Протеиназа K

Описание

Proteinase K is a proteolytic enzyme (a serine protease) naturally found in the mold Tritirachium album. In solution, it is stable over a pH range of 4.0–12.5 with an optimum of pH 8.0, and a temperature range of 25°C–65°C. The enzyme has two binding sites for Ca++, though in the absence of this divalent cation some catalytic activity is retained. Maximum proteinase K activity is observed with the inclusion of 1 mM Ca++ in the reaction buffer. Proteinase K digestion is routinely performed at 50°C, and occasionally in the presence of EDTA to inhibit labile, Mg++-dependent nucleases. Proteinase K is prepared commonly as a 20 mg/mL stock solution in sterile water (stable for 1 year at ?20°C) or in a solution of 50 mM Tris, pH 8.0, 1 mM CaCl2 (stable for months at 4°C). It is generally used at a working concentration of up to 50 μg/mL in any of a number of buffer formulations, including those that contain as much as 0.5% SDS[1].

Химические свойства

Powder beige

Использование

A stable and highly reactive serine protease; used for protein and nucleic acid isolation.Proteinase K is used in the purification of RNA and DNA from tissues or cell lines. It finds application in protein modification, determination of enzyme localization and inactivation of RNases/DNases during nucleic acid extraction. It is also used in mitochondria isolation and removal of endotoxins bound to cationic proteins like lysozyme and ribonuclease A. Further, it is involved in the treatment of paraffin embedded tissue sections in order to expose antigen binding sites for antibody labeling.

Определение

Proteinase K cleaves peptide bonds next to the carboxyl-terminal of aromatic amino acids, hydrophobic amino acids and sulfuric amino acids, within the polypeptide chain. Hence Proteinase K can act as an endoprotease on a wide range of protein substrates. Because of its proteolytic preferences, it is a member of the subtilisin group of serine proteases. The K in Proteinase K refers to its ability to digest Keratin protein found in hairs, nails and hooves. Proteinase K is an essential reagent in molecular biology labs because it is useful in isolating DNA and RNA molecules by degrading and inactivating proteins. Fungus Tritirachium album Limber naturally produces proteinase K.

Общее описание

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.

Механизм действия

Proteinase K is a serine protease that cleaves peptide bonds in proteins. Its mechanism of action involves several steps:
Binding: Proteinase K first binds to the protein or nucleic acid substrate through non-specific hydrophobic interactions.
Activation: Once bound, the enzyme undergoes an activation step where a catalytic serine residue is activated by a histidine residue and a water molecule. This leads to the forming of an active site that can cleave peptide bonds.
Cleavage: The active site of proteinase K cleaves the peptide bond on the carboxylic acid side of the amino acid residues that contain aromatic, aliphatic or hydrophobic amino acid residues. The enzyme can also cleave peptide bonds on the amide side of glycine residues.
Product release: After cleavage, the products of the reaction (i.e., peptides) are released from the enzyme.
The mechanism of action of proteinase K is similar to other serine proteases. Still, it is notable for its ability to function under harsh conditions, such as high temperatures and in the presence of detergents.