P5568

Proteinase K aus Tritirachium album

≥500 units/mL, buffered aqueous glycerol solution

• Synonym(e): Endopeptidase K
• CAS-Nummer: 39450-01-6
• UNSPSC Code: 12352204
• eCl@ss: 32160410
• NACRES: NA.54
• EG-Nummer: 3.4.21.64 (BRENDA, IUBMB)
• MDL number: MFCD00132129
• Biological source: microbial (T.album; T. ALBUM)
• Concentration: ≥10 mg/mL, ≥500 units/mL

Eigenschaften

• biological source: microbial (T.album; T. ALBUM)
• Quality Segment: 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

Beschreibung

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

Nützlich zur proteolytischen Inaktivierung von Nukleasen bei der Isolierung von DNA und RNA.
Entfernt Endotoxine, die an kationische Proteine wie Lysozym und Ribonuklease A binden.
Berichten zufolge nützlich für die Isolierung von Leber-, Hefe- und Mungbohnen-Mitochondrien
Bestimmung der Enzymlokalisierung auf Membranen
Behandlung von Paraffinschnitten zur Freilegung von Antigenbindungsstellen zur Antikörpermarkierung.
Verdauung von Proteinen aus Hirngewebeproben für Prionen in der TSE-Forschung.

Proteolytische Deaktivierung von Nucleasen während der Isolation von DNA und RNA

Proteinase K from Tritirachium album has been used:

• to break down cardiac muscle during histopathology studies[1]
• during the digestion of HEK-293 cells[2]

The enzyme from Sigma has been used in the digestion of sealed cytosolic side out ER vesicles.[2] 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.[3]

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).

Biochem/physiol Actions

Proteinase K ist eine stabile und hochreaktive Serinprotease. Ergebnisse von Kristall- und Molekularstrukturstudien lassen darauf schließen, dass das Enzym zur Subtilisin-Familie mit einer katalytischen Triade (Asp³⁹-His⁶⁹-Ser²²⁴) im aktiven Zentrum gehört. Es ist in einer Reihe von Umgebungen stabil: pH, Puffersalze, Detergenzien (SDS) und Temperatur. In Gegenwart von 0,1–0,5 % SDS bleibt Proteinase K aktiv und verdaut zahlreiche Proteine und Nukleasen in DNA-Präparationen, ohne die Integrität der isolierten DNA zu beeinträchtigen.

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.[4][5] The optimum pH is between 7.5-9.0[4] and the isoelectric point is 8.9.[4] Ca²⁺ (1-5 mM) is required for activation. Proteinase K is inhibited by DIFP or PMSF.

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.[4]

Other Notes

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).

Sicherheitshinweise

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