determine the optimum conditions. Conditions for trial
digests are described in Section 3.6.
3.4. Monitoring of Digestion
3.4.1. Limited Digestion
Conditions for controlled proteolysis are far more difficult to pre-
dict than the rather obvious extreme conditions used for total proteoly-
sis and are highly dependent on the experimental conditions chosen by
the user. Since relatively large peptides are to be generated, the pro-
duction of peptides in trial digests is best monitored by SDS gel elec-
trophoresis (see vol. 1 of this series, Chapter 6). Following addition of
the enzyme (1:100 to 1:1000 [w/w]) to either the native or denatured
protein at 30-37°C, remove samples containing about 5 l.tg of protein
at regular intervals (say every 15 min for 4 h) and add to SDS sample
buffer. Since many proteolytic enzymes (e.g., clostripain, thermolysin,
endoproteinase Glu-C, elastase, papain, and chymotrypsin) are still
active in 0.1% SDS, it is essential to boil the sample immediately to
inactivate the protease. When all samples have been taken, they are run
on the gel and the time of digestion that gives the required pattern of
peptides used for the large-scale digest. If digestion was too rapid, a
repeat trial should be carried out at a lower enzyme-to-substrate ratio
and/or a lower temperature. However, if cleavage of the native protein
is being attempted, considerably higher enzyme-to-substrate ratios
may be needed, possibly as high as 1:1 (w/w).
3.4.2. Total Digestion
Following addition of the enzyme (1:10 to 1:100 [w/w]) at 37°C,
remove samples containing about 5 ktg of protein at 0, 1, 2, 4, and 8 h.
A further addition of protease is made (to replace enzyme lost by
autolysis), and incubation continued overnight. A further sample is