Peptide Production 295
morpholine acetate (pH 8.5). For pepsin, 5% acetic acid or 10 mM HC1
is used. Ammonium acetate (0.1M) can be used in the pH range 4.5-
5.5. If samples are to be taken for SDS gel electrophoresis, potassium
ions should be avoided, since these precipitate with dodecyl sulfate
and can aggregate peptides in the electrophoresis wells.3.2. Condition of the Substrate
The physical state of the substrate is of great importance in deter-
mining the degree of digestion. Cleavage of the native protein will
often result in large proteolytic fragments, since only these cleavage
sites on the surface of the protein will be accessible to the enzyme.
However, if it is required that the enzymic digestion go to completion,
then the substrate protein should be denatured and disulfide bridges
reduced, thus making all potential cleavage sites accessible to the enzyme.
Denaturation can be achieved by boiling the protein solution, precipi-
tation in 5% trichloroacetic acid, or treatment in 8M urea or 6M
guanidium hydrochloride. The reduction and alkylation of sulfhydryl
groups have been described in vol. 3 of this series, Chapter 7. Dena-
tured proteins are often insoluble, and although proteolytic enzymes
will cleave insoluble substrates, it is desirable that the precipitate be
as finely divided as possible to allow maximum access of the enzyme.
This can be achieved by dissolving the precipitate in 8M urea and then
removing the urea by dialysis against buffer, or dissolving the precipi-
tate at low pH and then slowly titrating back to the required pH. Many
of the enzymes described above remain active in the presence of SDS
or urea, and therefore, these agents may be used to denature the protein
substrate (which remains soluble) and then enzyme may be added
directly. For glycoproteins, the sugar backbone can sterically hinder
enzyme access to the polypeptide backbone and severely reduce diges-
tion. Deglycosylation prior to enzymic cleavage can be achieved using
appropriate enzymes.
3.3. Reaction Conditions
When attempting to achieve complete digestion, an enzyme to sub-
strate ratio of 1-2% (w/w) is commonly used, usually at 37°C for 2-
4 h. However, longer times (24-48 h) with repeated addition of enzyme
may have to be used with particularly resistant substrates. In these
cases, one should be aware of the increased possibility of generating