Trypsin

Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen. 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.

Function
Trypsin in the duodenum acts to hydrolyse peptides into amino acids. This is a necessary step in protein absorption because peptides (though smaller than proteins) are too big to be absorbed through the lining of the small intestine. Trypsin catalyses the hydrolysis of peptide bonds.

Trypsin is produced in the pancreas in the form of the inactive zymogen trypsinogen. 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 it into trypsin by proteolytic cleavage. Trypsin can then function to activate additional trypsinogen (autocatalysis), so only a small amount of enteropeptidase is necessary to start the reaction. This activation mechanism is common for most serine proteases, and serves to prevent autodegradation of the pancreas.

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. These three residues form a charge relay that serves to make the active site serine nucleophilic. This is achieved by modifying the electrostatic environment of the serine. The enzymatic reaction that trypsins catalyze 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 trypsins 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 N-terminal proline. Trypsins are considered endopeptidases, i.e., the cleavage occurs within the polypeptide chain rather than at the terminal amino acids located at the ends of polypeptides.

Properties
Trypsins have an optimal operating pH of about 8 and optimal operating temperature of about 37°C.

The activity of trypsins is not affected by the 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.

Trypsins should be stored at very cold temperatures (between &minus;20°C and &minus;80°C) to prevent autolysis, which may also be impeded by storage of trypsins 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:

Clinical significance
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 trypsins and other proteases, then passed in faeces.

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, trypsins are used to re-suspend cells adherent to the cell culture dish wall during the process of harvesting cells. 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. When used for this purpose, the trypsin solution must only be allowed to be in contact with the cells for a short period of time (< 60 seconds), otherwise the trypsin may damage the cells.

Trypsin can also be used to dissociate dissected cells (for example, prior to cell fixing and sorting).

Trypsins 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.

Trypsin is used in baby food to pre-digest it. It can break down the protein molecules, which helps the baby to digest it, as its stomach is not strong enough to digest bigger protein molecules.