308 Sweeney and Walker
Stock solutions of the enzyme are normally prepared in water at 10-
25 mg/mL and stored at -20°C. However, proteinase K may be stored
in 50 mM Tris-HC1, pH 8.0, containing 1 mM CaC12 and is stable for
at least 12 mo at 4°C (28).
- Inhibitors
Proteinase K appears to be a serine protease, being inhibited by
diisopropyl phosphofluoridate (DFP or Dip F) and phenylmethane-
sulfonyl fluoride (PMSF) (1). PMSF is normally added to a final con-
centration of 5 mM from a 100 mM dry stock solution in DMSO or
isopropyl alcohol. It is not, however, inhibited by tosyl lysyl
chloromethyl ketone (TLCK or Tos-Lys-CH2CI ), an inhibitor of trypsin-
like serine proteases, or tosyl phenylalanyl chloromethyl ketone (TPCK
or Tos-Phe-CH2C1), an inhibitor of chymotrypsin-like serine proteases.
It is not inhibited by sulfhydryl reagents.
- Experimental Procedures
3.1. Nucleic Acid Isolation
A stock solution of the enzyme is diluted typically to 50-200 lag/mL
in the solution to be digested, normally in the pH range 7.5-8.0 and at
37°C. Incubation times vary depending on the nature of the experi-
ment, but can range from 30 min to 18 h. Although inhibitors of pro-
teinase K are known (see Section 2.7.) they are generally not used as
the proteinase K is usually denatured by subsequent phenol extrac-
tions. Nucleic acid purification protocols involving the use of protein-
ase K are described in detail in earlier volumes of this series (2-8).
3.2. Protein Studies
When studying protein translocation, membrane topology, and enzyme
compartmentation, proteinase K is added to membrane preparations
(or in vitro translation systems supplemented with membrane vesicles)
to degrade those portions of the proteins that are accessible. Thus,
cytoplasmic proteins are completely degraded, completely translo-
cated proteins should be fully protected, whereas transmembrane pro-
teins and those that cross the membrane more than once will be degraded
to yield discrete fragments (11-19). Following proteolytic digestion,
proteins are analyzed by SDS gel electrophoresis and patterns com-
pared before and after proteolysis to identify protected polypeptides.
