trypsin n : an enzyme of pancreatic origin; catalyzes the hydrolysis of proteins to smaller polypeptide units

English

Noun

1. A digestive enzyme that cleaves peptide bonds (a serine protease).

Trypsin () is a serine protease found in the digestive system, where it breaks down proteins. Trypsin predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine and 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.

Chemistry and function

Trypsin is secreted into the duodenum, where it acts to hydrolyse peptides into its smaller building blocks, namely amino acids (these peptides are the result of the enzyme pepsin breaking down the proteins in the stomach). This is necessary for the uptake of protein in the food as though peptides are smaller than proteins, they are still too big to be absorbed though the lineing of the ileum. Trypsin catalyses the hydrolysis of peptide bonds. The enzymatic mechanism is like all other serine proteases: A catalytic triad 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"). Trypsins have an optimal operating pH of about 8 and optimal operating temperature of about 37°C.

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 followed by 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.

Trypsin is produced in the pancreas in the form of inactive zymogen, trypsinogen. It is then secreted into the small intestine, where the enzyme enteropeptidase activates it into trypsin by proteolytic cleavage. The resulting trypsins themselves activate more trypsinogens (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 autodigestion of the pancreas.

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.

Involvement in disease

One consequence of inheriting the autosomal recessive disease cystic fibrosis is a deficiency 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 feces.

Storage

Trypsins should be stored at very cold temperatures (between −20°C and −80°C) to prevent autolysis (self-cleavage). Autolysis may also be prevented by storage of trypsins at pH 3 or by using trypsin modified by e.g. reductive methylation. When the pH is adjusted back to pH 8 activity returns.

Applications

Trypsin is available in high quantities 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.

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

Trypsins can be used to breakdown 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.

See also

• Trypsin inhibitor

References

1. Promega PDF - 58kB

External links

• Trypsin Inhibitors and Trypsin Assay Method at Sigma-Aldrich