Enzyme Activity
Activated by calcium (1–5 mM), the enzyme digests proteins preferentially after hydrophobic amino acids (aliphatic, aromatic and other hydrophobic amino acids). Although calcium ions do not affect the enzyme activity, they do contribute to its stability. Proteins will be completely digested, if the incubation time is long and the protease concentration high enough. Upon removal of the calcium ions, the stability of the enzyme is reduced, but the proteolytic activity remains. Proteinase K has two binding sites for Ca2+, which are located close to the active center, but are not directly involved in the catalytic mechanism. The residual activity is sufficient to digest proteins, which usually contaminate nucleic acid preparations. Therefore, the digestion with Proteinase K for the purification of nucleic acids is usually performed in the presence of EDTA (inhibition of calcium-dependent enzymes such as nucleases).
Proteinase K is also stable over a wide pH range (4–12), with a pH optimum of pH 8.0. An elevation of the reaction temperature from 37 °C to 50–60 °C may increase the activity several times, like the addition of 0.5–1% sodium dodecyl sulfate (SDS) or Guanidinium chloride (3 M), Guanidinium thiocyanate (1 M) and urea (4 M). Temperatures above 65 °C, trichloroacetic acid (TCA) or the serine protease-inhibitors AEBSF, PMSF or DFP inhibit the activity. Proteinase K will not be inhibited by Guanidinium chloride, Guanidinium thiocyanate, urea, Sarkosyl, Triton X-100, Tween 20, SDS, citrate, iodoacetic acid, EDTA or, interestingly, by other serine protease inhibitors like Nα-Tosyl-Lys Chloromethyl Ketone (TLCK) and Nα-Tosyl-Phe Chloromethyl Ketone (TPCK).
Protease K activity in commonly used buffers
| Buffer (pH = 8.0, 50°C, 1.25 µg/mL protease K, 15 min incubation) | Proteinase K activity (%) |
|---|---|
| 30 mM Tris·Cl | 100 |
| 30 mM Tris·Cl; 30 mM EDTA; 5% Tween 20; 0.5% Triton X-100; 800 mM GuHCl | 313 |
| 36 mM Tris·Cl; 36 mM EDTA; 5% Tween 20; 0.36% Triton X-100; 735 mM GuHCl | 301 |
| 10 mM Tris·Cl; 25 mM EDTA; 100 mM NaCl; 0.5% SDS | 128 |
| 10 mM Tris·Cl; 100 mM EDTA; 20 mM NaCl; 1% Sarkosyl | 74 |
| 10 mM Tris·Cl; 50 mM KCl; 1.5 mM MgCl2; 0.45% Tween 20; 0.5% Triton X-100 | 106 |
| 10 mM Tris·Cl; 100 mM EDTA; 0.5% SDS | 120 |
| 30 mM Tris·Cl; 10 mM EDTA; 1% SDS | 203 |
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