Article: Trypsin

Trypsin(EC 3.4.21.4) family of serine proteases are enzymes that cleave proteins at the carboxyl side (or "C-terminus") of the basic amino acids lysine and arginine except when these two residues are followed by proline ^[1]. 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.

Chemistry and Function

The reaction that trypsins catalyze is thermodynamically favorable but requires significant activation energy (that is to say, the reaction is kinetically unfavorable). 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. Trypsins have an optimal operating pH of about 8 and optimal operating temperature of about 50°C. One of the consequences 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 - obstruction of the intestine (ileus) due to overly thick meconium, the dark sticky stuff that is normally present in the intestine at birth and, after trypsin and other enzymes from the pancreas have acted on it, is normally defecated.

The activity of trypsins is not affected by tosyl phenylalanyl chloromethyl ketone TPCK, which deactivates chymotrypsin. This is important because, in some applications, like mass spectrometry, the specificity of cleavage is important.

Production

Trypsins are produced in the pancreas in the form of inactive trypsinogens, and are then transported to the small intestine, where after trypsin autocatalytic activation of trypsinogen to trypsin they begin the digestion of proteins to polypeptides. A small amount of another enzyme, enterokinase, is required to catalyze the initial reaction of trypsinogen to trypsin.

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. When the pH is adjusted back to pH 8 activity returns.

Applications

In a tissue culture lab, trypsins are used to re-suspend cells adherent to the petri dish wall during the process of harvesting cells.

Trypsins can be used to breakdown casein in 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 can also be used to dissociate dissected cells. For example, prior to cell fixing and sorting.