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- QUESTION: What is Proteinase K?
ANSWER: In molecular biology Proteinase K (also protease K or endopeptidase 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. The molecular weight of Proteinase K is 28,900 daltons (28.9 kDa).
- QUESTION: What is the function of proteinase K in DNA extraction?
ANSWER: During the extraction of DNA (or nucleic acids in general), there is a lot of contaminating proteins present. These contaminants must be removed. Proteinase K, which is a broad spectrum serine protease, is used in many DNA extraction protocols to digest these contaminating proteins.
In addition, there may be nucleases (enzymes that degrade nucleic acids) present. The addition of proteinase K degrades these nucleases and protects the nucleic acids from nuclease attack. In addition, proteinase K is stable over a wide pH range and is well suited for use in DNA extraction.
- QUESTION: What are proteinase K applications?
ANSWER: 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 nucleases 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 used for the destruction of proteins in cell lysates (tissue, cell culture cells) and for the release of nucleic acids, since it very effectively inactivates DNases and RNases. Some examples for applications: 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. Purification of genomic DNA from bacteria (miniprep): bacteria from a saturated liquid culture are lysed and proteins are removed by a digest with 100 μg/ml Proteinase K for 1 h at 37 °C. 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.
- QUESTION: Why is proteinase K digestion performed at 50°C?
ANSWER: Proteinase K activity is greatly increased by addition of denaturing agents like SDS or urea (Hilz et al., 2008), indicating that the denaturation of the substrates helps Proteinase K to degrade them. Increasing the temperature to 50°C will also unfold some proteins already, making it easier for the Proteinase K to degrade them. The proteinase K seems to be a pretty stable enzyme, and can still work at this temperature.
- QUESTION: What are temperatures proteinase k inactivated?
ANSWER: Proteinase K is inactivated by heat, eg. Incubating at >55 °C.
- QUESTION: What is the quickest most effective way to inactivate proteinase K?
ANSWER: As with most protein enzymes change the temperature or change the pH significantly.
- QUESTION: What is the Enzyme activity of proteinase K?
ANSWER: Activated by calcium (1 – 5 mM), the enzyme digests proteins preferentially after hydrophobic amino acids (aliphatic, aromatic and other hydrophobic amino acids). Although calcium ions do not affect the enzyme activity, they do contribute to its stability. Proteins will be completely digested, if the incubation time is long and the protease concentration high enough. Upon removal of the calcium ions, the stability of the enzyme is reduced, but the proteolytic activity remains. Proteinase K has two binding sites for Ca2+, which is located close to the active center, but is not directly involved in the catalytic mechanism. Removal of the Ca2+ ions reduces the catalytic activity of Proteinase K by 80 %. The residual activity is sufficient to digest proteins, which usually contaminate nucleic acid preparations. Therefore, the digest with Proteinase K for the purification of nucleic acids is performed in the presence of EDTA (inhibition of magnesium-dependent enzymes). If the presence of Ca2+ required, Ca2+ is added up to a concentration of 1 mM and is removed by the addition of EGTA (pH 8.0; final conc. 2 mM) later on.
- QUESTION: What are buffers according to Proteinase K activity?
|Buffer (pH 8.0, 50°C, 1.25 µg/ml protease K, 15 min incubation)||Proteinase K activity (%)|
|30 mM Tris·Cl||100|
|30 mM Tris·Cl; 30 mM EDTA; 5% Tween 20; 0.5% Triton X-100; 800 mM GuHCl||313|
|36 mM Tris·Cl; 36 mM EDTA; 5% Tween 20; 0.36% Triton X-100; 735 mM GuHCl||301|
|10 mM Tris·Cl; 25 mM EDTA; 100 mM NaCl; 0.5% SDS||128|
|10 mM Tris·Cl; 100 mM EDTA; 20 mM NaCl; 1% Sarkosyl||74|
|10 mM Tris·Cl; 50 mM KCl; 1.5 mM MgCl2; 0.45% Tween 20; 0.5% Triton X-100||106|
|10 mM Tris·Cl; 100 mM EDTA; 0.5% SDS||120|
|30 mM Tris·Cl; 10 mM EDTA; 1% SDS||203|
- QUESTION: What are the guidelines for using Proteinase K?
1.Isolation of high molecular weight DNA
Chromosomal DNA that has been embedded in agarose plugs can be treated with Proteinase K to inactivate rare-cutting restriction enzymes used to digest the DNA. Proteinase K is used for this method at a concentration of 1mg/ml in a buffer containing 0.5M EDTA and 1% N-lauroylsarcosine (v/v). Incubate 24-48 hours at 37°C.
2.Isolation of plasmid and genomic DNA
Genomic or plasmid DNA can be isolated from liquid nitrogen frozen cells or cultured cells using Proteinase K. Incubate 50-100 mg of tissue or 1×108 cells in 1 ml of buffer containing 0.5% SDS (w/v) with ProteinaseK at a concentration of 1mg/ml, for 12-18 hours at 50°C.
3.Isolation of RNA
For cytoplasmic RNA isolation, centrifuge the cell lysate, remove the supernate and add 200ug/ml Proteinase K and SDS to 2%(w/v). Incubate for 30 minutes at 37°C. Total RNA can be isolated by passing the lysate through a needle fitted to a syringe prior Proteinase K treatment.
4.Inactivation of RNases , DNases and enzymes in reactions
Proteinase K is active in a wide variety of buffers (see FAQ “What is the Proteinase K activity in commonly used buffers?”). The enzyme should be used at a ratio of approximately 1:50 (w/w, proteinase K:enzyme ). Incubation is at 37°C for 30 minutes.
- QUESTION: How to determine if the proteinase K is working?
ANSWER: One can use an artificial substrate like benzoyl arginine -p-nitroanilide that when cleaved by the proteinase yield sa yellow colored p-nitroaniline that absorbs at ~ 410 nm. You can then determine the activity of the proteinase K by determinh how many micromoles of the p-nitroanilide are produced per minute. The by dividing by the total amount of protein in the solution you can determine the specific activity of the enzyme activity = units ( one unit equals 1 umole of p-nitroanilide produced /min ), specific activity = units of enzyme activity /mg total protein. Alternatively, prepare a 1.25 % agar containing 2% casein in pH eight buffer and pour into a petri dish. Punch 4mm diameter wells in the gel about 20 mm apart. In the wells place various concetrations of your proteinase K solution. Allow to incubate at room temp (humidified)for 6-8 hrs. Look for the clear zones around the wells. The size of the clear zone is oproortional to the concentration of the proteinase K and gives a visual appraisal of active digestion of a protein rather than a synthetic substrate.
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- Betzel C, Singh TP, Visanji M, et al. (July 1993). “Structure of the complex of proteinase K with a substrate analogue hexapeptide inhibitor at 2.2-A resolution”. The Journal of Biological Chemistry 268 (21): 15854–15858. PMID 8340410.
- Ebeling W, Hennrich N, et al. (1974). “Proteinase K from Tritirachium album Limber”. European Journal of Biochemistry 47 (1): 91–97. doi:10.1111/j.1432-1033.1974.tb03671.x. PMID 4373242.
- Müller A, Hinrichs W, Wolf WM, Saenger W (1994). “Crystal structure of calcium-free proteinase K at 1.5-Å resolution”. The Journal of Biological Chemistry 269: 23108-23111. PMID 8083213.