Proteinase K

Proteinase K is a typical member of the subtilisin family of proteases (S8). The amino acid sequence is obtained by Edman degradation and gene sequence. The polypeptide chain consists of 278 amino acids with a molecular weight of 28,930 and a pI of 8.9. Proteinase K is an endopeptidase and carries catalytic triads Asp39, His69, Ser224 at its active site. Full enzyme activity requires the presence of calcium ions. Maximum activity is in the pH range of 7.5-12.0. When one unit hydrolyzes 1μmol of Ac-Tyr-OEt per minute in Tris-HCl buffer at pH 9.3 and 30°C, the specific activity is about 300 Umg21. Peptidyl chloromethane and Hg21 act as inhibitors, remaining 7% Hg21 remains active even in excess. Proteinase K dissolves at low concentrations (~0.01mg/ml), but is more stable at concentrations above 1.0mg/ml.

Figure 1. Structure of Proteinase K.

Inhibitor

Protease K is a serine protease, so its effect can be inhibited with benzylsulfonyl fluoride, which is an inhibitor of serine protease.

Figure 2. Chemical structure of benzylsulfonyl fluoride.

Functions

It is worth noting that proteinase K is usually used in pre-hybridization processing in in situ hybridization technology. It has the effect of digesting and surrounding the target DNA protein to increase the chance of the probe binding to the target nucleic acid and improve the hybridization signal. But proteinase K's When the concentration is too high, the digestion time is too long, or the incubation temperature is too high, it will damage the structure of the cells to some extent, leading to the shedding of tissue sections and the disappearance of the nucleus, which will affect the results of hybridization. EDTA buffer can replace the role of proteinase K. Solve the above problems and achieve the ideal dyeing effect.

Features

Proteinase K is a serine protease with a wide range of cleavage activities. It cleaves carboxy-terminal peptide bonds of aliphatic and aromatic amino acids. This enzyme has been purified to remove RNase and DNase activity. Since proteinase K is stable in urea and SDS and has the ability to degrade natural proteins, it is widely used, including the preparation of chromosomal DNA for pulse electrophoresis, Western blotting, and the removal of nucleases in DNA and RNA preparation. The general working concentration of proteinase K is 50-100 μg / ml. It is active over a wide pH range (pH 4-12.5).

Recommended reaction buffer: 50mM Tris-HCl (pH7.5), 10mM CaCl₂.

Proteinase K preparation standard: 20mg/ml proteinase K preparation standard: Add 200mg of proteinase K to 9.5ml of water and shake gently until proteinase K is completely dissolved. Do not vortex to mix. Add water to make up to 10ml, then aliquot and store at -20°C.

Storage of proteinase K: Store in a refrigerator at -20°C, avoid repeated freeze-thaw cycles, and effectively guarantee 12 months.

The incubation temperature is 55 to 65°C, the ideal incubation temperature is 58°C, the incubation time is 15 minutes to 48 hours; the ideal incubation time is 2 hours.

Applications

Proteinase K has been widely used in leather, fur, silk, medicine, food and brewing. Hair removal and softening in the leather industry have made extensive use of proteases, saving time and improving labor hygiene. Protease can also be used for silk degumming, meat tenderization, and wine clarification. It can be used clinically, such as pepsin to treat indigestion, bronchitis with acid protease, vasculitis with elastase, and purification of surgical purulent wounds with trypsin and chymotrypsin, and interthoracic serosa. Treatment of adhesions. Enzyme-containing washing powder is a new product in detergents. It contains alkaline protease, which can remove blood stains and protein dirt on clothing. However, be careful not to contact the skin when using it, so as not to damage the protein on the skin surface, causing allergies such as rash and eczema.

Reference

1. Betzel C, et al. Structure of the complex of proteinase K with a substrate analogue hexapeptide inhibitor at 2.2-A resolution. J. Biol. Chem. 1993,268 (21): 15854–8.