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About Proteinase K
In molecular biology, proteinase K (EC 3.4.21.64, Protease K, Endopeptidase K, Tritirachium alkaline proteinase, Tritirachium album serine proteinase, Tritirachium album 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 can digest hair (keratin), hence the name "proteinase K". The predominant cleavage site is the peptide bond near the carboxyl group of aliphatic and aromatic amino acids with alpha-amino blocking groups. It is often used because of its broad specificity. Proteinase K belongs to the S8 (subtilisin) family of peptidases. The molecular weight of proteinase K is 28,900 daltons (28.9 kDa).
Proteinase K activity
Activated by calcium, the enzyme proteinase K digests proteins preferentially for hydrophobic amino acids (aliphatic, aromatic, and other hydrophobic amino acids). Calcium ions do not affect the activity of the enzyme but contribute to its stability. Proteins are fully digested if the incubation time is long and the protease concentration is sufficiently high. If calcium ions are removed, the stability of the enzyme is reduced, but proteolytic activity is maintained. Proteinase K has two Ca2+ binding sites, which are close to the active site, but are not directly involved in the catalytic mechanism. The residual activity is sufficient to digest proteins that normally contaminate nucleic acid preparations. Therefore, proteinase K digestion for nucleic acid purification is usually performed in the presence of EDTA (inhibition of metal ion-dependent enzymes such as nuclease).
Stability of proteinase K
Proteinase K is stable over a wide pH range (4-12), the optimum being pH 8.0. Increasing the reaction temperature from 37 °C to 50-60 °C can increase the activity several-fold, as can the addition of 0.5-1 % sodium dodecyl sulfate (SDS) or guanidinium chloride (3 M), guanidinium thiocyanate (1 M) and urea (4 M). The above conditions increase the activity of proteinase K by making its substrate cleavage sites more accessible. Temperatures above 65 °C, trichloroacetic acid (TCA) or the serine protease inhibitors AEBSF, PMSF or DFP inhibit activity. Proteinase K is not inhibited by guanidinium chloride, guanidinium thiocyanate, urea, sarcosyl, Triton X-100, Tween 20, SDS, citrate, iodoacetic acid, EDTA or other serine protease inhibitors such as Nα-tosyl-Lys chloromethylketone (TLCK) and Nα-tosyl-Phe chloromethylketone (TPCK).
Applications of proteinase K
Proteinase K is widely used in molecular biology to digest proteins and remove impurities from nucleic acid preparations. By adding proteinase K to nucleic acid preparations, nucleases that might otherwise degrade DNA or RNA during purification are rapidly inactivated. It is suitable for this application because the enzyme is active in the presence of protein-denaturing chemicals such as SDS and urea, chelating agents such as EDTA, hydrogen sulfide reagents, and trypsin or chymotrypsin inhibitors. Proteinase K is used for protein destruction in cell lysates (tissues, cell culture cells) and for nucleic acid release, as it inactivates DNases and RNases with high efficiency. Some examples of applications: Proteinase K is very useful in the purification of highly natural and undamaged DNA or RNA, since most microbial or mammalian DNases and RNases are rapidly inactivated by the enzyme, especially in the presence of 0.5-1% SDS. The activity of the enzyme toward native proteins is stimulated by denaturants such as SDS. In contrast, when measuring with peptide substrates, denaturants inhibit the enzyme. The reason for this result is that denaturants unfold protein substrates and make them more accessible to the protease. Proteinase K Inhibitors Proteinase K has two disulfide bonds, but shows increased proteolytic activity in the presence of reducing agents (e.g., 5 mM DTT), indicating that the presumed reduction of its own disulfide bonds does not lead to its irreversible inactivation. Proteinase K is inhibited by serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), or 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). Proteinase K activity is not affected by sulfite-modifying reagents such as para-chloromercuribenzoic acid (PCMB), N-alpha-tosyl-L-lysyl chloromethyl ketone (TLCK), or N-alpha-tosyl-l-phenylalanine chloromethyl ketone (TPCK), although it is likely to be inhibited when these reagents are used in conjunction with reducing disulfide reagents that expose the normally unavailable thiols of proteinase K.
FAQs
Which CAS number does Proteinase K have?
The CAS number of Proteinase K is 39450-01-6.
CAS Proteinase K?
The CAS number of Proteinase K is 39450-01-6.
CAS 39450-01-6?
The CAS number 39450-01-6 is assigned to Proteinase K.
What is the process to deactivate Proteinase K?
One of the most frequent inquiries we get is possibly about the inactivation of Proteinase K. The solution is also quite straightforward. Proteinase K is frequently inactivated with heat. Proteinase K is inactivated after 10 minutes of heating at 95°C, despite the fact that its activity rises with temperature and is optimal at about 65°C. It should be emphasized, though, that boiling Proteinase K does not totally render the enzyme inactive. With this approach, some action is always left behind. AEBSF (Pefabloc®) and PMSF, two protease inhibitors, can also be employed to permanently inactivate Proteinase K. Note: There is debate regarding the precise inactivation temperature, which ranges from 70 to 95 °C. But, based on feedback from the general public and in-depth research, we have determined that 95 °C is the ideal temperature for inactivation.
Which temperature is ideal for activating Proteinase K?
The previous query said that Proteinase K activity rises with temperature (to some extent). Between 50 to 65 C, the ideal temperature for activity, is. Warmer temperatures facilitate protein cleavage and facilitate protein degradation by Proteinase K. Proteinase K optimization, however, might not be the most crucial step in your process. To get the best outcomes overall, particular approaches occasionally need temperatures that have been adjusted. Although the enzyme is still active at temperatures between 20 and 65 °C, you should also keep in mind that despite the wide reported temperature range for Proteinase K activity. This broad temperature flexibility can be advantageous for very specific processes you are undertaking. Proteinase K could become inactive at temperatures higher than 65 °C.
How do Proteinase K and calcium interact with each other?
In order to ensure the stability of the enzyme, particularly when exposed to high temperatures, Proteinase K binds two Ca2+ ions. Protein K is also shielded from autolysis by calcium. Although not necessary for proteolytic activity, calcium and heat assist sustain Proteinase K's thermostability. Richard Tullis and Harvey Rubin assert that the presence of DNase I makes this link even more intriguing. Although DNase I is protected by Proteinase K (1 mg/mL concentration) when Ca2+ is present, Proteinase K is known to inactivate DNases and RNases. On the other hand, Ca2+ is not necessary for RNase to become inactive. Your findings point to a way to either purify the highly polymerized RNA or treat the contaminated RNase without using DNaseI.
What effect does EDTA have on the inactivation of Proteinase K?
While talking about Proteinase K, this question also tends to come up regularly. Proteinase K's enzymatic activity is not directly impacted by chelating chemicals like EDTA or EGTA. The removal of calcium is typically the purpose of combining EDTA with Proteinase K during DNA or RNA purification. The addition of EDTA may, however, impact calcium and, as a result, somewhat lessen Proteinase K activity since calcium and its relationship to calcium are related to the stability of Proteinase K.
Are there any Proteinase K activators?
Urea and SDS (sodium dodecyl sulfate) are both Proteinase K activators. In general, buffers containing these activators make Proteinase K more stable and active.
In what way is Proteinase K used in cell lysis?
To begin with, we must define Proteinase K in order to respond to this question. It is a broad-spectrum protease that can break down many different native proteins. By digesting surface proteins, Proteinase K can be used in the lysis processes of a cell, particularly in the subsequent DNA isolation and purification. Proteinase K helps to break down proteins that might otherwise cause the sample to degrade later on in the process when it's time to resuspend and lyse the nuclei in a buffer that contains it.
On what criterion do many recipes for DNA extraction buffers call for the use of Proteinase K and RNase?
RNases are known to be digested by Proteinase K. Why then combine the two in a lysis buffer? In order to destroy the contaminating RNA during DNA purification, you should first add RNase. Proteinase K should be used since it can break down harmful proteins, DNases, and RNases. Timing and optimization are key factors in determining the solution to this issue. Some scientists advise adding RNase first and allowing it time to function. The undesirable proteins can then be broken down with Proteinase K and SDS. Some techniques call for SDS, Proteinase K, and RNase to be incubated at 37°C for a while. At this temperature, Proteinase K activity is not as optimal, which presumably provides the RNase more time to function. The protocol's subsequent steps advise a second incubation at 55 °C for a longer amount of time since that temperature promotes Proteinase K activity and enables it to break down additional undesirable proteins.
In which applications is Proteinase K used?
A broad-spectrum serine protease, Proteinase K is a member of the subtilisin-like protease family (there are two types of serine proteases, chymotrypsin-like and subtilisin-like). It is mostly utilized for the isolation of nucleic acids, such as genomic DNA, cytoplasmic RNA, high-nativity DNA and RNA, etc. because it is a broad-spectrum protease. Because Proteinase K can digest proteins and inactivate DNases and RNases that would otherwise degrade a desired DNA or RNA sample, it is perfect for these applications. Proteinase K is used for a variety of processes, including the digestion of unwanted proteins in molecular biology applications, the release of endotoxins from cationic proteins like lysozyme and RNaseA, the removal of nucleases for in situ hybridization, the study of prion diseases such as transmissible spongiform encephalopathies (TSEs), protease footprinting, mitochthon purification, genomic DNA purification, cytoplasmic.
What is the best way to store Proteinase K and how long does it last?
Standard stock solutions are typically stable at -20 °C for up to a year. Freeze-dried powder is typically dried for up to two years at -20 C. The provisions of the individual manufacturers' certificates of analysis, which might not agree with this assertion, nonetheless, apply.
Which is the best way to dissolve Proteinase K? / What should be considered when dissolving Proteinase K?
In addition to being easily dissolved in water, Proteinase K can also be done so in Tris or PBS. However, dealing with PBS can be a little challenging, probably due to the pH. Proteinase K powder may often be dissolved in PBS by adding it to the solution and stirring it.
How are nuclease inactivated by Proteinase K?
Nucleic acids are known to be shielded from protein breakdown by Proteinase K. This occurs as a result of Proteinase K's capacity to break down proteins that otherwise cause harm to your sample.
Are there alternatives to the use of Proteinase K in DNA extractions?
The ability of Proteinase K to breakdown a range of damaging nucleases makes it useful for DNA extraction. Moreover, it is quite effective at breaking down proteins on cell membranes. Nevertheless, phenol-chloroform extraction is another viable choice for extracting proteins from a solution if this inquiry is especially intended to isolate DNA from other proteins. This approach is more harmful, though.
What relevance does Proteinase K have to TSEs or prion diseases?
Normal PrP C (prion/resistant protein) and PrPSC are differentiated by Proteinase K. (disease-causing isoform). The molecular weight of PrPC and PrPSC is the same, however PrPSC is Proteinase K resistant. Proteinase K is used to treat samples that may contain both. This enzyme eliminates PrPC and changes PrPSC into PrPRES, which has a lower molecular weight and can be detached and subsequently separated.
How high is the molar mass of Proteinase K?
The molecular weight of Proteinase K is 28.5 kDa.
At what pH is the optimum value for Proteinase K?
Proteinase K is active at a pH between 7.5 and 12.0.
How is the primary sequence of Proteinase K structured?
GAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGYSTIIAGMDFVASDKNNRNCPVVASLSLGGGYSVNSAAARLQSSGVMVAAGNNADARNYSPASIPSVCTVGASDRYRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPFGTVNLLAYNNYQA
What exactly is Proteinase K?
A broad-spectrum serine protease from the subtilisin family, Proteinase K. For its capacity to deactivate RNases and DNases that can harm desired nucleic acid samples during extraction, it is well-known in the scientific community. Its capacity to hydrolyze keratin, which was first found, gave rise to its name.
What is the importance of Proteinase K in DNA extraction?
When extracting DNA, Proteinase K is utilized to break down several contaminants that are present. Nucleases that might be present during DNA extraction are also degraded, and nucleic acids are shielded from nuclease attack.
In what kind of applications is Proteinase K used?
Uses of microarray and next-generation sequencing (NGS) technologies Improvements in cloning efficiency of PCR products, sample preparation for quantifying DNA adduct levels by accelerator mass spectrometry, inactivation of enzyme cocktails in ribonuclease protection assays, and complementing extraction procedures to optimize RNA yield from primary mammary tumors for microarray studies are all examples of nucleic acid purification by nuclease inactivation. Applications in molecular biology include the identification of proteins associated with bovine spongiform encephalopathy that display unusual resistance to proteolytic breakdown. An alternate sample preparation method for quantitative analysis by liquid chromatography-tandem mass spectrometry is tissue digestion (protein denaturation). To evaluate membrane structures for protein localisation and to create protein fragments for functional research, specific modifications of cell surface proteins are made.
Are there guidelines for the use of Proteinase K?
Chromosomal DNA embedded in agarose plugs can be treated with Proteinase K to render the restriction enzymes that were employed to digest the DNA inactive, allowing for the isolation of high molecular weight DNA. In this approach, the enzyme is employed at a concentration of 1 mg/mL in a buffer containing 1% N-lauroylsarcosine (v/v) and 0.5 M EDTA. 24-48 hours of incubation at 37 °C. DNA from cultivated cells or cells that have been frozen in liquid nitrogen can be used to isolate genomic and plasmid DNA, respectively. 50-100 mg of tissue or 1x108 cells should be incubated for 12–18 hours at 50 °C in 1 ml of buffer containing 0.5% SDS (w/v) and Proteinase K at a concentration of 1 mg/mL. RNA Isolation: Centrifuge the cell lysate, take off the supernatant, add 200 ug/mL Proteinase K, and 2% (w/v) SDS for cytoplasmic RNA isolation. At 37 °C, incubate for 30 minutes. By separating the lysate prior to enzyme treatment, total RNA can be separated and extracted using a syringe and needle. RNases, DNases, and enzymes are inactivated during reactions by Proteinase K, which is active in a range of buffers. It is recommended to utilize the enzyme at a ratio of roughly 1:50 (w/w, Proteinase K: enzyme). Incubate for 30 minutes at 37 °C. How come digestion starts at 50 °C? Certain proteins are unfolded to allow Proteinase K to more readily break them down by raising the temperature to 50 °C. Its activity rises and the enzyme is steady. In the presence of denaturants like SDS and urea, the enzyme is stable and exhibits a considerable increase in activity.
With which method can Proteinase K be inactivated in the fastest and most efficient way?
The most effective technique to deactivate the enzyme is to raise the temperature or dramatically alter the pH, just like with other proteins. Heat (such as incubation at 55°C) inactivates Proteinase K. How can you determine whether the enzyme is active? You can carry out the following Two actions to see if the enzyme is active: Calculate the p-nitroanilide production rate in micromoles per minute. Divide the result by the overall protein concentration in the solution. In this manner, the specific activity of the enzyme can be calculated as units of enzyme activity/mg of total protein, where one unit equals 1 mole of p-nitroanilide generated per minute.
Which cleavage site is used for Proteinase K?
The carboxyl group of hydrophobic, N-substituted aliphatic, and aromatic amino acids is also broken down by Proteinase K. Moreover, it breaks down peptide amides.
During which lifetime is Proteinase K active and functional?
Since it is extremely stable, Proteinase K has a shelf life of 6 months when kept in a dry location at 4–8 °C. Proteinase K's activity and stability won't be impacted by brief room-temperature storage. The certificates of analysis' conditions set forth by the manufacturer, however, are applicable.
What is the purpose of Proteinase K related to the Covid-19 assay?
When preparing the samples for the Covid-19 PCR test, Proteinase K is of crucial importance. Proteinase K has the task of cleaving the proteins of the sample, especially the nuclease, which would otherwise promote the degradation of the DNA and RNA of the sample and falsify the results of the test.
What is a natural source of protease and where can it be found in nature?
In 1974, Proteinase was first found in fungus Engyodontium album extracts (formerly Tritirachium album).
Literature
[1] Betzel C, Singh TP, Visanji M, Peters K, Fittkau S, Saenger W, Wilson KS (July 1993). "Structure of a proteinase K complex with a hexapeptide inhibitor analogous to the substrate at 2,2-A resolution". J. Biol. Chem. 268 (21): 15854-8. [2] Morihara K, Tsuzuki H (1975). "Specificity of proteinase K from Tritirachium album Limber for synthetic peptides". Agriculture. Biol. Chem. 39 (7): 1489-1492. [3] Kraus E, Kiltz HH, Femfert UF (February 1976). "The specificity of proteinase K against oxidized insulin B-chain". Hoppe-Seyler Z. Physiol. Chem. 357 (2): 233-7. [4] Jany KD, Lederer G, Mayer B (1986). "Amino acid sequence of proteinase K from the mold Tritirachium album Limber". FEBS Lett. 199 (2): 139-144. [5] Ebeling W, Hennrich N, Klockow M, Metz H, Orth HD, Lang H (August 1974). "Proteinase K from Tritirachium album Limber". Eur. J. Biochem. 47 (1): 91-7. [6] Müller A, Hinrichs W, Wolf WM, Saenger W (September 1994). "Crystal structure of calcium-free proteinase K at 1.5 A resolution". J. Biol. Chem. 269 (37): 23108-11. [7] Hilz H, Wiegers U, Adamietz P (1975). "Stimulation of proteinase K action by denaturants: application to nucleic acid isolation and degradation of 'masked' proteins". European Journal of Biochemistry. 56 (1): 103-108. [8] Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. & Struhl, K. (eds.) (1995) Current Protocols in Molecular Biology. Greene Publishing & Wiley-Interscience, New York [9] Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition page B16. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. [10] Müller, A. et al. (1994) J. Biol. Chem. 269, 23108-23111 Crystal structure of free Calcium-Proteinase K at 1.5 A resolution. [11] Wallace, D.M. (1987) Enzymol Methods. 152, 41-48 Small and large scale phenol extraction. [11] Breyer, J., Wemheuer, W. M., Wrede, A., Graham, C., Benestad, S. L., Brenig, B., . Schulz-Schaeffer, W. J. (2012). Detergents alter PrPSc proteinase K resistance in several transmissible spongiform encephalopathies (TSEs). Veterinary Microbiology, 157(1-2), 23-31. [12] Charette, S. J., & Cosson, P. (2004, September). Rapid preparation of genomic DNA for PCR analysis. Tullis, R. H., & Rubin, H. (1980). Calcium protects DNase I from proteinase K: a new method for removal of RNase contaminating DNase I. Analytical Biochemistry, 107(1), 260-264. [13] Valeria Genoud, Martin Stortz, Ariel Waisman, Bruno G. Berardino, Paula Verneri, Virginia Dansey, Melina Salvatori, Federico Remes Lenicov, Valeria Levi (2021) Non-extraction protocol combining proteinase K and thermal inactivation for the detection of SARS-CoV-2 by RT-qPCR. https://doi.org/10.1371/journal.pone.0247792
