Isolation and characterization of chymotrypsin inhibitor and trypsin inhibitors from seeds of momordica cochinchinensis.

• Other Authors: Wong, Ricardo Chi Ho.
• Format: Others
• Language: English; Chinese
• Published: 2000
• Subjects: Serine Proteinase Inhibitors > isolation & purification; Chymotrypsin > antagonists & inhibitors; Trypsin Inhibitors
• Online Access: http://library.cuhk.edu.hk/record=b5890258; http://repository.lib.cuhk.edu.hk/en/item/cuhk-323235

by Ricardo Wong Chi Ho. === Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. === Includes bibliographical references (leaves 128-138). === Abstracts in English and Chinese. === Acknowledgments --- p.i === Abstract --- p.ii === 論文摘要 --- p.iv === Table of Contents --- p.vi === List of Figures --- p.xi === List of Tables --- p.xiii === List of Abbreviations --- p.xiv === Chapter Chapter 1 --- Introduction --- p.1 === Chapter 1.1 --- Overview of Serine Protease Inhibitors --- p.1 === Chapter 1.2 --- Classification of Serine Protease Inhibitors --- p.2 === Chapter 1.2.1 --- Kunitz Type Serine Protease Inhibitors --- p.7 === Chapter 1.2.2 --- Bowman-Birk Type Serine Protease Inhibitors --- p.11 === Chapter 1.2.3 --- Squash Type Serine Protease Inhibitors --- p.16 === Chapter 1.3 --- Role of Serine Protease Inhibitors in Plants --- p.20 === Chapter 1.4 --- Nutritional Fact of Serine Protease Inhibitors --- p.22 === Chapter 1.5 --- Possible Applications of Serine Protease Inhibitors --- p.25 === Chapter 1.5.1 --- Medical Applications --- p.25 === Chapter 1.5.2 --- Agricultural Applications --- p.29 === Chapter 1.6 --- Rationale of the Present Study --- p.31 === Chapter Chapter 2 --- Screening of Seeds for Inhibitory Activities Against Serine Proteases --- p.33 === Chapter 2.1 --- Introduction --- p.33 === Chapter 2.2 --- Materials and Methods --- p.37 === Chapter 2.2.1 --- Materials --- p.37 === Chapter 2.2.2 --- Extraction Method --- p.37 === Chapter 2.2.3 --- Assays for Proteases Inhibitory Activities --- p.38 === Chapter 2.2.3.1 --- Assay for Chymotrypsin Activity --- p.38 === Chapter 2.2.3.2 --- Assay for Trypsin Activity --- p.38 === Chapter 2.2.3.3 --- Assay for Elastase Activity --- p.39 === Chapter 2.2.3.4 --- Assay for Subtilisin Activity --- p.39 === Chapter 2.2.3.5 --- Assays for Protease Inhibitory Activities --- p.40 === Chapter 2.2.4 --- Determination of Protein Concentration --- p.41 === Chapter 2.3 --- Results --- p.42 === Chapter 2.3.1 --- Extraction --- p.42 === Chapter 2.3.2 --- Serine Proteases Inhibitory Activities --- p.42 === Chapter 2.4 --- Discussion --- p.47 === Chapter Chapter 3 --- Isolation of Chymotrypsin Inhibitor and Trypsin Inhibitors from Momordica cochinchinensis Seeds --- p.49 === Chapter 3.1 --- Introduction --- p.49 === Chapter 3.2 --- Materials and Methods --- p.56 === Chapter 3.2.1 --- Materials --- p.56 === Chapter 3.2.2 --- Protein Extraction --- p.57 === Chapter 3.2.3 --- SP-Sepharose Chromatography --- p.57 === Chapter 3.2.4 --- Reversed Phase High Pressure Liquid Chromatography --- p.58 === Chapter 3.2.5 --- Assays for Chymotrypsin and Trypsin Inhibitory Activities --- p.60 === Chapter 3.2.6 --- Titration of Chymotrypsin --- p.61 === Chapter 3.2.7 --- Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.62 === Chapter 3.2.8 --- Coupling of Trypsin-Sepharose 4B Affinity Column --- p.63 === Chapter 3.2.9 --- Affinity Chromatography on Trypsin-Sepharose 4B --- p.64 === Chapter 3.3 --- Results --- p.65 === Chapter 3.3.1 --- SP-Sepharose Chromatography --- p.65 === Chapter 3.3.2 --- Reversed Phase High Pressure Liquid Chromatography --- p.67 === Chapter 3.3.3 --- Summary of Purification --- p.71 === Chapter 3.3.4 --- Titration of Chymotrypsin --- p.74 === Chapter 3.3.5 --- Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.74 === Chapter 3.3.6 --- Affinity Chromatography on Trypsin-Sepharose 4B --- p.78 === Chapter 3.4 --- Discussion --- p.81 === Chapter Chapter 4 --- Characterization of Chymotrypsin Inhibitor and Trypsin Inhibitors --- p.88 === Chapter 4.1 --- Introduction --- p.88 === Chapter 4.2 --- Materials and Methods --- p.90 === Chapter 4.2.1 --- Materials --- p.90 === Chapter 4.2.2 --- Determination of Molecular Weight --- p.90 === Chapter 4.2.3 --- Amino Acid Sequence Analysis --- p.91 === Chapter 4.2.4 --- Surface Plasmon Resonance Measurement --- p.92 === Chapter 4.2.4.1 --- Immobilization of Ligands on the Surface of Optical Biosensors --- p.92 === Chapter 4.2.4.2 --- Determination of Kinetics Constants --- p.93 === Chapter 4.2.4.3 --- pH Dependence of the Inhibition by Chymotrypsin Inhibitor --- p.93 === Chapter 4.2.4.4 --- Data Analysis --- p.94 === Chapter 4.2.5 --- Effect of Chymotrypsin Inhibitor on the Estereolytic Activity and Proteolytic Activity of Chymotrypsin --- p.95 === Chapter 4.2.6 --- Specificities of the Inhibitors % --- p.96 === Chapter 4.2.7 --- Binding Ratio of CI to Different Proteases --- p.97 === Chapter 4.2.8 --- Effects of the Proteases on Their Corresponding Inhibitors --- p.97 === Chapter 4.2.8.1 --- Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.97 === Chapter 4.2.8.2 --- Assay for Chymotrypsin Inhibitory Activity --- p.98 === Chapter 4.3 --- Results --- p.99 === Chapter 4.3.1 --- Molecular Weight of the Inhibitors --- p.99 === Chapter 4.3.2 --- N-terminal Amino Acid Sequence --- p.99 === Chapter 4.3.3 --- Surface Plasmon Resonance Measurement --- p.102 === Chapter 4.3.3.1 --- Kinetics of Chymotrypsin Inhibitor --- p.102 === Chapter 4.3.3.2 --- Kinetics of Trypsin Inhibitors --- p.106 === Chapter 4.3.3.3 --- pH Dependence of the Inhibition by Chymotrypsin Inhibitor --- p.106 === Chapter 4.3.4 --- Effect of Chymotrypsin Inhibitor on the Estereolytic Activity and Proteolytic Activity of Chymotrypsin --- p.106 === Chapter 4.3.5 --- Specificities of the Inhibitors --- p.110 === Chapter 4.3.6 --- Binding Ratio of CI to Different Proteases --- p.112 === Chapter 4.3.7 --- Effects of the Proteases on Their Corresponding Inhibitors --- p.112 === Chapter 4.4 --- Discussion --- p.119 === Chapter Chapter 5 --- Conclusion --- p.125 === References --- p.128