Proteinase K
Encyclopedia
Proteinase K is a broad-spectrum serine protease
. The enzyme was discovered in 1974 in extracts of the fungus
Engyodontium album (formerly Tritirachium album). Proteinase K is able to digest native keratin (hair), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8.
Molecular weight of Proteinase K is 28,900 daltons (~28.9 KDa). Although calcium ions do not affect the enzyme activity, they do contribute to its stability when present at a concentration of 1 - 5 μmoles. An interesting characteristic of proteinase K is that it retains its activity in the presence of sodium dodecyl sulphate (SDS) or urea. (0.5 - 1% SDS and 1 - 4 M urea). Raising the temperature of the reaction from 37°C to 50°C - 60°C can increase the activity several folds. A special feature of proteinase K is its ability to digest native proteins, thereby inactivating enzymes such as DNase and RNase without recourse to a denaturation process.
In addition to cleavage of peptide bonds, it also catalyzes peptide amide hydrolysis. Proteinase K is inactivated by diisopropyl fluorophosphate (DFP) or phenyl methane sulphonyl fluoride (PMSF). Chelating agents such as citrate and EDTA have no effect on the enzyme activity.
Proteinase K is very useful in the isolation of highly native, undamaged DNAs or RNAs, since most microbial or mammalian DNases and RNases are rapidly inactivated by the enzyme, particularly in the presence of 0.5 - 1% SDS.
Proteinase K is commonly used in molecular biology
to digest protein and remove contamination from preparations of nucleic acid
. Addition of proteinase K to nucleic acid preparations rapidly inactivates nuclease
s that might otherwise degrade the DNA or RNA during purification. It is highly-suited to this application since the enzyme is active in the presence of chemicals that denature
proteins, such as SDS
and urea
, chelating agents such as EDTA
, sulfhydryl reagents, as well as trypsin
or chymotrypsin
inhibitors. Proteinase K is also stable over a wide pH
range (4-12), with a pH optimum of pH 7.5-12.
The enzyme's activity towards native proteins is stimulated by denaturants such as SDS. In contrast, when measured using peptide
substrates, denaturants inhibit
the enzyme. The reason for this result is that the denaturing agents unfold the protein substrates and make them more accessible to the protease.
Serine protease
Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site.They are found ubiquitously in both eukaryotes and prokaryotes...
. The enzyme was discovered in 1974 in extracts of the fungus
Fungus
A fungus is a member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and molds , as well as the more familiar mushrooms. These organisms are classified as a kingdom, Fungi, which is separate from plants, animals, and bacteria...
Engyodontium album (formerly Tritirachium album). Proteinase K is able to digest native keratin (hair), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8.
Molecular weight of Proteinase K is 28,900 daltons (~28.9 KDa). Although calcium ions do not affect the enzyme activity, they do contribute to its stability when present at a concentration of 1 - 5 μmoles. An interesting characteristic of proteinase K is that it retains its activity in the presence of sodium dodecyl sulphate (SDS) or urea. (0.5 - 1% SDS and 1 - 4 M urea). Raising the temperature of the reaction from 37°C to 50°C - 60°C can increase the activity several folds. A special feature of proteinase K is its ability to digest native proteins, thereby inactivating enzymes such as DNase and RNase without recourse to a denaturation process.
In addition to cleavage of peptide bonds, it also catalyzes peptide amide hydrolysis. Proteinase K is inactivated by diisopropyl fluorophosphate (DFP) or phenyl methane sulphonyl fluoride (PMSF). Chelating agents such as citrate and EDTA have no effect on the enzyme activity.
Proteinase K is very useful in the isolation of highly native, undamaged DNAs or RNAs, since most microbial or mammalian DNases and RNases are rapidly inactivated by the enzyme, particularly in the presence of 0.5 - 1% SDS.
Proteinase K is commonly used in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
to digest protein and remove contamination from preparations of nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
. Addition of proteinase K to nucleic acid preparations rapidly inactivates nuclease
Nuclease
A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Older publications may use terms such as "polynucleotidase" or "nucleodepolymerase"....
s that might otherwise degrade the DNA or RNA during purification. It is highly-suited to this application since the enzyme is active in the presence of chemicals that denature
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
proteins, such as SDS
Sodium dodecyl sulfate
Sodium dodecyl sulfate , sodium laurilsulfate or sodium lauryl sulfate is an organic compound with the formula CH311OSO3Na). It is an anionic surfactant used in many cleaning and hygiene products...
and urea
Urea
Urea or carbamide is an organic compound with the chemical formula CO2. The molecule has two —NH2 groups joined by a carbonyl functional group....
, chelating agents such as EDTA
EDTA
Ethylenediaminetetraacetic acid, widely abbreviated as EDTA , is a polyamino carboxylic acid and a colourless, water-soluble solid. Its conjugate base is named ethylenediaminetetraacetate. It is widely used to dissolve limescale. Its usefulness arises because of its role as a hexadentate ligand...
, sulfhydryl reagents, as well as trypsin
Trypsin
Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when...
or chymotrypsin
Chymotrypsin
Chymotrypsin is a digestive enzyme that can perform proteolysis. Chymotrypsin preferentially cleaves peptide amide bonds where the carboxyl side of the amide bond is a tyrosine, tryptophan, or phenylalanine. These amino acids contain an aromatic ring in their sidechain that fits into a...
inhibitors. Proteinase K is also stable over a wide pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
range (4-12), with a pH optimum of pH 7.5-12.
The enzyme's activity towards native proteins is stimulated by denaturants such as SDS. In contrast, when measured using peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
substrates, denaturants inhibit
Enzyme inhibitor
An enzyme inhibitor is a molecule that binds to enzymes and decreases their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used as herbicides and pesticides...
the enzyme. The reason for this result is that the denaturing agents unfold the protein substrates and make them more accessible to the protease.