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P4850

Sigma-Aldrich

Proteinase K from Tritirachium album

buffered aqueous glycerol solution, for molecular biology, ≥800 units/mL

Synonym(s):
Endopeptidase K
CAS Number:
Enzyme Commission number:
MDL number:
NACRES:
NA.54

Quality Level

grade

for molecular biology

form

buffered aqueous glycerol solution

mol wt

28.93 kDa

concentration

≥10 mg/mL
≥800 units/mL

impurities

≤0.5 ppm DNA (PicoGreen® assay)

foreign activity

DNase, Nickase and RNase, none detected

storage temp.

2-8°C

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Application

Proteinase K from Tritirachium album has been used:
  • in bovine endometrial epithelial cells for herpes viral DNA extraction
  • for viral RNA extraction from nasal swabs
  • as a component of phase lock and direct PCR lysis buffer

The product has been used to study its pre-treatment effects on the silk fibroin. The aspects analysed in this study included the crystallographic properties of hydroxyapatite (HAp), and the microstructure and microhardness of the composites. The enzyme has also been used to facilitate the access of probes to rRNA using FISH techniques to detect pathogenic Staphylococcus aureus.
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 α-amino groups. The molecular weight of proteinase K from amino acid sequence is found to be 28,930 Da and from SDS-PAGE, it is found to be 28,500 Da. The optimum pH is between 7.5-9.0 and its isoelectric point is 8.9. Ca2+ (1-5 mM) is required for its activation. Proteinase K is inhibited by DIFP (diisopropylfluorophosphate) or PMSF (phenylmethylsulfonyl fluoride).
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% glycerol (v/v) containing 10 mM Tris-HCl, pH 7.5, with 1 mM calcium acetate.

Legal Information

PicoGreen is a registered trademark of Life Technologies

Pictograms

Health hazard

Signal Word

Danger

Hazard Statements

Precautionary Statements

Hazard Classifications

Resp. Sens. 1

Storage Class Code

10 - Combustible liquids

WGK Germany

WGK 1

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Personal Protective Equipment

dust mask type N95 (US),Eyeshields,Gloves

Certificate of Analysis

Certificate of Origin

Bovine herpes virus type 4 alters TNF-alpha and IL-8 profiles and impairs the survival of bovine endometrial epithelial cells
Chanrot M, et al.
Reproductive Biology, 17(3), 225-232 (2017)
Shin-Rong J Wu et al.
Scientific reports, 9(1), 11434-11434 (2019-08-09)
The highly conserved SNARE protein SEC22B mediates diverse and critical functions, including phagocytosis, cell growth, autophagy, and protein secretion. However, these characterizations have thus far been limited to in vitro work. Here, we expand our understanding of the role Sec22b...
M H Marana et al.
Fish & shellfish immunology, 105, 16-23 (2020-07-04)
Enteric redmouth disease (ERM), caused by the Gram negative enterobacterium Yersinia ruckeri, affects farming of salmonids, but vaccination against ERM confers a certain degree of protection dependent on the administration route. Recent studies on oral vaccination of rainbow trout suggest...
Three-dimensional porous network structure developed in hydroxyapatite-based nanocomposites containing enzyme pretreated silk fibroin.
Wang Li, et al.
Journal of Nanoparticle Research, 6, 91-98 (2004)
K D Jany et al.
Biological chemistry Hoppe-Seyler, 366(5), 485-492 (1985-05-01)
The molecular mass of proteinase K was determined by gel electrophoresis in the presence of sodium dodecyl sulfate and by active site labelling with diisopropyl fluorophosphate. Both methods indicate molecular masses in the range of 27 000-29 000 Da. These...

Articles

Enzymatic Assay of Proteinase K with Hemoglobin Substrate

Proteinase K (EC 3.4.21.64) activity can be measured spectrophotometrically using hemoglobin as the substrate. Proteinase K hydrolyzes hemoglobin denatured with urea, and liberates Folin-postive amino acids and peptides. One unit will hydrolyze hemoglobin to produce color equivalent to 1.0 μmol of tyrosine per minute at pH 7.5 at 37 °C (color by Folin & Ciocalteu's Phenol Reagent).

Protocols

Enzymatic Assay of Proteinase K (EC 3.4.21.64)

Proteinase K (EC 3.4.21.64) activity can be measured spectrophotometrically using hemoglobin as the substrate. Proteinase K hydrolyzes hemoglobin denatured with urea, and liberates Folin-postive amino acids and peptides. One unit will hydrolyze hemoglobin to produce color equivalent to 1.0 μmol of tyrosine per minute at pH 7.5 at 37 °C (color by Folin & Ciocalteu's Phenol Reagent).

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