Proteinase K exhibits broad substrate specificity. It degrades many proteins in the native state even in the presence of detergents. Proteinase K is isolated from a fungus, Engyodontium album (formerly Tritirachium album) which is able to grow on keratin. Consequently, proteinase K is able to digest native keratin (hair), hence, the name "Proteinase K".1 The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity.2,3,4
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Proteinase K is a stable S8 family serine alkaline protease containing two disulfide bridges and one free Cys near His at the active site.24
28,930 Da (amino acid sequence)21
28,500 Da (SDS-PAGE)22
pH range: 7.5 to 12.0 (urea-denatured hemoglobin as substrate), but most often used in pH range 7.5-9.0.2,3
Temperature profile: maximum activity at 37 °C (activity is >80% of maximum between 20 to 60 °C)3
Extinction coefficient: E1% = 14.2 (280 nm, 10 mM NaCl and 5 mM CaCl2, pH 8.0)2
Activators: 1-5 mM Ca2+ is required for activation. When calcium is removed from the enzyme (addition of EDTA) 25% of the catalytic activity is lost. However, if the EDTA-Ca2+ complex is removed from the enzyme solution by gel filtration, a total of 80% of the enzyme activity is lost and only a small activation will occur upon addition of excess Ca2+ to the Ca2+-free enzyme.23
Proteinase K is active in 1% TRITON™ X-100 and fully active in 0.5% (w/v) SDS. SDS and urea will denature protein substrates resulting in increased digestion rates. Proteinase K itself is denatured much more slowly by these agents.3,19,20
Unit Definition: One unit will hydrolyze urea-denatured hemoglobin to produce color equivalent to 1.0 mmole (181 mg) of tyrosine per minute at pH 7.5 at 37 °C.
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Proteinase K is inhibited by DIFP or PMSF (the latter used at final concentration 5 mM).3 It is partly inactivated, but not inhibited, by EDTA (see Activators). Proteinase K is not inhibited by iodoacetic acid, the trypsin-specific inhibitor TLCK, the chymotrypsin-specific inhibitor TPCK, and p-chloromercuribenzoate.
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- Mitochondria Isolation
- Protein digestion for nucleic acid purification Proteinase K is frequently used in molecular biology applications to digest unwanted proteins, such as nucleases from DNA or RNA preparations from microorganisms, cultured cells, and plants.5-11 The enzyme is typically used at 50-200 µg/ml in nucleic acid preparations at pH 7.5-8.0 and 37 °C. Incubation times vary from 30 minutes to 18 hours. Proteinase K is usually denatured by subsequent phenol extractions, although it can autodigest during long incubations.3
- Proteinase K has been used to remove endotoxins bound to cationic proteins such as lysozyme and ribonuclease A.12
- Determination of enzyme localization on membranes13
- Treatment of paraffin embedded tissue sections to expose antigen binding sites for antibody labeling14
- Remove nucleases for in situ hybridization.15
- Research on prions in Transmissible Spongiform Encephalopathies (TSE) and proposed diagnostic tests utilize Proteinase K digestion of proteins from brain tissue samples.16,17
- Protease footprinting by Proteinase K digestion can reveal protein-protein surface interactions.18
Proteinase K is soluble in water (1 mg/ml), yielding a clear colorless solution.
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Solubility and Solution Stability
Sigma recommends storage at –20 °C lyophilized for powders. The product is stable for at least 2 years.
A Proteinase K solution is stable over a broad pH range (4.0-12.5, optimum pH 8.0) and is also stable over the temperature range of 25 to 65 °C during use. At pH 8.0, solutions will be stable for at least 12 months at 4 °C.3 At pH 4-11.5, solutions containing Ca2+ (1-6 mM) are expected to be stable for several weeks. An 80% ammonium sulfate suspension stored at 4 °C is stable for at least 12 months.2
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||Add to Cart
||Proteinase K from Tritirachium album lyophilized powder, =30 units/mg protein
||Proteinase K from Tritirachium album =30 units/mg protein (biuret), lyophilized powder
||Proteinase K from Tritirachium album 3-15 unit/mg solid, lyophilized powder
||Proteinase K from Tritirachium album =500 units/mL, buffered aqueous glycerol solution
||Proteinase K from Tritirachium album for molecular biology, >30 units/mg protein, lyophilized powder
||Proteinase K from Tritirachium album for molecular biology, >800 units/mL, buffered aqueous glycerol solution, DNAse, Nickase and RNAse, none detected
Sigma-Aldrich is a large-scale manufacturer of Proteinase K. We supply custom formulated preparations to diagnostic manufacturing and biotechnology customers. Inquire with your local SAFC representative.
- Betzel, C., Three Dimensional Structure of Proteinase K at 0.15 nm Resolution. Eur. J. Biochem., 178, 155-171 (1988).
- Ebeling, W., et al., Proteinase K from Tritirachium album Linder, Eur. J. Biochem., 47, 91 (1974).
- Enzymes of Molecular Biology, vol. 16, Burrell, M.M., ed. Humana Press (Totowa, NJ: 1993), p. 307. Kraus, E., and Femfert, U., Proteinase K from the Mold Tritirachium album Limber, Specificity and Mode of Action. Z. Physiol. Chem., 357, 937 (1976).
- Lizardi, P.M., and Engelberg, A., Rapid Isolation of RNA Using Proteinase K and Sodium Perchlorate. Anal. Biochem., 98, 116 (1979).
- Gross-Bellard, et al., Isolation of High Molecular Weight DNA from Mammalian Cells, Eur. J. Biochem., 36, 32-38 (1973).
- Molecular Cloning: A Laboratory Handbook, 2nd ed., Sambrook et al., eds., Cold Spring Harbor Press (Cold Spring Harbor, NY: 1989) p. 1.61 and p. B.16.
- Kasche, V., et al., A Two-step Procedure for Quantitative Isolation of Pure Double-strand DNA from Animal Tissues and Cell Cultures. Prep. Biochem., 11, 233 (1981).
- Hansen, J.N., Isolation of Higher Molecular Weight DNA from Bacillus cereus T Using Proteinase K. Prep. Biochem., 4, 473 (1974).
- Holm, C., et al., A Rapid, Efficient Method for Isolating DNA from Yeast. Gene, 42, 169 (1986).
- La Claire, J.W., and Herrin, D.L., Co-isolation of High-Quality DNA and RNA from Coenocytic Green Algae. Plant Mol. Biol. Reporter, 15, 263 (1997).
- Petsch, P., et al., Proteinase K Digestion of Proteins Improves Detection of Bacterial Endotoxins by the Limulus Amebocyte Assay: Application for Endotoxin removal from Cationic Proteins. Anal. Biochem., 259, 42 (1998).
- Brdiczyka, D., and Krebs, W., Localization of Enzymes by Means of Proteases. Biochem. Biophys. Acta, 297, 203 (1973).
- Short, B.G., et al., Automated Double Labeling of Proliferation and Apoptosis in Glutathione S-transferase-positive Hepatocytes in Rats. J. Histochemistry and Cytochemistry, 45, 1299 (1997).
- Angerer, L.M., et al., Identification of Tissue-Specific Gene Expression by in-situ Hybridization. Methods in Enzymology, 152, 649 (1987).
- Sakaguchi, S., et al., Accumulation of Proteinase K-Resistant Prion Protein (PrP) is Restricted by the Expression Level of Normal PrP in Mice Inoculated with a Mouse-Adapted strain of the Creutzfeldt-Jakob Disease Agent. J. Virology, 69, 7586 (1995).
- Bennion, B.J., and Daggett, V., Protein Conformation and Diagnostic Tests: the Prion Protein. Clinical Chemistry, 48, 2105 (2002).
- Hori, R., and Carey, M., Protease Footprinting Analysis of Ternary Complex Formation by Human TFIIA. J. Biol. Chem., 272, 1180 (1997).
- Hilz, H., et al., Stimulation of Proteinase K action by Denaturing Agents: Application to the Isolation of Nucleic Acids and the degradation of “Masked” Proteins. Eur. J. Biochem., 56, 103 (1975).
- Methods of Enzymatic Analysis, 3rd Edition, Bergmeyer, H.U., ed., Academic Press (New York, NY: 1983) vol. 2, p. 299.
- Jany, K.D., et al., Amino Acid Sequence of Proteinase K from the Mold, Tritirachium album Linder. Proteinase K; a Subtilisin-related Enzyme with Disulfide Bonds. FEBS Letters, 199, 139 (1986).
- Jany, K.D., and Mayer, B., Proteinase K from Tritirachium album linder, Molecular Mass and Sequence Around the Active Serine Residue. Biol. Chem. Hoppe-Seyler, 366, 485 (1985).
- Bajorath, J., et al., The Enzymatic Efficiency of Proteinase K is Controlled by Calcium. Eur. J. Biochem., 176, 441-447 (1988).
- IUBMB Enzyme Nomenclature: http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/64.html
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