P5568

Proteinasa K from Tritirachium album

≥500 units/mL, buffered aqueous glycerol solution

• Sinónimos: Endopeptidasa K
• Número de CAS: 39450-01-6
• Comisión internacional de enzimas: 3.4.21.64 (BRENDA, IUBMB)
• MDL number: MFCD00132129
• UNSPSC Code: 12352204
• eCl@ss: 32160410
• NACRES: NA.54

Propiedades

• 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

Descripción

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

Útil para la inactivación proteolítica de nucleasas durante el aislamiento de ADN y ARN.
Elimina las endotoxinas que se unen a las proteínas catiónicas como la lisozima y la ribonucleasa A.
Se ha comunicado que es útil para el aislamiento de mitocondrias hepáticas, de levadura y de frijol mung
Determinación de la localización de las enzimas en las membranas
Tratamiento de cortes de tejido incluidos en parafina para exponer los sitios de unión de antígenos para el marcado con anticuerpos.
Digestión de proteínas de muestras de tejido cerebral para investigación de los priones en las encefalopatías espongiformes transmisibles (EET).

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.

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.

La proteinasa K es una serín proteasa estable y muy reactiva. La evidencia procedente de estudios de la estructura cristalina y molecular indica que la enzima pertenece a la familia subtilisina con una tríada catalítica en el sitio activo (ASP³⁹-HIS⁶⁹-ser²²⁴). Es estable en una amplia variedad de entornos: pH, sales amortiguadoras, detergentes (SDS) y temperatura. En presencia de SDS al 0,1 - 0,5 %, la proteinasa K conserva su actividad y digerirá una variedad de proteínas y nucleasas en preparaciones de ADN sin comprometer la integridad del ADN aislado.

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.

Información de seguridad

• 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