Trypsin का स्रोत देखें

{{Short description|Family of digestive enzymes}}
{{Pfam box
| Symbol = Trypsin
| Name = Trypsin
| image = ChimeraX rendering of bovine trypsin (PDB 1UTN).png
| 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 | display-authors = 6 | 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 | doi-access = free }}</ref>
| Pfam = PF00089
| InterPro = IPR001254
| SMART = SM00020
| PROSITE = PDOC00124
| MEROPS = S1
| SCOP = 1c2g
| TCDB = 
| CDD = cd00190
| OPM family = 
| OPM protein = 
}}'''Trypsin''' is an [[enzyme]] in the first section of the small intestine that starts the digestion of [[protein]] molecules by cutting these long chains of amino acids into smaller pieces. It 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 | pages = 19–61 | year = 1994 | pmid = 7845208 | doi = 10.1016/0076-6879(94)44004-2 | pmc = 7133253 | url = https://archive.org/details/proteolyticenzym0000unse/page/19 | url-access = registration | isbn = 978-0-12-182145-6 }}</ref><ref>The German physiologist [[Wilhelm Kühne]] (1837-1900) discovered trypsin in 1876.  See:  {{cite journal | vauthors=Kühne W|date=1877|title=Über das Trypsin (Enzym des Pankreas) | url= https://books.google.com/books?id=jzdMAAAAYAAJ&pg=PA194 |journal=Verhandlungen des Naturhistorisch-medicinischen Vereins zu Heidelberg|series=new series|volume=1|issue=3|pages=194–198|via=Google Books}}{{Open access}}</ref> Trypsin is formed in the [[small intestine]] when its [[zymogen|proenzyme]] form, the [[trypsinogen]] produced by the [[pancreas]], is activated. Trypsin cuts [[peptide]] chains mainly at the [[carboxyl]] side of the [[amino acids]] [[lysine]] or [[arginine]]. It is used for numerous [[biotechnological]] processes. The process is commonly referred to as trypsin [[proteolysis]] or [[trypsinisation|trypsinization]], and proteins that have been digested/treated with trypsin are said to have been trypsinized.<ref>{{cite book | vauthors = Engelking LR |chapter=Chapter 7 - Protein Digestion |title=Textbook of Veterinary Physiological Chemistry |date=2015-01-01 |location=Boston |publisher=Academic Press |edition=Third |pages=39–44 | doi = 10.1016/B978-0-12-391909-0.50007-4 |isbn=978-0-12-391909-0 }}</ref> Trypsin was discovered in 1876 by [[Wilhelm Kühne]] and was named from the [[Ancient Greek]] word for rubbing since it was first isolated by rubbing the pancreas with [[glycerin]].<ref>{{cite book| vauthors = Kühne W | chapter=Ueber das Trypsin (Enzym des Pankreas)|trans-chapter=About trypsin (enzyme of the pancreas) |pages=194–8 |chapter-url=https://archive.org/stream/verhandlungendes7477natu#page/194/mode/2up|via=Archive.org |title=Verhandlungen des Naturhistorisch-medizinischen Vereins zu Heidelberg |trans-title=Negotiations by the Natural History Medical Association in Heidelberg<!--Google Translate--> |language=de |date=March 6, 1876 |publisher=Carl Winter's Universitätsbuchhandlung |editor-last=Naturhistorisch-medizinischen Verein |location=Heidelberg, Germany |publication-date=1877 }}</ref>{{enzyme
| Name = Trypsin
| EC_number = 3.4.21.4
| CAS_number = 9002-07-7
| IUBMB_EC_number = 3/4/21/4
| GO_code = 0004295
| image = 
| width = 
| caption =
|name=Trypsin}}
== 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 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 | date=July 14, 2015<!--last mod date from page info-->|website=Elective course (Clinical biochemistry)|publisher=Ternopil National Medical University|access-date=April 11, 2020}}</ref>

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|enterokinase]] (also called enteropeptidase) activates trypsinogen into trypsin by [[proteolytic cleavage]]. The trypsin then activates additional trypsin, chymotrypsin and carboxypeptidase.

== 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.<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 | s2cid = 3343824 }}</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 | title = Biochemistry | vauthors = Voet D, Voet JG |date=2011 |publisher=John Wiley & Sons |isbn=978-0-470-57095-1 |edition=4th |location=Hoboken, NJ |oclc=690489261}}</ref>{{Page needed|date=April 2020}}  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 stabilization 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 negative 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 | publisher = www.promega.com | 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 | citeseerx = 10.1.1.163.4052 }}</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 ==
Human trypsin has an optimal [[operating temperature]] of about 37 °C.<ref>{{cite journal | vauthors = Chelulei Cheison S, Brand J, Leeb E, Kulozik U | title = Analysis of the effect of temperature changes combined with different alkaline pH on the β-lactoglobulin trypsin hydrolysis pattern using MALDI-TOF-MS/MS | journal = Journal of Agricultural and Food Chemistry | volume = 59 | issue = 5 | pages = 1572–81 | date = March 2011 | pmid = 21319805 | doi = 10.1021/jf1039876 }}</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 | s2cid = 42480996 }}</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.

The activity of trypsin is not affected by the [[enzyme inhibitor]] tosyl phenylalanyl chloromethyl ketone, [[tosyl phenylalanyl chloromethyl ketone|TPCK]], which deactivates [[chymotrypsin]]. 

Trypsin should be stored at very cold temperatures (between &minus;20 and &minus;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 ==

These human genes encode proteins with trypsin enzymatic activity:
{|
|- valign="top"
|{{infobox protein
|Name=[[Trypsin 1|protease, serine, 1 (trypsin 1)]]
|caption= 
|image=
|width=
|HGNCid=9475
|Symbol=[[Trypsin 1|PRSS1]]
|AltSymbols=TRY1
|EntrezGene=5644
|OMIM=276000
|RefSeq=NM_002769
|UniProt=P07477
|PDB=
|ECnumber=3.4.21.4
|Chromosome=7
|Arm=q
|Band=32
|LocusSupplementaryData=-qter
}}
|{{infobox protein
|Name=[[PRSS2|protease, serine, 2 (trypsin 2)]]
|caption=
|image=
|width=
|HGNCid=9483
|Symbol=[[PRSS2]]
|AltSymbols= TRYP2
|EntrezGene=5645
|OMIM=601564
|RefSeq=NM_002770
|UniProt=P07478
|PDB=
|ECnumber=3.4.21.4
|Chromosome=7
|Arm=q
|Band=35
|LocusSupplementaryData=
}}
|{{infobox protein
|Name=[[PRSS3|protease, serine, 3 (mesotrypsin)]]
|caption=
|image=
|width=
|HGNCid=9486
|Symbol=[[PRSS3]]
|AltSymbols=PRSS4
|EntrezGene=5646
|OMIM= 613578
|RefSeq=NM_002771
|UniProt=P35030
|PDB=
|ECnumber=3.4.21.4
|Chromosome=9
|Arm=p
|Band=13
|LocusSupplementaryData=
}}
|}
Other [[isoform]]s 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.<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 = https://cdr.lib.unc.edu/downloads/4b29bf69m }}</ref>

==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.<ref>{{cite web|url=http://www.stemcell.com/en/Products/All-Products/TrypsinEDTA-025.aspx|title=Trypsin-EDTA (0.25%) |publisher=Stem Cell Technologies |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 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 veterinary medicine, trypsin is an ingredient in wound spray products, such as Debrisol, to dissolve dead tissue and pus in wounds in horses, cattle, dogs, and cats.<ref>{{Cite web | url = https://www.drugs.com/international/debrisol.html | title = Debrisol | publisher = drugs.com }}</ref>

=== 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:<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 | url-status = dead | archive-url = https://web.archive.org/web/20150224164346/http://www.gmo-compass.org/eng/database/enzymes/94.protease.html | archive-date = 2015-02-24 }}</ref>
* 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 [[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==
{{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-style = vanc | title = Biochemistry | pages = [https://archive.org/details/biochemistry00voet_0/page/396 396–400] | edition = 2nd | year = 1995 | publisher = John Wiley & Sons | isbn = 978-0-471-58651-7 | url = https://archive.org/details/biochemistry00voet_0/page/396 }}</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>

== See also ==
* {{Portal-inline|Biology}}

== References ==
{{Reflist}}

== Further reading ==
{{refbegin}}
* {{cite journal | vauthors = Brosens JJ, Salker MS, Teklenburg G, Nautiyal J, Salter S, Lucas ES, Steel JH, Christian M, Chan YW, Boomsma CM, Moore JD, Hartshorne GM, Sućurović S, Mulac-Jericevic B, Heijnen CJ, Quenby S, Koerkamp MJ, Holstege FC, Shmygol A, Macklon NS | display-authors = 6 | title = Uterine selection of human embryos at implantation | journal = Scientific Reports | volume = 4 | pages = 3894 | date = February 2014 | pmid = 24503642 | pmc = 3915549 | doi = 10.1038/srep03894 | bibcode = 2014NatSR...4E3894B | url = https://www.livescience.com/43157-embryo-implant-signals-pregnancy.html | access-date = 15 March 2019 | postscript = . Article on the role of trypsin in the implantation of human embryos. | id = Article number 3894 }}
{{refend}}

== 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]
* [https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-trypsin-inhibitor Trypsin Inhibitors] and [http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Enzyme_Explorer/Analytical_Enzymes/Trypsin.html Trypsin Assay Method] at [[Sigma-Aldrich]]
* {{MeshName|Trypsin}}

{{Serine endopeptidases}}
{{Enzymes}}

[[Category:Cell culture reagents]]
[[Category:EC 3.4.21]]
[[Category:Proteases]]