306 Sweeney and Walkermethod (8-10). Because of its broad specificity and its ability to func-
tion in the presence of detergent, proteinase K has also been used
extensively to study membrane protein topology and protein translo-
cation across membranes (11-19). These various uses of proteinase K
are further discussed in Section 3. The use of proteinase K to degrade
polypeptides to produce a mixture of oligopeptides 2-6 residues in
length that is suitable for sequence analysis by GC-MS has also been
reported (20).
The chromosomal gene for proteinase K has been characterized and
expression of the eDNA in E. coli has been achieved (21). The crystal
and molecular structure of the enzyme have also been determined by
X-ray diffraction studies to 0.15 nm resolution (22).
- Enzyme Data
2.1. Specificity
The enzyme is an endoprotease having a fairly broad specificity but
with a preference for cleavage of peptide bonds C-terminal to aliphatic
and aromatic amino acids, especially alanine (1). Although commonly
used for its broad specificity, the enzyme has also been used to gener-
ate a single cleavage in a native protein. Lebherz et al. used proteinase
K to cleave an isoenzyme of creatine kinase at a single position between
two alanine residues (11). X-ray and model building studies on the speci-
ficity of the active site of proteinase K have been described (23).
2.2. Molecular Mass
Early work based on gel filtration studies suggested a mol mass of
18,500 + 500 Da (1). Later studies using SDS gel electrophoresis gave
values of 27,000 Da (24). The gene sequence was later determined and
gave a true value for the mol mass of proteinase K of 28,930 Da (25).
2.3. Isoelectric Point
The isoelectric point of the protein occurs at pH 8.9 (1).
2.4. pH Optimum
The pH activity curve of proteinase K, determined for the hydroly-
sis of urea-denatured hemoglobin, showed optimal activity in the pH
range 7.5-12.0 (1). However, the enzyme is normally used in pH range
7.5-9.0 (1,26,27).