Trypsin is an

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

in the first section of the small intestine that starts the digestion of

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

molecules by cutting long chains of

amino acids Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the Proteinogenic amino acid, 22 α-amino acids incorporated into p ...

into smaller pieces. It is a serine protease from the PA clan superfamily, found in the

digestive system The human digestive system consists of the gastrointestinal tract plus the accessory organs of digestion (the tongue, salivary glands, pancreas, liver, and gallbladder). Digestion involves the breakdown of food into smaller and smaller compone ...

of many

vertebrates Vertebrates () are animals with a vertebral column (backbone or spine), and a cranium, or skull. The vertebral column surrounds and protects the spinal cord, while the cranium protects the brain. The vertebrates make up the subphylum Vertebra ...

, where it hydrolyzes

s. Trypsin is formed in the

small intestine The small intestine or small bowel is an organ (anatomy), organ in the human gastrointestinal tract, gastrointestinal tract where most of the #Absorption, absorption of nutrients from food takes place. It lies between the stomach and large intes ...

when its proenzyme form, the trypsinogen produced by the

pancreas The pancreas (plural pancreases, or pancreata) is an Organ (anatomy), organ of the Digestion, digestive system and endocrine system of vertebrates. In humans, it is located in the abdominal cavity, abdomen behind the stomach and functions as a ...

, is activated. Trypsin cuts

peptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty am ...

chains mainly at the carboxyl side of the

lysine or

arginine Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidinium, guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) a ...

. It is used for numerous biotechnological processes. The process is commonly referred to as trypsinogen

proteolysis Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Protein degradation is a major regulatory mechanism of gene expression and contributes substantially to shaping mammalian proteomes. Uncatalysed, the hydrolysis o ...

or trypsinization, and proteins that have been digested/treated with trypsin are said to have been trypsinized. Trypsin was discovered in 1876 by Wilhelm Kühne. Although many sources say that Kühne named trypsin from the

Ancient Greek Ancient Greek (, ; ) includes the forms of the Greek language used in ancient Greece and the classical antiquity, ancient world from around 1500 BC to 300 BC. It is often roughly divided into the following periods: Mycenaean Greek (), Greek ...

word for rubbing, 'tripsis', because the enzyme was first isolated by rubbing the pancreas with glass powder and alcohol, in fact Kühne named trypsin from the Ancient Greek word 'thrýpto' which means 'I break' or 'I break apart'.

Function

In the

duodenum The duodenum is the first section of the small intestine in most vertebrates, including mammals, reptiles, and birds. In mammals, it may be the principal site for iron absorption. The duodenum precedes the jejunum and ileum and is the shortest p ...

, trypsin catalyzes the

hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...

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

. 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

) via the pancreatic duct. Once in the small intestine, the enzyme enterokinase (also called enteropeptidase) activates trypsinogen into trypsin by proteolytic cleavage. The trypsin then activates additional trypsin, chymotrypsin and

carboxypeptidase A carboxypeptidase ( EC number 3.4.16 - 3.4.18) is a protease enzyme that hydrolyzes (cleaves) a peptide bond at the carboxy-terminal (C-terminal) end of a protein or peptide. This is in contrast to an aminopeptidases, which cleave peptide b ...

Mechanism

The enzymatic mechanism is similar to that of other serine proteases. These enzymes contain a catalytic triad consisting of

histidine Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an Amine, α-amino group (which is in the protonated –NH3+ form under Physiological condition, biological conditions), a carboxylic ...

-57, aspartate-102, and serine-195. 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. 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 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

s at the carboxyl side (or " C-terminal side") of the

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...

s lysine and arginine except when either is bound to a C-terminal proline, although large-scale mass spectrometry data suggest cleavage occurs even with proline. 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. In contrast, the

Atlantic cod The Atlantic cod (: cod; ''Gadus morhua'') is a fish of the family Gadidae, widely consumed by humans. It is also commercially known as '' cod'' or ''codling''.poikilotherm fish to survive at different body temperatures. Cod trypsins include trypsin I with an activity range of and maximal activity at , as well as trypsin Y with a range of and a maximal activity at . 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. 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 Pancreatitis is a condition characterized by inflammation of the pancreas. The pancreas is a large organ behind the stomach that produces digestive enzymes and a number of hormone A hormone (from the Ancient Greek, Greek participle , "se ...

. One consequence of the autosomal recessive disease

cystic fibrosis Cystic fibrosis (CF) is a genetic disorder inherited in an autosomal recessive manner that impairs the normal clearance of Sputum, mucus from the lungs, which facilitates the colonization and infection of the lungs by bacteria, notably ''Staphy ...

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.

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. Some cell types adhere to the sides and bottom of a dish when cultivated ''

in vitro ''In vitro'' (meaning ''in glass'', or ''in the glass'') Research, studies are performed with Cell (biology), cells or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in ...

''. 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. In India, Trypsin - Chymotrypsin is widely prescribed for inflammation reduction during laryngitis and surgery recovery. Use outside India is not well documented and most papers written on its effectivity in the aforementioned situations have been funded by Torrent Pharmaceuticals which is one major brand that makes these tablets in India.

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: * 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. 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. Trypsin inhibitors can serve as tools when addressing metabolic and obesity disorders. Metabolic disorders, obesity, and being overweight are known to increase non-communicable chronic disease prevalence. It is of public health policy interest to explore various ways to mitigate this occurrence including use of trypsin inhibitors. These inhibitors have capabilities of reducing colon, breast, skin, and prostate cancer by way of radioprotective and anticarcinogenic activity. Trypsin inhibitors can act as regulatory mechanisms to control release of neutrophil proteases and avoid significant tissue damage. In regards to cardiovascular conditions associated with unproductive serine protease activity, trypsin inhibitors can block their activity in platelet aggregation, fibrinolysis, coagulation, and blood coagulation. The multifunctionality of trypsin inhibitors includes being potential protease inhibitors for AMP activity. While the antibacterial action mechanisms of trypsin inhibitors are unclear, studies have aimed to study their mechanisms as potential applications in bacterial infection treatments. Research and scanning microscopy showed antibacterial effects on bacterial membranes from ''

Staphylococcus aureus ''Staphylococcus aureus'' is a Gram-positive spherically shaped bacterium, a member of the Bacillota, and is a usual member of the microbiota of the body, frequently found in the upper respiratory tract and on the skin. It is often posi ...

''. Trypsin inhibitors from amphibian skin showed bacterial death promotion that affected the cell wall and membrane of ''Staphylococcus aureus''. Studies also analyzed antibacterial actions in trypsin inhibitor peptides, proteins, and '' E. coli''. The results showed sufficient bacterial growth prevention. However, trypsin inhibitors have to meet certain criteria to be utilized in foods and medical treatments.

Trypsin alternatives

Trypsin digestion of extra cellular matrix is a common practice in cell culture. However, this enzymatic degradation of the cells can negatively effect cell viability and surface markers, especially in stem cells. There are gentler alternatives than trypsin such as Accutase which doesn't effect surface markers such as cd14, cd117, cd49f, cd292. However Accutase decreases the surface levels of FasL and Fas receptor on

macrophage Macrophages (; abbreviated MPhi, φ, MΦ or MP) are a type of white blood cell of the innate immune system that engulf and digest pathogens, such as cancer cells, microbes, cellular debris and foreign substances, which do not have proteins that ...

s, these receptors are associated with cell cytotoxicity in the immune system and can also facilitate

apoptosis Apoptosis (from ) is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemistry, Biochemical events lead to characteristic cell changes (Morphology (biol ...

-related cell death. ProAlanase could also serve as an alternative to Trypsin in proteomic applications. ProAlanase is an ''Aspergillus niger'' fungus protease that can achieve high proteolytic activity and specificity for digestion under the correct conditions. ProAnalase, the acidic prolyl-endopeptidase protease, previously studied as An-PEP, has been observed in various experiments to define its specificity. ProAnalase performed optimally in LC-MS applications with short digestion times and highly acidic pH.

References

Sigma-Aldrich Sigma-Aldrich (formally MilliporeSigma) is an American chemical, life science, and biotechnology company owned by the multinational chemical conglomerate Merck Group. Sigma-Aldrich was created in 1975 by the merger of Sigma Chemical Company and ...

* {{Enzymes Cell culture reagents EC 3.4.21 Proteases