Proteinase K is commonly recommended for fast digestion. Endogenous RNAses and DNAses are deactivated during the earliest phases of nucleic acid separation.
What Is a Protease?
In biotechnology and molecular biology laboratories, proteases are essential reagents. In order to inactivate protein contaminants in our prepared samples, we frequently utilize proteases.
Proteases are enzymes that catalyze the breakdown of proteins into their constituent amino acids. As a nucleophile, these enzymes use an amino residue or an activated water molecule to break down a protein.
An amino acid atom that transfers an electron to another molecule that causes modifications in another amino acid is known as a nucleophile.
What Is Proteinase K?
Proteinase K is a serine protease endopeptidase that belongs to the subtilisin family. Proteinase K is used in molecular biology laboratories to degrade and inactivate proteins during the production of DNA or RNA samples.
Serine proteases (also known as serine endopeptidases) break peptide bonds in proteins. Serine is the nucleophilic amino acid in the active site of the enzyme. They can be present in eukaryotes and prokaryotes alike.
Proteinase K is a serine protease. Serine proteases are distinguished by the presence of a catalytic triad.
The catalytic triad is a cluster of three amino acids that form the catalytic center and consists of serine, aspartic acid, and histidine amino acids, which can often vary but all of these enzymes have a nucleophile serine and the same catalytic mechanism.
Chemical Structure of Proteinase K
Proteinase K, isolated from the Tritirachium album, is utilized for protease digestion during the synthesis of DNA and RNA. It is a serine protease with a wide cleavage specificity.
Proteinase K is a member of the subtilisin proteinase family. The amino acid sequence was generated by Edman degradation from the gene sequencing. The polypeptide chain contains 278 amino acids, a molecular mass of 28 930, and a pI of 8.9.
Proteinase K is an endopeptidase with the catalytic triad Asp39, His69, and Ser224 at its active site. The presence of calcium ions is required for full enzymatic action.
Proteinase K has the catalytic triad consisting of Ser 224, His 69, and Asp 39. The substrate recognition sites are two peptide chains, 99-104 and 132-136. It also has a free Cys 73 near His 69.
Proteinase K is made up of five cysteines. Four cysteine residues create two disulfide linkages (34-124 and 179-248, respectively), and one is located beneath one of the catalytic triads.
Proteinase K frequently binds two calcium ions, both of which are necessary for complete enzymatic action.
What Is Mechanism of Action of Proteinase K?
Serine proteases share a nucleophile serine and the same catalytic mechanism, according to homology studies and investigations of the active site.
The catalytic mechanism of all serine proteases is commonly represented by chymotrypsin. The reason for this is that chymotrypsin’s structure and mechanism are well understood.
Chymotrypsin is a protein cleaver that catalyzes the breakage of peptide bonds around an aromatic amino acid’s carboxyl group. Ser 195, His 57, and Asp 102 comprise the catalytic triad of chymotrypsin.
Chymotrypsin’s step-by-step catalytic mechanism:
- The active Serine (Ser 195) attacks the peptide’s carbonyl carbon atom with a nucleophilic assault.
- A tetrahedral intermediate is formed as a result of the nucleophilic assault.
- The tetrahedral intermediate undergoes transformation into an acyl-enzyme intermediate. The first amine product is replaced by the solvent water.
- The initial amine product exits and is replaced by the solvent water.
- A second tetrahedral intermediate is formed in the process.
- The final reaction results in the synthesis of the active enzyme and the release of the second amine product.
Proteinase K Vs. Protease: What’s The Difference?
Proteinase K and protease are different in that proteinase K is used to digest proteins and remove contaminants from nucleic acid preparations, whereas protease is used for biological processes such as protein digestion, catabolism, and cell signaling.
Protein cleaving enzymes such as proteinase K and protease are two examples. Proteinase K and protease, in other words, can break peptide bonds in proteins.
What Is Function of Proteinase K?
Proteinase K is active in a wide range of circumstances, making it a great choice for use with diverse buffers and cell lysis conditions. This versatility allows it to be used in a wide range of applications.
For example, chromosomal DNA preparation for pulsed-field gel electrophoresis, protein fingerprinting, and, more often, nuclease removal from DNA and RNA preparations.
Prior to DNA or RNA separation, mechanical or detergent treatment of cells destroys cellular ultrastructure and releases nucleases such as DNAse or RNAse, which can degrade DNA/RNA before isolation.
Proteinase K digests all proteins and nucleases, allowing undamaged DNA/RNA molecules to be isolated.
How To Use Proteinase K?
Proteinase K is typically used at a concentration of 50–100 g/ml. One Anson Unit (AnsonU) is defined as the quantity of proteinase K required to release Folin-positive amino acids and peptides.
It is equivalent to 1 mol tyrosine under assay conditions in 1 minute using hemoglobin as a substrate, under assay conditions.
Following proteinase K digestion of cellular lysates, phenol extractions are required to isolate high molecular weight DNA. Proteinase K is removed throughout the procedure. Phenol extraction also allows for phase separation of lipids, proteins, and DNA.
Because of their hydrophobic nature, lipids remain in the organic phase, whereas DNA dwells in the aqueous phase. The organic/aqueous interphase is where protein (including proteinase K) remains. The aqueous layer is then used to extract clean DNA.
Is Proteinase K Stable at Room Temperature?
Proteinase K is thermostable due to the presence of calcium, which is not required for catalysis. Proteinase K can hydrolyze a variety of peptide bonds under a variety of circumstances, including those that would render other proteases inert.
Proteinase K, for example, is active over a wide variety of temperatures, including up to 65°C and a pH range of 7.5 to 12.0.
Proteinase K activity is somewhat increased in the presence of chaotropic substances such as SDS (up to 1-2%) and Urea (4 M).. As a result, Proteinase K stays active in the presence of EDTA. Proteinase K is best stored at -20°C, however, it can also be kept at 4°C.
What Makes Protease K Temperature Resistant?
Proteinase K is a protein, however, it is extremely resistant to heat denaturation. Chaotropic salts and detergents (e.g., SDS, sarkosyl) will continue to work in their presence as long as the temperature/concentration does not exceed a certain temperature/concentration threshold (up to 65°C).
Because of its strong temperature tolerance, it is frequently employed at higher temperatures (more than 50°C), where many of the nucleases that would chew up your DNA would be inactivated.
What Is the Best Temperature to Use and Activate Proteinase K?
Up to the 65°C maximum limits, proteinase K activity rises with temperature. As a result, Proteinase K is most active at temperatures between 50°C and 65°C.
As contaminated proteins and nucleases unfold at higher temperatures, Proteinase K’s capacity to break down these contaminant and undesirable proteins improve.
Temperatures lower than those stated above may be required for certain procedures and unique methods. As a result, even at temperatures as low as 37°C, Proteinase K is active.
This adaptability might come in handy for the specific or specialized procedures you’re working on, especially if they need distinct operational settings.
What Is Role of Proteinase K In DNA Extraction Protocol?
Proteinase K finds its most important application in DNA extraction in mammals. To isolate DNA from mammalian cells and leucocytes, the first step is to lyse the cells and spin them down to collect a pellet.
Detergent (SDS) is added to the solution to break the nuclear membrane. After being digested by proteinase K, both histone and non-histone proteins release DNA. In addition, ribonuclease in the solution is digested and inactivated by proteinase K.
Following proteinase K digestion, phenol extractions separate DNA from lipids and proteins. The organic phase is occupied by hydrophobic lipids, whereas the aqueous phase is occupied by DNA.
The organic/aqueous interphase is where protein (including proteinase K) remains. The aqueous layer is used to extract DNA. Proteinase K must be removed from high molecular weight DNA, which necessitates phenol extractions.
Why Is Proteinase K Used to Isolate Mammalian DNA?
All kinds of material, including dangerous nucleases, are released into the lysate after the addition of a detergent. A protease, in this instance proteinase K, is utilized to preserve DNA against destruction.
Proteinase K is an excellent choice since it is very stable at higher temperatures and has a broad range of targets.
What Is Use of Proteinase K In Next-Generation Sequencing (NGS) And Microarray Technology Applications?
When extracting DNA and RNA from yeast, bacteria, mammalian cell lysates, and plant cell lysates, nucleases are inactivated. PCR product cloning efficiency is improved.
Sample preparation for accelerator mass spectrometry measurement of DNA adduct levels.Enzyme cocktails inactivation in ribonuclease protection experiments
To improve RNA yields from primary breast tumors for microarray investigations, it was added to extraction techniques.
How Proteinase K Is Used in Molecular Biology?
It is used to identify Bovine Spongiform Encephalopathy proteins that are resistant to proteolytic degradation.
This enzyme digests tissues (denatures proteins) as an alternative sample preparation method for quantitative analysis utilizing liquid chromatography-tandem mass spectrometry.
These enzymes particularly change cell surface proteins in order to investigate membrane architecture for protein localization. This enzyme generates protein fragments for use in functional research.
What Is Use of Proteinse K In PCR?
Proteinase K, recombinant, PCR grade, efficiently digests native proteins. As a result, it can be utilized to inactivate endogenous RNases and DNases quickly during nucleic acid separation.
Proteinase K is particularly well suited to the separation of natural RNA and DNA from tissues and cell lines because of this feature.
By activating a bacterial autolytic factor, the enzyme also induces cell lysis. Proteinase K is also used to analyze membrane architecture on cell surfaces by altering proteins and glycoproteins.
The solution is ideal for separating PCR and RT-PCR templates since it is checked for the presence of RNases and DNases and is essentially DNA-free.
Proteinase K may also be used to remove cellular debris from colony lifts and to prepare tissue slices for in situ hybridization to guarantee effective probe penetration.
What Role Does Proteinase K Play in Prion Disorders And TSE?
Proteinase K helps distinguish between normal PrP C (prion protein/protease-resistant protein) and PrPSC (disease-causing isoform). PrPC and PrPSC have the same molecular weight, however, PrPSC is proteinase K resistant.
Proteinase K is used to treat samples that may include both, removing PrPC and converting PrPSC to PrPRES, which has a smaller molecular weight and may be pelleted, making it easier to differentiate.
What Is Role Does Proteinase K Play in Cell Lysis?
Proteinase K can be used in the lysis phase of cell lysis, especially for downstream DNA isolation and purification, by digesting surface proteins.
When it comes time to resuspend and lyse the nuclei in a solution containing proteinase K later in the operation, the proteinase K will aid in the digestion of proteins that would otherwise destroy the sample.
How Proteinase K Can Be Inactivated?
Proteinase K is temperature stable over a wide range of temperatures. Proteinase K is therefore inactivated at high temperatures by heating it to 95°C for 10 minutes.
It’s worth noting that Proteinase K can’t be totally deactivated by heat. However, additional washing stages in protocols for research such as nucleic acid separation and PCR sample preparation will eliminate the residual enzymes swiftly and effectively.
Protease inhibitors like PMSF and AEBSF can also be used to inactivate the virus.
Frequently Asked Questions (FAQs)
Can Proteinase K Be Used to Isolate RNA?
Proteinase K degrades and inactivates even trace levels of ribonuclease in the presence of substrate during RNA separation. The extra phenol, chloroform, and isopropanol precipitation stage decreases protein contamination and makes the final pellet simpler to dissolve.
Why Is Proteinse K Digestion Carried Out At 50°C?
When the temperature is raised to 50°C, certain proteins unfold, making it simpler for Proteinase K to destroy them. The enzyme is stable and denaturing chemicals such as SDS and urea considerably boost its activity.
Where Does Proteinase K Cleaves ?
Proteinase K cleaves peptide bonds between N-substituted hydrophobic, aliphatic, and aromatic amino acids and their carboxyl groups. It may also break down peptide amides.
Is Proteinase K a Good Choice for RNA Isolation?
In the presence of substrate, proteinase K degrades and inactivates even minute levels of ribonuclease during RNA isolation. Protein contamination is reduced, and the final pellet is simpler to re-dissolve, thanks to the extra phenol-chloroform and isopropanol precipitation stage.
What Is the Shelf Life of Proteinase K?
Because it is a highly stable product, it has a shelf life of 12 months when stored in a dry area at 4–8°C. The enzyme’s activity and stability are not harmed by short-term storage at room temperature.
What Is Proteinase K Lyophilization?
Lyophilization is a method of “freeze-drying” proteins or other materials. The substance is frozen at lower pressure, which allows water to sublimate and preserves the activity while also extending the shelf life.
In many circumstances, lyophilization minimizes storage temperature limits, and metered aliquots reduce material loss.
Proteinase K is a fantastic and versatile enzyme. Proteinase K is a protein, however, it is very resistant to denaturation by heat, chaotropic salts, and detergents (e.g., SDS, sarkosyl) and will continue to operate in its presence as long as the temperature/concentration does not exceed a certain temperature/concentration (up to 65°C).
The good news is that it can be kept and used later. It has a wide range of uses, including cell lysis, molecular biology, and DNA and RNA extraction. It’s also utilized to lyse cells. Temperature changes might also deactivate it.