by EM Science ®
For the isolation of native, high molecular weight nucleic acids: DNA, RNA
A highly active stable endopeptidase with a broad spectrum of action was isolated by E. Mercks Darmstadt Biochemical Research Department in 1970 from a culture filtrate of the fungus, Tritirachium album Limer. This fungus is able to grow on keratin (e.g. wool, horn particles) as the sole source of carbon and nitrogen. The isolated protease was, therefore, given the K designation.
Following isolation and chromatographic purification, Proteinase K becomes a homogenous crystallizable protein.
Mass: 30,000 daltons
Isoelectric point (isoelectric focusing): pI 8.9
pH optimum (denatured hemoglobin as substrate): pH 7.5 -12.00
Isolation of native high molecular weight nucleic acids (DNA,RNA) Proteinase K preparations are free of ribonuclease (Rnase) and deoxyribonuclease (Dnase). After 60 minutes incubation of radio labeled adenovirus-DNA with Proteinase K at 37ºC, only 0.0005% of the original radioactivity was found in the acid-soluble portion. Dnases and Rnases from most microorganisms and mammalian cells are rapidly inactivated by Proteinase K, particularly in the presence of 0.5-1.0% of SDS. Addition of Proteinase K during the cell digestion enables the isolation of native undamaged high molecular DNA or RNA. This method has been established as a standard method, as documented in numerous publications and textbooks. Analysis of membrane structure Proteinase K is very useful in specific modification of proteins and glycoproteins on cell membranes. Structural investigations on proteins Because of the cleavage specificity of Proteinase K, characteristic fragments of proteins are obtained which are useful in determining the structure and function of proteins, particularly enzymes. Specificity Proteinase K cleaves peptide bonds mostly after the carboxyl group of N-substituted hydrophobic aliphatic and aromatic amino acids, as shown by specificity trials with amino acid-4-nitroanilides. Thus, it shows similarities with alkaline Aspergillus proteases. However, unlike the latter, Proteinase K also cleaves peptide amides, comparable to the alkaline serine-proteases from Bacillus species. The specificity of ester cleavage is also high. Inhibition Proteinase K belongs to the group of serine proteases with an easily esterifed serine fragment at the active center and, as with other proteases in this group, e.g. trypsin, chymotrypsin, is inactivated by diisopropylfluorophosphate or phenlymethane sulfonyl fluoride. Metallic ion complexing agents, e.g. chelate formers such as EDTA and sulfhydryl reagents, have no effect on the activity of Proteinase K. Activity Towards denatured haemoglobin as substrate, this highly purified Proteinase K demonstrates a specific activity of 32 mAnson units/mg and is, therefore, 6 times as active, weight for weight, as the Streptomyces protease pronase and about 3 times as active as beef trypsin. Stability The enzyme is stable for a number of years in the solid form when stored dry in an airtight container at approximately 4ºC. Aqueous solutions containing Ca²+ (1.5 mmol/L) are stable for several weeks at pH 4.0 - 11.5 at room temperature and are resistant to autolysis. Compared with denaturation agents such as dodecyl sodium salt (SDS) or urea, Proteinase K has become available in solution form stable for many months even at room temperature. This convenient, ready-to-use form omits the steps needed to bring a specified quantity into solution and avoids the potential hazard of inadvertent contact.
Specifications: Activity (Haemoglobin acc. to Anson, pH 7.5, 25oC) >/= 30 mAnson units per mg
Foreign activities: Ribonuclease not detectable Deoxyribonuclease not detectable
Applications: Highly active protease with low specificity. Aqueous solutions containing Ca²+ (1.5 mmoL/L) are stable for weeks at pH 4.0-11.5, even at room temperature. A remarkable property of Proteinase k is its ability to rapidly deactivate native proteins, particularly enzymes by hydrolysis. Addition of either 0.5-1% of sodium dodecyl sulphate or 1-4 mmoL/L of urea increases the activity, because the substrates are more easily attacked when denatured. Proteinase K itself is denatured much more slowly by these agents. Proteinase K is chromatographically purifed and lyophilized in bottles ready for reconstitution in TRIS HCl.
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