Lai Chi-wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 93-100). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.II / 摘要 --- p.IV / ABBREVIATION --- p.VI / TABLE OF CONTENTS --- p.VII / LIST OF FIGURES --- p.XII / LIST OF TABLES --- p.XIV / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Catalases --- p.2 / Chapter 1.2 --- Classification of catalases --- p.3 / Chapter 1.2.1 --- Catalase peroxidase (HPI) --- p.3 / Chapter 1.2.2 --- Monofunctional catalases (HPII) --- p.6 / Chapter 1.2.2.1 --- NADPH in catalases --- p.9 / Chapter 1.2.3 --- Mn-catalases --- p.11 / Chapter 1.3 --- Sources and cytotoxic effects of hydrogen peroxide --- p.13 / Chapter 1.4 --- The Chironomidae --- p.14 / Chapter 1.4.1 --- Life cycle of Chironomidae --- p.14 / Chapter 1.4.2 --- Bloodworms --- p.18 / Chapter 1.4.3 --- Sources of bloodworms --- p.19 / Chapter 1.5 --- Aim of the project --- p.22 / Chapter 1.6 --- Application of the project --- p.22 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.24 / Chapter 2.1 --- Protein determination --- p.25 / Chapter 2.2 --- In vitro activity assays --- p.27 / Chapter 2.2.1 --- Catalase activity assay --- p.27 / Chapter 2.2.2 --- Peroxidase activity assay --- p.27 / Chapter 2.3 --- Screening of catalase in different subcellular fractions --- p.28 / Chapter 2.3.1 --- Preparation of mitochondrial fractions --- p.28 / Chapter 2.3.2 --- Preparation of microsomal fractions --- p.29 / Chapter 2.3.3 --- Preparation of cytosolic fractions --- p.29 / Chapter 2.3.4 --- Preparation of post-mitochondrial fractions --- p.29 / Chapter 2.4 --- Purification of post-mitochondrial catalase --- p.29 / Chapter 2.4.1 --- Preparation of post-mitochondrial fractions --- p.30 / Chapter 2.4.2 --- Ethanol-chloroform precipitation --- p.30 / Chapter 2.4.3 --- Affinity chromatography --- p.30 / Chapter 2.4.4 --- Cation exchange chromatography --- p.31 / Chapter 2.5 --- Molecular mass determination --- p.34 / Chapter 2.6 --- Isoelectric focusing --- p.39 / Chapter 2.7 --- Kinetic studies of the purified enzyme --- p.42 / Chapter 2.7.1 --- Optimal pH --- p.42 / Chapter 2.7.2 --- Thermal stability --- p.42 / Chapter 2.7.3 --- Km and Vmax --- p.42 / Chapter 2.7.4 --- Inhibition studies --- p.43 / Chapter 2.7.4.1 --- "3-amino-1,2,4-triazole" --- p.43 / Chapter 2.7.4.2 --- Potassium cyanide and sodium azide --- p.43 / Chapter 2.8 --- Spectroscopic analysis --- p.44 / Chapter 2.8.1 --- Native enzyme --- p.44 / Chapter 2.8.2 --- Denatured enzyme --- p.44 / Chapter 2.8.3 --- Determination of pyridine hemochrome --- p.44 / Chapter 2.9 --- N-terminal amino acid sequence analysis for blotted protein --- p.45 / Chapter 2.9.1 --- Semi-dry electroblotting --- p.45 / Chapter 2.9.2 --- Protein staining on PVDF membrane --- p.46 / Chapter 2.9.3 --- N-terminal amino acid sequence analysis --- p.46 / Chapter 2.9.4 --- N-terminal deblocking of protein bound on PVDF membrane… --- p.47 / Chapter 2.9.5 --- BLAST® search --- p.48 / Chapter CHAPTER 3 --- RESULTS --- p.49 / Chapter 3.1 --- Catalase in different sub-cellular fractions --- p.50 / Chapter 3.2 --- Purification of post-mitochondrial catalase --- p.51 / Chapter 3.2.1 --- Ethanol-chloroform precipitation --- p.51 / Chapter 3.2.2 --- Affinity chromatography --- p.51 / Chapter 3.2.3 --- Cation exchange chromatography --- p.52 / Chapter 3.3 --- Determination of molecular mass --- p.57 / Chapter 3.4 --- Determination of isoelectric point --- p.57 / Chapter 3.5 --- Kinetic studies of the catalase --- p.62 / Chapter 3.5.1 --- Optimal pH --- p.62 / Chapter 3.5.2 --- Thermal stability --- p.62 / Chapter 3.5.3 --- Km and Vmax --- p.65 / Chapter 3.5.4 --- Inhibition studies --- p.65 / Chapter 3.5.4.1 --- "3-amino-1,2,4-triazole" --- p.65 / Chapter 3.5.4.2 --- Potassium cyanide and sodium azide --- p.65 / Chapter 3.5.5 --- Catalase peroxidase activity --- p.66 / Chapter 3.6 --- Spectroscopic analysis --- p.73 / Chapter 3.6.1 --- Native enzyme --- p.73 / Chapter 3.6.2 --- Denatured enzyme --- p.73 / Chapter 3.6.2.1 --- Potassium cyanide --- p.73 / Chapter 3.6.2.2 --- Sodium azide --- p.73 / Chapter 3.6.3 --- Pyridine hemochrome characterization --- p.73 / Chapter 3.7 --- N-terminal amino acid sequence analysis --- p.79 / Chapter CHAPTER 4 --- DISCUSSION --- p.81 / Chapter 4.1 --- Subcellular locations of catalase in bloodworms --- p.82 / Chapter 4.2 --- Purification of post-mitochondrial catalase --- p.82 / Chapter 4.3 --- Physical properties of the purified enzyme --- p.84 / Chapter 4.3.1 --- Native and subunit molecular mass --- p.84 / Chapter 4.3.2 --- Isoelectric point --- p.85 / Chapter 4.4 --- Kinetic properties of the purified enzyme --- p.85 / Chapter 4.4.1 --- Optimal pH --- p.85 / Chapter 4.4.2 --- Thermal stability --- p.85 / Chapter 4.4.3 --- Km and Vmax --- p.87 / Chapter 4.4.4 --- Inhibition studies --- p.87 / Chapter 4.4.5 --- Catalase peroxidase activity --- p.87 / Chapter 4.5 --- Spectroscopic analysis --- p.88 / Chapter 4.5.1 --- Native and denatured enzyme --- p.88 / Chapter 4.5.2 --- Pyridine hemochrome characterization --- p.88 / Chapter 4.6 --- N-terminal amino acid analysis --- p.89 / Chapter 4.7 --- Conclusions --- p.89 / REFERENCES --- p.93