What is Proteinase K? What Does Proteinase K Do in DNA Extraction?

Proteinase K is an important enzyme used in molecular biology labs for digesting and removing proteins from nucleic acid preparations. This proteolytic enzyme has key properties that make it exceptionally useful for research applications. In this post, we’ll explore what exactly proteinase K is, how it functions, and its many uses in the lab.

What is Proteinase K?

Proteinase K is a broad-spectrum serine protease isolated from the fungus Tritirachium album. It can rapidly digest proteins into smaller peptide fragments and amino acids.

The name “proteinase K” comes from its ability to digest keratin, a tough structural protein found in hair, nails, feathers, etc. The fungus Tritirachium album secretes proteinase K when keratin is provided as its sole nitrogen source.

What are the facts about Proteinase K?

Some key facts about proteinase K:

• Classification: Belongs to the peptidase family S8 (subtilisin family)
• Catalytic Type: Serine protease
• Optimal pH:5 – 12.0
• Optimal Temperature:50°C – 65°C
• Molecular Weight:28,930 Da

Proteinase K contains a catalytic triad of three amino acids – aspartate, histidine, and serine. The serine acts as a nucleophile that cleaves peptide bonds. Calcium ions are important for full enzymatic activity.

How Does Proteinase K Work?

As a serine protease, proteinase K catalyzes the hydrolysis of peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids.

Its mechanism of action resembles that of other serine proteases like chymotrypsin and trypsin. Here are the key steps:

1. The enzyme’s catalytic serine initiates a nucleophilic attack on the peptide bond carbonyl carbon.
2. This forms a tetrahedral intermediate stabilized by hydrogen bonding.
3. The nitrogen-carbon bond then breaks, releasing the first product and forming an acyl-enzyme intermediate.
4. A water molecule deactivates the enzyme, releasing the second product.
5. This regenerates the active serine site for another round of catalysis.

Proteinase K cleaves proteins at interior peptide bonds as an endopeptidase. It does not have strict specificity but prefers large hydrophobic residues. This allows it to rapidly break down most proteins into smaller fragments.

What is Proteinase K Used For?

Proteinase K has become an essential reagent for molecular biology research. Here are some of its most common uses:

Digesting Contaminating Proteins

When isolating DNA or RNA from cells and tissues, proteinase K is used to digest away contaminating proteins. This removes impurities from nucleic acid preparations. Adding detergents like SDS helps expose proteins for enhanced digestion.

Inactivating Nucleases

Many nucleases that can degrade DNA and RNA are proteins themselves. Proteinase K rapidly inactivates these harmful nucleases by proteolytic cleavage. This protects isolated nucleic acids from degradation.

Tissue Digestion

Proteinase K can help dissociate cells and solubilize tissues, releasing nucleic acids for extraction. It’s included in tissue lysis buffers for DNA/RNA purification protocols.

Protein Analysis

The protease produces specific peptide fragments that provide information about protein structure. Proteinase K digestion patterns are useful for studying protein domains, modification sites, etc.

Inactivating Infectious Prions

As an endopeptidase, proteinase K can digest the misfolded prion proteins responsible for transmissible spongiform encephalopathies like Creutzfeldt-Jakob disease. It’s used to inactivate prion contamination.

DNA Sequencing

Some DNA sequencing methods use proteinase K to remove the DNA polymerase after sequence extension, preparing the DNA for electrophoresis.

In Situ Hybridization

The enzyme can gently permeabilize fixed cells/tissue for the hybridization of nucleotide probes while preserving RNA and DNA integrity.

What Conditions Does Proteinase K Require?

Following are the conditions that Proteinase K requires to function properly.

Temperature

Proteinase K exhibits high activity from 50°C to 65°C, with an optimal temperature of around 60°C. It retains some proteolytic capability even at 37°C. The higher temperatures likely make protein structures more accessible.

pH

This enzyme functions over a wide pH range from pH 4 to pH 12, with maximal activity between pH 7.5 and 12. Alkaline conditions are best, but neutral pH also works well.

Detergents

Adding ionic detergents like SDS (sodium dodecyl sulfate) can enhance proteinase K activity by denaturing protein substrates. Non-ionic detergents like Triton X-100 also work.

Divalent Cations

Proteinase K requires calcium ions for full enzymatic activity and stability. EDTA chelates calcium and can inhibit the enzyme. However, proteinase K still retains some function without calcium.

How is Proteinase K Used in the Lab?

Proteinase K has important functions in Lab and it is used in the lab in the following ways:

Proteinase K Buffer Recipe

A typical proteinase K digestion buffer contains:

• 10 mM Tris-HCl pH 8.0
• 100 mM NaCl
• 5% SDS
• 50 μg/mL proteinase K

The Tris maintains an alkaline pH of around 8.0. NaCl provides some ionic strength. SDS denatures proteins for digestion. The buffer can be modified as needed.

Digestion Protocol

• Add proteinase K to the buffer just before use.
• Incubate samples in the buffer for 30 min to 2 hrs at 50°C to 65°C.
• Inactivate proteinase K by heating at 95°C for 10 min or using PMSF.

Longer digests or a higher temperature can increase activity. The sample context dictates optimal conditions.

Caution!

Proteinase K is a very potent protease. Care must be taken to avoid contaminating samples or degrading proteins meant to be analyzed. Always wear gloves and use dedicated supplies.

How is Proteinase K Inactivated?

Once proteinase K has served its purpose, it must be inactivated to stop further digestion. Here are some methods:

• Heating: Temperatures above 90°C quickly inactivate the enzyme. However, heating does not fully eliminate residual activity.
• PMSF: This irreversible serine protease inhibitor covalently modifies the catalytic serine, permanently blocking activity.
• AEBSF: Another serine protease inhibitor that permanently inactivates proteinase K.
• TCA: Trichloroacetic acid denatures and precipitates the enzyme.

Heating is the most convenient method, but serine protease inhibitors provide complete inactivation.

What are Some Alternatives to Proteinase K?

Some alternatives of Proteinase K include:

• Pronase: A nonspecific protease mix isolated from Streptomyces griseus.
• Trypsin: A serine protease with specificity for lysine and arginine.
• Pepsin: An aspartic protease from mammalian gastric juice. Works best under acidic conditions.
• Papain: A cysteine protease from papaya. Often used for cell dissociation.

Each has some overlap with proteinase K in degrading proteins, but their specific properties differ. Proteinase K is still the most widely used for general lab purposes.

Conclusion

The powerful serine protease proteinase K is indispensable for molecular biology research and diagnostics. Its remarkable ability to rapidly digest proteins while remaining active in harsh conditions makes it the go-to enzyme for removing contaminating proteins and protecting nucleic acids.

Understanding what proteinase K is, how it functions, and its optimal working conditions allows researchers to harness its full potential in the lab. This versatile protease continues to enable countless techniques by eliminating interfering proteins and inactivating harmful nucleases to recover DNA and RNA of the highest purity.