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{{enzyme | {{enzyme | ||
| Name = | | Name = trypsin | ||
| EC_number = 3.4.21.4 | | EC_number = 3.4.21.4 | ||
| CAS_number = 9002-07-7 | | CAS_number = 9002-07-7 | ||
| Line 14: | Line 14: | ||
| image = 1UTN.png | | image = 1UTN.png | ||
| width = | | width = | ||
| caption = [[X-ray crystallography#Biological macromolecular crystallography|Crystal structure]] of [[cattle|bovine]] trypsin.<ref name="pmid15044735">{{PDB|1UTN}}; {{cite journal |vauthors=Leiros HK, Brandsdal BO, Andersen OA, Os V, Leiros I, Helland R, Otlewski J, Willassen NP, Smalås AO | title = Trypsin specificity as elucidated by LIE calculations, X-ray structures, and association constant measurements | journal = Protein | | caption = [[X-ray crystallography#Biological macromolecular crystallography|Crystal structure]] of [[cattle|bovine]] trypsin.<ref name="pmid15044735">{{PDB|1UTN}}; {{cite journal | vauthors = Leiros HK, Brandsdal BO, Andersen OA, Os V, Leiros I, Helland R, Otlewski J, Willassen NP, Smalås AO | title = Trypsin specificity as elucidated by LIE calculations, X-ray structures, and association constant measurements | journal = Protein Science | volume = 13 | issue = 4 | pages = 1056–70 | date = April 2004 | pmid = 15044735 | pmc = 2280040 | doi = 10.1110/ps.03498604 }}</ref> | ||
| Pfam = PF00089 | | Pfam = PF00089 | ||
| InterPro = IPR001254 | | InterPro = IPR001254 | ||
| Line 26: | Line 26: | ||
| OPM protein = | | OPM protein = | ||
}} | }} | ||
'''Trypsin''' ({{EC number|3.4.21.4}}) is a [[serine protease]] from the [[PA clan]] superfamily, found in the [[digestive system]] of many [[vertebrates]], where it [[hydrolysis| | '''Trypsin''' ({{EC number|3.4.21.4}}) is a [[serine protease]] from the [[PA clan]] superfamily, found in the [[digestive system]] of many [[vertebrates]], where it [[hydrolysis|hydrolyzes]] [[protein]]s.<ref name="pmid7845208">{{cite journal | vauthors = Rawlings ND, Barrett AJ | title = Families of serine peptidases | journal = Methods in Enzymology | volume = 244 | issue = | pages = 19–61 | year = 1994 | pmid = 7845208 | doi = 10.1016/0076-6879(94)44004-2 | isbn = 978-0-12-182145-6 | series = Methods in Enzymology }}</ref><ref>The German physiologist [[Wilhelm Kühne]] (1837-1900) discovered trypsin in 1876. See: W. Kühne (1877) "[https://books.google.com/books?id=jzdMAAAAYAAJ&pg=PA194&ie=ISO-8859-1&output=html Über das Trypsin (Enzym des Pankreas)]", ''Verhandlungen des naturhistorisch-medicinischen Vereins zu Heidelberg'', new series, vol. 1, no. 3, pages 194-198.</ref> Trypsin is formed in the [[small intestine]] when its [[zymogen|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]]. 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.<ref>{{Cite book|url=http://www.sciencedirect.com/science/article/pii/B9780123919090500074|title=Textbook of Veterinary Physiological Chemistry (Third Edition)|last=Engelking|first=Larry R.|date=2015-01-01|publisher=Academic Press|isbn=9780123919090|location=Boston|pages=39–44}}</ref> Trypsin was discovered in 1876 by [[Wilhelm Kühne]].<ref>{{Cite web|url=https://archive.org/stream/verhandlungendes7477natu#page/190/mode/2up|title=Verhandlungen des Naturhistorisch-medizinischen Vereins zu Heidelberg|website=archive.org|access-date=2017-04-24}}</ref> | ||
== Function == | == Function == | ||
In the [[duodenum]], trypsin [[catalysis|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]].<ref>{{ | In the [[duodenum]], trypsin [[catalysis|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]].<ref name = "tdmu.edu.ua">{{cite web | url = http://intranet.tdmu.edu.ua/data/kafedra/internal/distance/lectures_stud/English/2%20course/Elective%20course%20(Clinical%20biochemistry)/10.%20METABOLISM%20OF%20AMINOACIDS.%20DIGESTION%20OF%20%20PROTEINS..htm|title= Digestion of Proteins |website=intranet.tdmu.edu.ua|access-date=2017-04-24}}</ref> | ||
Trypsin is produced as the inactive zymogen trypsinogen in the | 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]]. Autocatalysis does not happen with trypsin, as trypsinogen is a poor substrate, therefore enzymatic damage to the pancreas is avoided.<ref name = "tdmu.edu.ua" /> | ||
== Mechanism == | == Mechanism == | ||
The enzymatic mechanism is similar to that of other | 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.<ref name="pmid16003488">{{cite journal | vauthors = Polgár L | title = The catalytic triad of serine peptidases | journal = Cellular and Molecular Life Sciences | volume = 62 | issue = 19-20 | pages = 2161–72 | date = October 2005 | pmid = 16003488 | doi = 10.1007/s00018-005-5160-x }}</ref> This catalytic triad was formerly called a charge relay system, implying the abstraction of protons from serine to histidine and from histidine to aspartate, but owing to evidence provided by NMR that the resultant alkoxide form of serine would have a much stronger pull on the proton than does the imidazole ring of histidine, current thinking holds instead that serine and histidine each have effectively equal share of the proton, forming short [[low-barrier hydrogen bond]]s therewith.<ref>{{cite book | url = https://www.worldcat.org/oclc/690489261 | title = Biochemistry | first1 = Donald | last1 = Voet | first2 = Judith G | last2 = Voet | name-list-format = vanc |date=2011|publisher=John Wiley & Sons |isbn=9780470570951|edition=4th|location=Hoboken, NJ|oclc=690489261}}</ref> By these means, the [[nucleophile|nucleophilicity]] of the [[active site]] serine is increased, facilitating its attack on the amide carbon during proteolysis. The enzymatic reaction that trypsin catalyzes is [[thermodynamics|thermodynamically]] favorable, but requires significant [[activation energy]] (it is "[[enzyme kinetics|kinetically]] unfavorable"). In addition, trypsin contains an "oxyanion hole" formed by the backbone amide hydrogen atoms of Gly-193 and Ser-195, which through hydrogen bonding stabilize the negative charge which accumulates on the amide oxygen after nucleophilic attack on the planar amide carbon by the serine oxygen causes that carbon to assume a tetrahedral geometry. Such stabilisation of this tetrahedral intermediate helps to reduce the energy barrier of its formation and is concomitant with a lowering of the free energy of the transition state. Preferential binding of the transition state is a key feature of enzyme chemistry. | ||
The aspartate residue (Asp 189) located in the catalytic pocket (S1) of trypsin is responsible for attracting and stabilizing positively charged | 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 [[protein]]s at the [[carboxyl]] side (or "[[C-terminal]] side") of the [[amino acid]]s lysine and arginine except when either is bound to a C-terminal [[proline]],<ref name="urlwww.promega.com">{{cite web | url = http://www.promega.com/tbs/9piv511/9piv511.pdf | title =Sequencing Grade Modified Trypsin | date = 2007-04-01 | format = | work = | publisher = www.promega.com | pages = | quote = | access-date = 2009-02-08}}</ref> although large-scale mass spectrometry data suggest cleavage occurs even with proline.<ref name="pmid18067249">{{cite journal | vauthors = Rodriguez J, Gupta N, Smith RD, Pevzner PA | title = Does trypsin cut before proline? | journal = Journal of Proteome Research | volume = 7 | issue = 1 | pages = 300–5 | date = January 2008 | pmid = 18067249 | doi = 10.1021/pr0705035 | url = http://proteomics.ucsd.edu/pavel/papers/does_trypsin_cut_before_proline.pdf }}</ref> 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 [[peptide|polypeptides]]. | ||
== Properties == | == Properties == | ||
Human trypsin has an optimal [[operating temperature]] of about 37 °C.<ref>{{cite web|url=http://cdn.intechopen.com/pdfs-wm/29629.pdf|title=A Critical Review of Trypsin Digestion for LC-MS Based Proteomics| | Human trypsin has an optimal [[operating temperature]] of about 37 °C.<ref>{{cite web|url=http://cdn.intechopen.com/pdfs-wm/29629.pdf|title=A Critical Review of Trypsin Digestion for LC-MS Based Proteomics| first1 = Hanne Kolsrud | last1 = Hustoft | first2 = Helle | last2 = Malerod | first3 = Steven Ray | last3 = Wilson | first4 = Leon | last4 = Reubsaet | first5 = Elsa | last5 = Lundanes | first6 = Tyge | last6 = Greibrokk | name-list-format = vanc | page = 80 | publisher = [[University of Oslo]] | location = Norway }}</ref> In contrast, the [[Atlantic cod]] has several types of trypsins 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).<ref>{{cite journal | vauthors = Gudmundsdóttir A, Pálsdóttir HM | title = Atlantic cod trypsins: from basic research to practical applications | journal = Marine Biotechnology | volume = 7 | issue = 2 | pages = 77–88 | year = 2005 | pmid = 15759084 | doi = 10.1007/s10126-004-0061-9 }}</ref> | ||
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. | 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, [[tosyl phenylalanyl chloromethyl ketone|TPCK]], which deactivates [[chymotrypsin]]. This is important because, in some applications, like [[mass spectrometry]], the specificity of cleavage is important. | The activity of trypsin is not affected by the [[enzyme inhibitor]] tosyl phenylalanyl chloromethyl ketone, [[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 | Trypsin should be stored at very cold temperatures (between −20 and −80 °C) to prevent [[Autolysis (biology)|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 == | == Isozymes == | ||
These human genes encode proteins with trypsin enzymatic activity: | |||
{| | {| | ||
|- valign="top" | |- valign="top" | ||
| Line 115: | Line 115: | ||
== Clinical significance == | == 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 | 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]], which involves intestinal obstruction ([[ileus]]) due to overly thick [[meconium]], which is normally broken down by trypsin and other proteases, then passed in feces.<ref name="pmid11729110">{{cite journal | vauthors = Noone PG, Zhou Z, Silverman LM, Jowell PS, Knowles MR, Cohn JA | title = Cystic fibrosis gene mutations and pancreatitis risk: relation to epithelial ion transport and trypsin inhibitor gene mutations | journal = Gastroenterology | volume = 121 | issue = 6 | pages = 1310–9 | date = December 2001 | pmid = 11729110 | doi = 10.1053/gast.2001.29673 | url = http://linkinghub.elsevier.com/retrieve/pii/S001650850176831X }}</ref> | ||
==Applications== | ==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. | 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 | In a [[tissue culture]] lab, trypsin is used to resuspend cells adherent to the cell culture dish wall during the process of harvesting cells.<ref>{{cite web|url=http://www.stemcell.com/en/Products/All-Products/TrypsinEDTA-025.aspx|title=Trypsin-EDTA (0.25%) |publisher=Stem Cell Technologies |date= |access-date=2012-02-23}}</ref> Some cell types adhere to the sides and bottom of a dish when cultivated ''[[in vitro]]''. Trypsin is used to cleave proteins holding the cultured cells to the dish, so that the cells can be removed from the plates. | ||
Trypsin can also be used to dissociate dissected cells (for example, prior to cell fixing and sorting). | 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 | 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 causes the milk to become [[translucent]]. The rate of reaction can be measured by using the amount of time needed 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 | 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 arginine or lysine residue. | ||
Trypsin can also be used to dissolve blood clots in its microbial form and treat inflammation in its pancreatic form. | Trypsin can also be used to dissolve blood clots in its microbial form and treat inflammation in its pancreatic form. | ||
=== In food === | === In food === | ||
Commercial protease preparations usually consist of a mixture of various protease enzymes that often includes trypsin. These preparations are widely | Commercial protease preparations usually consist of a mixture of various protease enzymes that often includes trypsin. These preparations are widely used in food processing:<ref name="urlProtease - GMO Database">{{cite web | url = http://www.gmo-compass.org/eng/database/enzymes/94.protease.html | title = Protease - GMO Database | date = 2010-07-10 | work = GMO Compass | publisher = European Union | access-date = 2012-01-01 | deadurl = yes | archiveurl = https://web.archive.org/web/20150224164346/http://www.gmo-compass.org/eng/database/enzymes/94.protease.html | archivedate = 2015-02-24 | df = }}</ref> | ||
* as a baking enzyme to improve the workability of dough | * as a baking enzyme to improve the workability of dough | ||
* in the extraction of seasonings and | * in the extraction of seasonings and flavorings from vegetable or animal proteins and in the manufacture of sauces | ||
* to control aroma formation in cheese and milk products | * to control aroma formation in cheese and milk products | ||
* to improve the texture of fish products | * to improve the texture of fish products | ||
* to tenderize meat | * to tenderize meat | ||
* during cold stabilization of beer | * during cold stabilization of beer | ||
* in the production of [[hypoallergenic]] food where proteases break down specific [[allergen]]ic proteins into nonallergenic peptides | * in the production of [[hypoallergenic]] food where proteases break down specific [[allergen]]ic 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 allergy|milk allergies]]. | ||
==Trypsin inhibitor== | ==Trypsin inhibitor== | ||
{{main|Trypsin inhibitor}} | {{main|Trypsin inhibitor}} | ||
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 [[Alpha 1-antitrypsin|α1-antitrypsin]] in the serum are present as part of the defense against its inappropriate activation. Any trypsin prematurely formed from the inactive trypsinogen is then bound by the inhibitor. The protein-protein interaction between trypsin and its inhibitors is one of the tightest bound, and trypsin is bound by some of its pancreatic inhibitors nearly irreversibly.<ref>{{cite book | first1 = Donald | last1 = Voet | first2 = Judith G. | last2 = Voet | name-list-format = vanc |title=Biochemistry |pages=396–400|edition=2nd |year=1995|publisher=John Wiley & Sons |isbn=978-0-471-58651-7 }}</ref> 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.<ref>{{cite journal | vauthors = Levilliers N, Péron M, Arrio B, Pudles J | title = On the mechanism of action of proteolytic inhibitors. IV. Effect of 8 M urea on the stability of trypsin in trypsin-inhibitor complexes | journal = Archives of Biochemistry and Biophysics | volume = 140 | issue = 2 | pages = 474–83 | date = October 1970 | pmid = 5528741 | doi = 10.1016/0003-9861(70)90091-3 }}</ref> | |||
== References == | == References == | ||
{{Reflist}} | {{Reflist}} | ||
==External links== | == External links == | ||
* The [[MEROPS]] online database for peptidases and their inhibitors: Trypsin 1 [http://merops.sanger.ac.uk/cgi-bin/merops.cgi?id=S01.151 S01.151], Trypsin 2 [http://merops.sanger.ac.uk/cgi-bin/merops.cgi?id=S01.258 S01.258], Trypsin 3 [http://merops.sanger.ac.uk/cgi-bin/merops.cgi?id=S01.174 S01.174] | * The [[MEROPS]] online database for peptidases and their inhibitors: Trypsin 1 [http://merops.sanger.ac.uk/cgi-bin/merops.cgi?id=S01.151 S01.151], Trypsin 2 [http://merops.sanger.ac.uk/cgi-bin/merops.cgi?id=S01.258 S01.258], Trypsin 3 [http://merops.sanger.ac.uk/cgi-bin/merops.cgi?id=S01.174 S01.174] | ||
* [http://www.sigmaaldrich.com/catalog/search/TablePage/15846780 Trypsin Inhibitors] and [http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Enzyme_Explorer/Analytical_Enzymes/Trypsin.html Trypsin Assay Method] at [[Sigma-Aldrich]] | * [http://www.sigmaaldrich.com/catalog/search/TablePage/15846780 Trypsin Inhibitors] and [http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Enzyme_Explorer/Analytical_Enzymes/Trypsin.html Trypsin Assay Method] at [[Sigma-Aldrich]] | ||
Latest revision as of 07:26, 10 January 2019
| Trypsin | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| File:1UTN.png | |||||||||
| Identifiers | |||||||||
| Symbol | Trypsin | ||||||||
| Pfam | PF00089 | ||||||||
| InterPro | IPR001254 | ||||||||
| SMART | SM00020 | ||||||||
| PROSITE | PDOC00124 | ||||||||
| MEROPS | S1 | ||||||||
| SCOP | 1c2g | ||||||||
| SUPERFAMILY | 1c2g | ||||||||
| CDD | cd00190 | ||||||||
| |||||||||
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 hydrolyzes 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. 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.[4] Trypsin was discovered in 1876 by Wilhelm Kühne.[5]
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.[6]
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. Autocatalysis does not happen with trypsin, as trypsinogen is a poor substrate, therefore enzymatic damage to the pancreas is avoided.[6]
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.[7] This catalytic triad was formerly called a charge relay system, implying the abstraction of protons from serine to histidine and from histidine to aspartate, but owing to evidence provided by NMR that the resultant alkoxide form of serine would have a much stronger pull on the proton than does the imidazole ring of histidine, current thinking holds instead that serine and histidine each have effectively equal share of the proton, forming short low-barrier hydrogen bonds therewith.[8] By these means, the nucleophilicity of the active site serine is increased, facilitating its attack on the amide carbon during proteolysis. 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 through hydrogen bonding stabilize the negative charge which accumulates on the amide oxygen after nucleophilic attack on the planar amide carbon by the serine oxygen causes that carbon to assume a tetrahedral geometry. Such stabilisation of this tetrahedral intermediate helps to reduce the energy barrier of its formation and is concomitant with a lowering of the free energy of the transition state. Preferential binding of the transition state is a key feature of enzyme chemistry.
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,[9] although large-scale mass spectrometry data suggest cleavage occurs even with proline.[10] 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.[11] In contrast, the Atlantic cod has several types of trypsins 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).[12]
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 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
These human genes encode proteins with trypsin enzymatic activity:
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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, which involves intestinal obstruction (ileus) due to overly thick meconium, which is normally broken down by trypsin and other proteases, then passed in feces.[13]
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 resuspend cells adherent to the cell culture dish wall during the process of harvesting cells.[14] Some cell types adhere to the sides and bottom of a dish when cultivated in vitro. Trypsin is used to cleave proteins holding the cultured cells to the dish, so that the cells can be removed from the plates.
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 causes the milk to become translucent. The rate of reaction can be measured by using the amount of time needed 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 arginine or lysine residue.
Trypsin can also be used to dissolve blood clots in its microbial form and treat inflammation in its pancreatic form.
In food
Commercial protease preparations usually consist of a mixture of various protease enzymes that often includes trypsin. These preparations are widely used in food processing:[15]
- as a baking enzyme to improve the workability of dough
- in the extraction of seasonings and flavorings 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
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 is then bound by the inhibitor. The protein-protein interaction between trypsin and its inhibitors is one of the tightest bound, and trypsin is bound by some of its pancreatic inhibitors nearly irreversibly.[16] 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.[17]
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 Science. 13 (4): 1056–70. doi:10.1110/ps.03498604. PMC 2280040. PMID 15044735.
- ↑ Rawlings ND, Barrett AJ (1994). "Families of serine peptidases". Methods in Enzymology. 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.
- ↑ 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.
- ↑ 6.0 6.1 "Digestion of Proteins". intranet.tdmu.edu.ua. Retrieved 2017-04-24.
- ↑ Polgár L (October 2005). "The catalytic triad of serine peptidases". Cellular and Molecular Life Sciences. 62 (19–20): 2161–72. doi:10.1007/s00018-005-5160-x. PMID 16003488.
- ↑ Voet D, Voet JG (2011). Biochemistry (4th ed.). Hoboken, NJ: John Wiley & Sons. ISBN 9780470570951. OCLC 690489261.
- ↑ "Sequencing Grade Modified Trypsin" (PDF). www.promega.com. 2007-04-01. Retrieved 2009-02-08.
- ↑ Rodriguez J, Gupta N, Smith RD, Pevzner PA (January 2008). "Does trypsin cut before proline?" (PDF). Journal of Proteome Research. 7 (1): 300–5. doi:10.1021/pr0705035. PMID 18067249.
- ↑ Hustoft HK, Malerod H, Wilson SR, Reubsaet L, Lundanes E, Greibrokk T. "A Critical Review of Trypsin Digestion for LC-MS Based Proteomics" (PDF). Norway: University of Oslo. p. 80.
- ↑ Gudmundsdóttir A, Pálsdóttir HM (2005). "Atlantic cod trypsins: from basic research to practical applications". Marine Biotechnology. 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.
- ↑ "Protease - GMO Database". GMO Compass. European Union. 2010-07-10. Archived from the original on 2015-02-24. Retrieved 2012-01-01.
- ↑ Voet D, Voet JG (1995). Biochemistry (2nd ed.). John Wiley & Sons. pp. 396–400. ISBN 978-0-471-58651-7.
- ↑ Levilliers N, Péron M, Arrio B, Pudles J (October 1970). "On the mechanism of action of proteolytic inhibitors. IV. Effect of 8 M urea on the stability of trypsin in trypsin-inhibitor complexes". Archives of Biochemistry and Biophysics. 140 (2): 474–83. 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)