Trypsin
| Trypsin | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| File:1UTN.png | |||||||||
| Identifiers | |||||||||
| Symbol | Trypsin | ||||||||
| Pfam | PF00089 | ||||||||
| InterPro | IPR001254 | ||||||||
| SMART | SM00020 | ||||||||
| PROSITE | PDOC00124 | ||||||||
| MEROPS | S1 | ||||||||
| SCOP | 1c2g | ||||||||
| SUPERFAMILY | 1c2g | ||||||||
| CDD | cd00190 | ||||||||
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Trypsin (EC 3.4.21.4) is a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyses proteins.[2][3] Trypsin is formed in the small intestine when its proenzyme form, the trypsinogen produced by the pancreas, is activated. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline.[4] It is used for numerous biotechnological processes. The process is commonly referred to as trypsin proteolysis or trypsinisation, and proteins that have been digested/treated with trypsin are said to have been trypsinized.[5] Trypsin was discovered in 1876 by Wilhelm Kühne.[6]
Function
In the duodenum, trypsin catalyzes the hydrolysis of peptide bonds, breaking down proteins into smaller peptides. The peptide products are then further hydrolyzed into amino acids via other proteases, rendering them available for absorption into the blood stream. Tryptic digestion is a necessary step in protein absorption as proteins are generally too large to be absorbed through the lining of the small intestine.[7]
Trypsin is produced as the inactive zymogen trypsinogen in the pancreas. When the pancreas is stimulated by cholecystokinin, it is then secreted into the first part of the small intestine (the duodenum) via the pancreatic duct. Once in the small intestine, the enzyme enteropeptidase activates trypsinogen into trypsin by proteolytic cleavage. Auto catalysis does not happen with trypsin, as trypsinogen is a poor substrate, therefore enzymatic damage to the pancreas is avoided. [8]
Mechanism
The enzymatic mechanism is similar to that of other serine proteases. These enzymes contain a catalytic triad consisting of histidine-57, aspartate-102, and serine-195.[9] These three residues form a charge relay that increases nucleophilicity of the active site serine. This is achieved by modifying the electrostatic environment of the serine. The enzymatic reaction that trypsin catalyzes is thermodynamically favorable but requires significant activation energy (it is "kinetically unfavorable"). In addition, trypsin contains an "oxyanion hole" formed by the backbone amide hydrogen atoms of Gly-193 and Ser-195, which serves to stabilize the developing negative charge on the carbonyl oxygen atom of the cleaved amides.
The aspartate residue (Asp 189) located in the catalytic pocket (S1) of trypsin is responsible for attracting and stabilizing positively charged lysine and/or arginine, and is, thus, responsible for the specificity of the enzyme. This means that trypsin predominantly cleaves proteins at the carboxyl side (or "C-terminal side") of the amino acids lysine and arginine except when either is bound to a C-terminal proline,[10] although large-scale mass spectrometry data suggest cleavage occurs even with proline.[11] Trypsin is considered an endopeptidase, i.e., the cleavage occurs within the polypeptide chain rather than at the terminal amino acids located at the ends of polypeptides.
Properties
Human trypsin has an optimal operating temperature of about 37 °C.[12] In contrast, the Atlantic cod has several types of trypsins in order for the poikilotherm fish to survive at different body temperatures. Cod trypsins include trypsin I with an activity range of 4 to 65 °C (40 to 150 °F) and maximal activity at 55 °C (130 °F), as well as trypsin Y with a range of 2 to 30 °C (36 to 86 °F) and a maximal activity at 21 °C (70 °F).[13]
As a protein trypsin has various molecular weights depending on the source. For example, a molecular weight of 23.3 kDa is reported for trypsin from bovine and porcine sources.
The activity of trypsin is not affected by the enzyme inhibitor tosyl phenylalanyl chloromethyl ketone, TPCK, which deactivates chymotrypsin. This is important because, in some applications, like mass spectrometry, the specificity of cleavage is important.
Trypsin should be stored at very cold temperatures (between −20 °C and −80 °C) to prevent autolysis, which may also be impeded by storage of trypsin at pH 3 or by using trypsin modified by reductive methylation. When the pH is adjusted back to pH 8, activity returns.
Isozymes
The following human genes encode proteins with trypsin enzymatic activity:
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Other isoforms of trypsin may also be found in other organisms.
Clinical significance
Activation of trypsin from proteolytic cleavage of trypsinogen in the pancreas can lead to a series of events that cause pancreatic self-digestion, resulting in pancreatitis. One consequence of the autosomal recessive disease cystic fibrosis is a deficiency in transport of trypsin and other digestive enzymes from the pancreas. This leads to the disorder termed meconium ileus. This disorder involves intestinal obstruction (ileus) due to overly thick meconium, which is normally broken down by trypsin and other proteases, then passed in feces.[14]
Applications
Trypsin is available in high quantity in pancreases, and can be purified rather easily. Hence it has been used widely in various biotechnological processes.
In a tissue culture lab, trypsin is used to re-suspend cells adherent to the cell culture dish wall during the process of harvesting cells.[15] Some cell types have a tendency to "stick" - or adhere - to the sides and bottom of a dish when cultivated in vitro. Trypsin is used to cleave proteins bonding the cultured cells to the dish, so that the cells can be suspended in fresh solution and transferred to fresh dishes.
Trypsin can also be used to dissociate dissected cells (for example, prior to cell fixing and sorting).
Trypsin can be used to break down casein in breast milk. If trypsin is added to a solution of milk powder, the breakdown of casein will cause the milk to become translucent. The rate of reaction can be measured by using the amount of time it takes for the milk to turn translucent.
Trypsin is commonly used in biological research during proteomics experiments to digest proteins into peptides for mass spectrometry analysis, e.g. in-gel digestion. Trypsin is particularly suited for this, since it has a very well defined specificity, as it hydrolyzes only the peptide bonds in which the carbonyl group is contributed either by an Arg or Lys residue.
Trypsin can also be used to dissolve blood clots in its microbial form and treat inflammation in its pancreatic form.
Atlantic cod trypsin is marketed under the trade name ColdZyme for prevention of common cold by the Enzymatica company, the same company that also made the underlying study wherein people administering Atlantic cod trypsin by oral spray on a daily basis were infected to a lesser degree if inoculated with rhinovirus.[16]
In food
Commercial protease preparations usually consist of a mixture of various protease enzymes that often includes trypsin. These preparations are widely utilized in food processing:[17]
- as a baking enzyme to improve the workability of dough;
- in the extraction of seasonings and flavourings from vegetable or animal proteins and in the manufacture of sauces;
- to control aroma formation in cheese and milk products;
- to improve the texture of fish products;
- to tenderize meat;
- during cold stabilization of beer;
- in the production of hypoallergenic food where proteases break down specific allergenic proteins into nonallergenic peptides. For example, proteases are used to produce hypoallergenic baby food from cow’s milk thereby diminishing the risk of babies developing milk allergies.
Trypsin inhibitor
In order to prevent the action of active trypsin in the pancreas which can be highly damaging, inhibitors such as BPTI and SPINK1 in the pancreas and α1-antitrypsin in the serum are present as part of the defense against its inappropriate activation. Any trypsin prematurely formed from the inactive trypsinogen would be bound by the inhibitor. The protein-protein interaction between trypsin and its inhibitors is one of the tightest found, and trypsin is bound by some of its pancreatic inhibitors essentially irreversibly.[18] In contrast with nearly all known protein assemblies, some complexes of trypsin bound by its inhibitors do not readily dissociate after treatment with 8M urea.[19]
See also
- Trypsinization
- PA clan of proteases
References
- ↑ PDB: 1UTN; Leiros HK, Brandsdal BO, Andersen OA, Os V, Leiros I, Helland R, Otlewski J, Willassen NP, Smalås AO (April 2004). "Trypsin specificity as elucidated by LIE calculations, X-ray structures, and association constant measurements". Protein Sci. 13 (4): 1056–70. doi:10.1110/ps.03498604. PMC 2280040. PMID 15044735.
- ↑ Rawlings ND, Barrett AJ (1994). "Families of serine peptidases". Methods Enzymol. Methods in Enzymology. 244: 19–61. doi:10.1016/0076-6879(94)44004-2. ISBN 978-0-12-182145-6. PMID 7845208.
- ↑ The German physiologist Wilhelm Kühne (1837-1900) discovered trypsin in 1876. See: W. Kühne (1877) "Über das Trypsin (Enzym des Pankreas)", Verhandlungen des naturhistorisch-medicinischen Vereins zu Heidelberg, new series, vol. 1, no. 3, pages 194-198.
- ↑ Reviews, C. T. I. (2016-09-26). Textbook of Veterinary Physiological Chemistry: Veterinary medicine, Veterinary medicine. Cram101 Textbook Reviews. ISBN 9781490289472.Template:Tertiary source
- ↑ Engelking, Larry R. (2015-01-01). Textbook of Veterinary Physiological Chemistry (Third Edition). Boston: Academic Press. pp. 39–44. ISBN 9780123919090.
- ↑ "Verhandlungen des Naturhistorisch-medizinischen Vereins zu Heidelberg". archive.org. Retrieved 2017-04-24.
- ↑ "DIGESTION OF PROTEINS". intranet.tdmu.edu.ua. Retrieved 2017-04-24.
- ↑ "DIGESTION OF PROTEINS". intranet.tdmu.edu.ua. Retrieved 2017-04-24.
- ↑ Polgár L (October 2005). "The catalytic triad of serine peptidases". Cell. Mol. Life Sci. 62 (19–20): 2161–72. doi:10.1007/s00018-005-5160-x. PMID 16003488.
- ↑ "Sequencing Grade Modified Trypsin" (PDF). www.promega.com. 2007-04-01. Retrieved 2009-02-08.
- ↑ Rodriguez J, Gupta N, Smith RD, Pevzner PA (2008). "Does trypsin cut before proline?" (PDF). J. Proteome Res. 7 (1): 300–305. doi:10.1021/pr0705035. PMID 18067249.
- ↑ Hanne Kolsrud Hustoft; Helle Malerod; Steven Ray Wilson; Leon Reubsaet; Elsa Lundanes; Tyge Greibrokk. "A Critical Review of Trypsin Digestion for LC-MS Based Proteomics" (PDF). page 80. University of Oslo, Norway
- ↑ Gudmundsdóttir A, Pálsdóttir HM (2005). "Atlantic cod trypsins: from basic research to practical applications". Mar. Biotechnol. 7 (2): 77–88. doi:10.1007/s10126-004-0061-9. PMID 15759084.
- ↑ Noone PG, Zhou Z, Silverman LM, Jowell PS, Knowles MR, Cohn JA (December 2001). "Cystic fibrosis gene mutations and pancreatitis risk: relation to epithelial ion transport and trypsin inhibitor gene mutations". Gastroenterology. 121 (6): 1310–9. doi:10.1053/gast.2001.29673. PMID 11729110.
- ↑ "Trypsin-EDTA (0.25%)". Stem Cell Technologies. Retrieved 2012-02-23.
- ↑ "Clinical study on the common cold". Enzymatica. 2014.
- ↑ "Protease - GMO Database". GMO Compass. European Union. 2010-07-10. Retrieved 2012-01-01.
- ↑ Voet & Voet (1995). Biochemistry (2nd ed.). John Wiley & Sons. pp. 396–400. ISBN 0-471-58651-X.
- ↑ N. Levilliers; M. Péron; B. Arrio; J. Pudles (October 1970). "On the mechanism of action of proteolyticinhibitors: IV. Effect of 8murea on the stability of trypsin in trypsin-lnhibitor complexes". Archives of Biochemistry and Biophysics. 140 (2): 474–483. doi:10.1016/0003-9861(70)90091-3. PMID 5528741.
External links
- The MEROPS online database for peptidases and their inhibitors: Trypsin 1 S01.151, Trypsin 2 S01.258, Trypsin 3 S01.174
- Trypsin Inhibitors and Trypsin Assay Method at Sigma-Aldrich
- Trypsin at the US National Library of Medicine Medical Subject Headings (MeSH)