trypsin活性测量方法.doc

Trypsin 背景介绍与实验设计 Trypsin, introduction form sigma /life-science/metabolomics/enzyme-explorer/analytical-enzymes/trypsin.html Physical Properties and In Vivo ProcessingTrypsin Enzyme Commission (EC) Number: Molecular Weight: 23.3 kDa1,2 (bovine porcine)Extinction Coefficient: E1% = 12.9 - 15.4 (280 nm)3,4pI: 10.1 - 10.52,5(bovine)Trypsinogen Molecular Weight: 24 kDa5 (bovine)Extinction Coefficient: E1% = 14.4 (280 nm)pI: 9.32 (bovine) Trypsinogen, the proenzyme (zymogen) form of trypsin, is produced in the acinar exocrine cells of the pancreas. Three isoforms are excreted from the human pancreas. The cationic and anionic forms are the predominant human isoforms. The inhibitor-resistant mesotrypsinogen is found only in trace amounts.6 The proenzyme is activated only after it reaches the lumen of the small intestine. Enterokinase activates pancreatic trypsinogen to trypsin by the hydrolysis of a hexapeptide(for bovine trypsin at the Lys6 - Ile7 peptide bond) from the NH2 terminus. Bovine trypsinogen consists of a single polypeptide chain of 229 amino acids and is cross linked by six disulfide bridges. Trypsin can autocatalytically activate more trypsinogen to trypsin. Trypsin consists of a single chain polypeptide of 223 amino acid residues. This native form of trypsin is refered to as β-trypsin. Autolysis of β-trypsin (which is cleaved at Lys131- Ser132 in the bovine sequence) results in α-trypsin which is held together by disulfide bridges. Trypsin is a member of the serine protease S1 family. The active site amino acid residues of trypsin include His46 and Ser183.2-5 Specificity, Kinetics, Substrates and Assay Methods Specificity and KineticsTrypsin will cleave peptides on the C-terminal side of lysine and arginine amino acid residues. The rate of hydrolysis is slower if an acidic residue is on either side of the cleavage site and no cleavage occurs if a proline residue is on the carboxyl side of the cleavage site. Trypsin will hydrolyze ester and