P5568

Proteinase K from Tritirachium album

≥500 units/mL, buffered aqueous glycerol solution

• Synonym(s): Endopeptidase K
• CAS Number: 39450-01-6
• Enzyme Commission number: 3.4.21.64 (BRENDA, IUBMB)
• MDL number: MFCD00132129
• UNSPSC Code: 12352204
• eCl@ss: 32160410
• NACRES: NA.54

Properties

• biological source: microbial (T.album; T. ALBUM)
• Quality Level: 200
• form: buffered aqueous glycerol solution
• mol wt: 28.93 kDa
• concentration: ≥10 mg/mL; ≥500 units/mL
• technique(s): DNA extraction: suitable
• storage temp.: 2-8°C

Description

General description

Proteinase K, an extracellular endopeptidase is synthesized by the mold, Tritirachium album Limber. Proteinase K belongs to a new subfamily of the subtilisins. It is a 277 amino acid protein and is characterized with an unhydrolyzed protein chain and autolyzed polypeptide chains.

Application

Proteinase K from Tritirachium album has been used:

• to break down cardiac muscle during histopathology studies
• during the digestion of HEK-293 cells

Proteinase K from Tritirachium album has been used in in situ detection of DNA fragmentation and in proteolysis experiments to measure the structural flexibility of interleukin 1ra (IL-1ra).

The enzyme from Sigma has been used in the digestion of sealed cytosolic side out ER vesicles. It has been used to deproteinize dissected brain and/or whole pupae sections of honey bee prior to in situ hybridisation. This was done during the study of neuropeptide Y-like signaling, and nutritionally-mediated gene expression and behaviour in the honey bee.

Useful for the proteolytic inactivation of nucleases during the isolation of DNA and RNA.
Removes endotoxins that bind to cationic proteins such as lysozyme and ribonuclease A.
Reported useful for the isolation of hepatic, yeast, and mung bean mitochondria
Determination of enzyme localization on membranes
Treatment of paraffin embedded tissue sections to expose antigen binding sites for antibody labeling.
Digestion of proteins from brain tissue samples for prions in Transmissible Spongiform Encephalopathies (TSE) research.

Biochem/physiol Actions

Proteinase K has a broad specificity and degrades many proteins even in the native state. It mainly cleaves the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha-amino groups. The optimum pH is between 7.5-9.0 and the isoelectric point is 8.9. Ca²⁺ (1-5 mM) is required for activation. Proteinase K is inhibited by DIFP or PMSF.

Proteinase K is a stable and highly reactive serine protease. Evidence from crystal and molecular structure studies indicates the enzyme belongs to the subtilisin family with an active-site catalytic triad (Asp³⁹-His⁶⁹-Ser²²⁴). It is stable in a broad range of environments: pH, buffer salts, detergents (SDS), and temperature. In the presence of 0.1-0.5% SDS, proteinase K retains activity and will digest a variety of proteins and nucleases in DNA preparations without compromising the integrity of the isolated DNA.

Unit Definition

One unit will hydrolyze urea-denatured hemoglobin to produce color equivalent to 1.0 μmole of tyrosine per min at pH 7.5 at 37 °C (color by Folin-Ciocalteu reagent).

Physical form

Solution in 40% (v/v) glycerol containing 10 mM Tris-HCl, pH 7.5, with 1 mM calcium acetate.

Preparation Note

Proteinase K in solution is stable over a pH range of 4.0-12.5 (optimum pH 8.0), and is also stable over the temperature range of 25°C to 65°C during use. At pH 8.0, solutions will be stable for at least 12 months at 4°C. 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.

Safety Information

• pictograms: GHS08
• signalword: Danger
• hcodes: H334
• pcodes: P261 - P284 - P501
• Hazard Classifications: Resp. Sens. 1
• Storage Class: 10 - Combustible liquids
• wgk_germany: WGK 1
• flash_point_f: Not applicable
• flash_point_c: Not applicable
• ppe: Eyeshields, Faceshields, Gloves, type ABEK (EN14387) respirator filter