Proteinase K 307
2.5. Assay
The assay is based on the hydrolysis ofN-acetyl-L-tyrosine ethylester
(ATEE). One unit will hydrolyze 1 ktmol of ATEE per minute at pH 9.0
and 30°C in Tris-HC1 buffer, pH 8.0. Hydrolysis is monitored at 237
nm. Using this assay, the enzyme is usually supplied with a specific
activity of approx 300 U/mg. Other suppliers use an assay whereby 1
Anson unit liberates 1 mmol of Folin-positive amino acids per minute
at pH 7.5 and 35°C using hemoglobin as substrate. In this case, the
enzyme is supplied with a specific activity of approx 20-30 U/mg of
protein. Alternatively, an assay based on the hydrolysis of Suc-(AIa)3-
NH-Np can be used. The assay is performed in 50 mM Tris-HC1, 5 mM
CaC12 (pH 8.0) using an enzyme concentration of 5-8 ~g/mL and 1
mM substrate in a final volume of 1 mL. The reaction mixture is
incubated for 60 min at 20°C and then stopped by the addition of 0.2
mL of glacial acetic acid. After a further 15 min, the absorbance at 410
nm of free nitroaniline is measured. Using this assay, the enzyme
shows a specific activity of approx 13 U/mg. One unit of activity is the
amount of enzyme that liberates 1 mmol ofp-nitroaniline per minute
of reaction (26,2 7).
2.6. Stability
Proteinase K is an unusually stable enzyme. Studies have shown
that the enzymic activity of proteinase K is controlled by calcium.
Hence, proteinase K is normally used in the presence of approx 2.5-
5 mM CaC12. If calcium is removed by EDTA, followed by gel filtra-
tion, enzymic activity drops to 20% of its original value within 6 h,
without autolysis. Addition of excess calcium results in an immediate
rise of residual activity to 28% of the original value but full activity is
not restored (27). The activity of the calcium bound enzyme is at a
maximum at 37°C. However, the enzyme demonstrates a broad tem-
perature profile with >80% of its maximum activity being retained
between 20 and 60°C. Autolysis of the enzyme occurs during sample
preparation for SDS gel electrophoresis and at low concentrations
(0.01 mg/mL) in aqueous solution. It does not occur at concentrations
1 mg/mL (26). Proteinase K is fully active in 0.5% (w/v) SDS and is
frequently used in the presence of SDS (2-8). It is also active in 1%
(w/v) Triton X-100.