酶学实验讲义.pdfVIP

生物化学大实验 实验讲义 酶学实验 指导教师：花宝光 教授 2014 年6 月 1 目录 背景介绍、Trypsin 实验一、胰蛋白酶活性测定 实验二、胰蛋白酶动力学测定 实验三、胰蛋白酶抑制类型判定及Ki 值测定 附录：实验报告要求 2 背景介绍 Trypsin Life Science Metabolomics Enzyme Explorer Analytical Enzymes Trypsin Physical Properties and In Vivo Processing 1. Trypsin Enzyme Commission (EC) Number: 3.4.21.4 Molecular Weight: 23.3 kDa 1,2 (bovine porcine) 1% 3,4 Extinction Coefficient: E = 12.9 - 15.4 (280 nm) ， （15.3 in this experiment） 2,5 pI: 10.1 - 10.5 (bovine) Trypsinogen Molecular Weight: 24 kDa5 (bovine) Extinction Coefficient: E1% = 15.3(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-trypsi