PROTEINASE K: CHARACTERISTICS, ENZYMATIC ACTIVITY, APPLICATIONS - BIOLOGY - 2025

The K proteinase is an enzyme belonging to the group of serine proteases, ie, it has in its middle a catalytically active amino acid serine and has the function of breaking the peptide bonds by hydrolysis. In turn, this enzyme belongs to the family of subtilisin proteins (peptidase S8).

Proteinase K has a molecular weight (MW) of 28,900 daltons and was isolated for the first time in 1974 in extracts of the fungus Engyodontium album, previously known as Tritirachium album Limber.

Molecular structure of Proteinase K. Source: Lykchiniadis

It has a high proteolytic capacity, demonstrated by being able to degrade the keratin present in the hair. The word keratin in English is spelled "keratin", hence it has been called "proteinase K".

Due to its high power to cleave native proteins, this enzyme is useful in various molecular biology techniques. It is primarily used to isolate and prepare high molecular weight (MW) nucleic acids.

Proteinase K works by releasing nuclear DNA, while destroying proteins and inactivates RNases and DNases, that is, it eliminates nucleases in DNA and RNA preparations.

On the other hand, it has been seen that proteinase K can hydrolyze some denatured native proteins, which has aroused the interest of researchers for its use in the study of prion proteins (PrPC).

However, despite their high proteolytic potency, there are proteins that are resistant to the action of proteinase K. Among them are some abnormal proteins called prions (PrPSc), associated with transmissible spongiform encephalopathies.

Proteinase K characteristics

Proteinase K has a tertiary structure made up of three layers, with a seven-chain β sheet interposed between two layers of helices. As it belongs to the S8 family of peptidases, it is characterized by having a catalytic triad in its active site, whose sequential order is (Asp, Her and Ser), which differentiates them from other families of peptidases.

This enzyme from the group of serine proteases is characterized by hydrolyzing the peptide bonds close to the carboxylic group of aliphatic and aromatic amino acids.

On the other hand, it is capable of acting in the presence of certain corrosive substances, such as sodium dodecyl sulfate (SDS), Tris-HCL and EDTA, which are used to help the denaturation of proteins, causing them to lose their native structure.

This is a preliminary step in preparing proteins for the electrophoresis technique. The pH range at which proteinase K acts is quite wide (2.0 to 12.0), with an optimal pH between 7.5 to 12.0, and its isoelectric point is 8.9. As can be seen, it is active against a very wide pH range.

Another characteristic that stands out in proteinase K is its stability in the presence of high temperatures (50 - 60 ° C).

Enzymatic activity

Proteinase K requires the presence of the calcium ion, although this does not affect its activity, if it is essential to maintain its stability.

For proteinase K to fully digest the substrate, a contact time of approximately 5 minutes to 2 hours is necessary.

However, in this sense, Daza et al. Compared the purity of the DNA obtained at various times of exposure against proteinase K, and concluded that a prolonged incubation (up to 24 h) significantly improves the quality of the DNA.

However, in relation to the concentration of the proteinase K enzyme used in the different protocols, it can be said that it is very varied.

It can be used from very low concentrations (5 µg / ml) to concentrations of 500 µg / ml. But the most common working concentrations range from 50–100μg / ml, especially for protein digestion and nuclease inactivation. Although for the treatment of tissues a concentration of 2 mg / ml is required.

Applications

Its applications are very broad and can be summarized as follows:

-It is used in the digestion of proteins and DNA extraction by various methods such as: salting-out, PK-SDS, cetyl-trimethyl ammonium bromide (CTAB), modified potassium acetate and extraction with sodium iodide.

-Inactivation of nucleases (RNases and DNases).

-In the in situ hybridization technique (HIS), to aid the release of nucleic acid, in addition to eliminating undesirable proteins.

-Modification of proteins.

-At the research level, in various studies.

Advantages of proteinase K

Several comparative studies have been carried out between DNA extraction techniques that use Proteinase K, with others that do not use it and all conclude that there are greater benefits when using the enzyme. Advantages include the following:

-DNA of high molecular weight, of high quality and purity is obtained.

-The extracted DNA is stable for up to 3 months.

The extracted DNA can be used in the following techniques: Southern blot, polymerase chain reaction (PCR), electrophoresis, among others.

Proteinase K resistant proteins

Various investigations have concluded that prions (abnormal toxic PrPSc proteins) differ from PrPC (native) proteins by being resistant to the action of proteinase K, while PrPCs are sensitive to its action.

Other authors have described that in the structure of PrPSc there are sensitive portions and others resistant to proteinase K. However, both parts are equally toxic and infectious.

On the other hand, Bastian et al. In 1987 isolated 4 proteins of 28, 30, 66 and 76 kda from a species of Spiroplasma mirum. All were found to be resistant to the action of proteinase K and also had a cross-reaction with some prions.

It is known that this species can cause cataracts and important neurological damage and due to Bastian's scientific findings, among other investigations, an attempt has been made to link this microorganism with transmissible spongiform encephalopathies.

However, the etiology of this degenerative neurological pathology continues to be attributed to prions today.

In this sense, Butler et al. In 1991 identified and characterized a class of protein resistant to proteinase K of 40 kda from two strains of Mycoplasma hyorhinis. This pathogen affects pigs, infecting their tissues, but in this case there was no cross-reaction with the prions tested.

More research is required to elucidate many unknowns in this regard.

References

1. Bastian F, Jennings R, and Gardner W. 1987. Antiserum to scrapie-associated fibril protein cross-reacts with Spiroplasma miru m fibril proteins. J. Clin. Microbiol. 25: 2430-2431.
2. Daza C, Guillen J, Rey J, Ruiz V. Evaluation of a DNA extraction and purification method from formaldehyde-fixed muscle tissue of unidentified cadavers. Med Magazine, 2014; 22 (1): 42-49,
3. Butler G, Kotani H, Kong L, Frick M, Evancho S, Stanbridge E, And Mcgarrity G. Identification and Characterization of Proteinase K-Resistant Proteins in Members of the Class Mollicutes. Infection and Immunity, 1991, 59 (3): 1037-1042
4. López M, Rivera M, Viettri M, Lares M, Morocoima A, Herrera L, et al. Comparison of two Trypanosoma cruzi DNA extraction protocols grown in axenic medium. Rev. Peru. Med. Exp. Public Health 2014; 31 (2): 222-227. Available at: scielo.org
5. Jiménez G, Villalobos M, Jiménez E and Palma W. Determination of the effectiveness of five DNA extraction protocols from paraffinized material for molecular studies. Rev Méd Univ Costa Rica. 2007; 1 (1): 10-19.