Trypsin from bovine pancreas

module.
Trypsin consists of a single-chain peptide of 223 amino acid residues, which is produced by removing the N-terminal hexapeptide from trypsinogen, which is cleaved at the Lys-lle peptide bond. The amino acid sequence is cross-linked by six disulfide bonds. This is the natural form of trypsin, β-trypsin. BETA- trypsin can be autolyzed and cleaved on Lys-Ser residue to produce α-trypsin. Trypsin is a member of the serine protease family.

Application.
For trypsin digestive peptides, trypsin: peptides are used in a ratio of about 1 to 1:20. Removing adherent cells from the culture surface is a typical application of this product. The concentration of trypsin needed to remove cells from its substrate mainly depends on the cell type and the age of the culture. Trypsin is also used in cell resuspension during cell culture, protein digestion in proteomics research and various in-gel digestion. Other applications include evaluating crystallization through membrane-based techniques in a study that determines that the presence of kinetic traps limits the folding rate and yield of proteins.

Biochemical / physiological behavior.
Trypsin cleaves peptides at the C-terminal of lysine and arginine residues. If the acid residue is located on either side of the cleavage site, the hydrolysis rate of the reaction slows down, and if the proline residue is located on the carboxyl side of the cleavage site, the hydrolysis stops. The best pH of trypsin activity was 7-9. Trypsin can also play the role of cleavage of ester and amide bonds of amino acid derivatives. EDTA is added to the trypsin solution as a chelating agent, which neutralizes calcium and magnesium ions, which blur the peptide bonds of trypsin. Removal of these ions increases enzyme activity. Serine protease inhibitors, including DFP,TLCK,APMSF,AEBSEF and aprotinin, inhibit trypsin.

Unit definition.
A unit of BAEE activity: using BAEE as the substrate, the absorbance of A253 will change by 0.001 per minute at 25 ℃ and pH 7.6. One BTEE unit = 320 ATEE units.

Points for attention in preparation.
A buffer salt solution without Ca2 + or Mg2 + should be used to dissolve trypsin. It is soluble in 1 mM HCl and the concentration is 1 mg / mL. It is also treated with TPCK and dialyzed. Treatment with LMel 1-toluenesulfonamide-2-phenylethyl chloromethyl ketone (TPCK) reduced the chymotrypsin activity commonly present in trypsin.

Warning.
The solution in 1mM HCl can be stable for one year when stored in small portions at-20 ℃. The presence of Ca2 + can also reduce the self-solubility of trypsin and maintain its stability in solution. Trypsin will also retain most of its activity in 2.0m urea, 2.0m guanidine hydrochloride or 0.1% (w / v) SDS.