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3278  structures 2833  species 0  interactions 76462  sequences 2023  architectures

Family: Trypsin (PF00089)

Summary: Trypsin

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Trypsin Edit Wikipedia article

EC number3.4.21.4
CAS number9002-07-7
IntEnzIntEnz view
ExPASyNiceZyme view
MetaCycmetabolic pathway
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO

Trypsin (EC 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. 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 and was named from the Ancient Greek word for rubbing since it was first isolated by rubbing the pancreas with glycerin.[5]


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.


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.


Human trypsin has an optimal operating temperature of about 37 Â°C.[citation needed] 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 Â°C (40 to 150 Â°F) and maximal activity at 55 Â°C (130 Â°F), as well as trypsin Y with a range of 2 to 30 Â°C (36 to 86 Â°F) and a maximal activity at 21 Â°C (70 Â°F).[11]

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 Â°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.


These human genes encode proteins with trypsin enzymatic activity:

protease, serine, 1 (trypsin 1)
Alt. symbolsTRY1
NCBI gene5644
Other data
EC number3.4.21.4
LocusChr. 7 q32-qter
protease, serine, 2 (trypsin 2)
Alt. symbolsTRYP2
NCBI gene5645
Other data
EC number3.4.21.4
LocusChr. 7 q35
protease, serine, 3 (mesotrypsin)
Alt. symbolsPRSS4
NCBI gene5646
Other data
EC number3.4.21.4
LocusChr. 9 p13

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.[12]


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.[13] 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, typsin is an ingredient in wound spray products, such as Debrisol, to dissolve dead tissue and pus in wounds in horses, cattle, dogs, and cats.[14]

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]

See also


  1. ^ PDB: 1UTN​; Leiros HK, Brandsdal BO, Andersen OA, Os V, Leiros I, Helland R, Otlewski J, Willassen NP, SmalÃ¥s 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.
  2. ^ Rawlings ND, Barrett AJ (1994). Families of serine peptidases. Methods in Enzymology. 244. pp. 19–61. doi:10.1016/0076-6879(94)44004-2. ISBN 978-0-12-182145-6. PMID 7845208.
  3. ^ The German physiologist Wilhelm Kühne (1837-1900) discovered trypsin in 1876. See: Kühne W (1877). "Über das Trypsin (Enzym des Pankreas)". Verhandlungen des naturhistorisch-medicinischen Vereins zu Heidelberg. new series. 1 (3): 194–198.
  4. ^ Engelking LR (2015-01-01). Textbook of Veterinary Physiological Chemistry (Third ed.). Boston: Academic Press. pp. 39–44. ISBN 9780123919090.
  5. ^ "Verhandlungen des Naturhistorisch-medizinischen Vereins zu Heidelberg". Retrieved 2017-04-24.
  6. ^ "Digestion of Proteins". Retrieved 2017-04-24.
  7. ^ 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.
  8. ^ Voet D, Voet JG (2011). Biochemistry (4th ed.). Hoboken, NJ: John Wiley & Sons. ISBN 9780470570951. OCLC 690489261.
  9. ^ "Sequencing Grade Modified Trypsin" (PDF). 2007-04-01. Retrieved 2009-02-08.
  10. ^ Rodriguez J, Gupta N, Smith RD, Pevzner PA (January 2008). "Does trypsin cut before proline?" (PDF). Journal of Proteome Research. 7 (1): 300–5. CiteSeerX doi:10.1021/pr0705035. PMID 18067249.
  11. ^ 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.
  12. ^ 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.
  13. ^ "Trypsin-EDTA (0.25%)". Stem Cell Technologies. Retrieved 2012-02-23.
  14. ^ "Debrisol".
  15. ^ "Protease - GMO Database". GMO Compass. European Union. 2010-07-10. Archived from the original on 2015-02-24. Retrieved 2012-01-01.
  16. ^ Voet D, Voet JG (1995). Biochemistry (2nd ed.). John Wiley & Sons. pp. 396–400. ISBN 978-0-471-58651-7.
  17. ^ 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.

Further reading

External links

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This tab holds the annotation information that is stored in the Pfam database. As we move to using Wikipedia as our main source of annotation, the contents of this tab will be gradually replaced by the Wikipedia tab.

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Literature references

  1. Rawlings ND, Barrett AJ; , Meth Enzymol 1994;244:19-61.: Families of Serine Peptidases PUBMED:7845208 EPMC:7845208

  2. Sprang S, Standing T, Fletterick RJ, Stroud RM, Finer-Moore J, Xuong NH, Hamlin R, Rutter WJ, Craik CS; , Science 1987;237:905-909.: The Three Dimensional Structure of Asnl02 Trypsin: Role of Aspl02 in Serine Protease Catalysis PUBMED:3112942 EPMC:3112942

Internal database links

External database links

This tab holds annotation information from the InterPro database.

InterPro entry IPR001254

This entry represents the active-site-containing domain found in the trypsin family members. The catalytic activity of the serine proteases from the trypsin family is provided by a charge relay system involving an aspartic acid residue hydrogen-bonded to a histidine, which itself is hydrogen-bonded to a serine. The sequences in the vicinity of the active site serine and histidine residues are well conserved in this family of proteases [ PUBMED:3136396 ]. A partial list of proteases known to belong to the trypsin family is shown below.

  • Acrosin.
  • Blood coagulation factors VII, IX, X, XI and XII, thrombin, plasminogen, and protein C.
  • Cathepsin G.
  • Chymotrypsins.
  • Complement components C1r, C1s, C2, and complement factors B, D and I.
  • Complement-activating component of RA-reactive factor.
  • Cytotoxic cell proteases (granzymes A to H).
  • Duodenase I.
  • Elastases 1, 2, 3A, 3B (protease E), leukocyte (medullasin).
  • Enterokinase (EC (enteropeptidase).
  • Hepatocyte growth factor activator.
  • Hepsin.
  • Glandular (tissue) kallikreins (including EGF-binding protein types A, B, and C, NGF-gamma chain, gamma-renin, prostate specific antigen (PSA) and tonin).
  • Plasma kallikrein.
  • Mast cell proteases (MCP) 1 (chymase) to 8.
  • Myeloblastin (proteinase 3) (Wegener's autoantigen).
  • Plasminogen activators (urokinase-type, and tissue-type).
  • Trypsins I, II, III, and IV.
  • Tryptases.

All the above proteins belong to family S1 in the classification of peptidases [ PUBMED:7845208 ] and originate from eukaryotic species. It should be noted that bacterial proteases that belong to family S2A are similar enough in the regions of the active site residues that they can be picked up by the same patterns. These proteases are listed below.

  • Achromobacter lyticus protease I.
  • Lysobacter alpha-lytic protease.
  • Streptogrisin A and B (Streptomyces proteases A and B).
  • Streptomyces griseus glutamyl endopeptidase II.
  • Streptomyces fradiae proteases 1 and 2.

Gene Ontology

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Domain organisation

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Curation and family details

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Seed source: SCOP and Prosite
Previous IDs: trypsin;
Type: Domain
Sequence Ontology: SO:0000417
Author: Lutfiyya LL, Sonnhammer ELL
Number in seed: 70
Number in full: 76462
Average length of the domain: 204.60 aa
Average identity of full alignment: 25 %
Average coverage of the sequence by the domain: 53.40 %

HMM information View help on HMM parameters

HMM build commands:
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 61295632 -E 1000 --cpu 4 HMM pfamseq
Model details:
Parameter Sequence Domain
Gathering cut-off 20.6 20.6
Trusted cut-off 20.6 20.6
Noise cut-off 20.5 20.5
Model length: 221
Family (HMM) version: 29
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Species distribution

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Archea Archea Eukaryota Eukaryota
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For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the Trypsin domain has been found. There are 3278 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein sequence.

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AlphaFold Structure Predictions

The list of proteins below match this family and have AlphaFold predicted structures. Click on the protein accession to view the predicted structure.

Protein Predicted structure External Information
A0A096P6M7 View 3D Structure Click here
A0A0A0MXZ4 View 3D Structure Click here
A0A0A0MY24 View 3D Structure Click here
A0A0B4JCS5 View 3D Structure Click here
A0A0B4JCV5 View 3D Structure Click here
A0A0B4JD89 View 3D Structure Click here
A0A0B4K687 View 3D Structure Click here
A0A0B4K7A7 View 3D Structure Click here
A0A0B4K7A9 View 3D Structure Click here
A0A0B4K7P3 View 3D Structure Click here
A0A0B4K875 View 3D Structure Click here
A0A0B4KFF2 View 3D Structure Click here
A0A0B4LG43 View 3D Structure Click here
A0A0B4LH86 View 3D Structure Click here
A0A0G2JX19 View 3D Structure Click here
A0A0G2K036 View 3D Structure Click here
A0A0G2K4I9 View 3D Structure Click here
A0A0G2K6M2 View 3D Structure Click here
A0A0G2K797 View 3D Structure Click here
A0A0G2K8W8 View 3D Structure Click here
A0A0G2KAN9 View 3D Structure Click here
A0A0G2KCJ9 View 3D Structure Click here
A0A0G2KT77 View 3D Structure Click here
A0A0G2KVK9 View 3D Structure Click here
A0A0G2L5F9 View 3D Structure Click here
A0A0G2L7P1 View 3D Structure Click here
A0A0N4STP7 View 3D Structure Click here
A0A0N4SVQ0 View 3D Structure Click here
A0A0R4IA13 View 3D Structure Click here
A0A0R4ID97 View 3D Structure Click here
A0A0R4IDY5 View 3D Structure Click here
A0A0R4IGA1 View 3D Structure Click here
A0A0R4IHG1 View 3D Structure Click here
A0A0R4IHM1 View 3D Structure Click here
A0A0R4IIJ1 View 3D Structure Click here
A0A0R4IIK0 View 3D Structure Click here
A0A0R4IL35 View 3D Structure Click here
A0A0R4INC3 View 3D Structure Click here
A0A0R4INR4 View 3D Structure Click here
A0A0R4IRW8 View 3D Structure Click here