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Title
CERTIFICATE
DECLARATION
ACKNOWLEDGEMENT
ABBREVIATIONS
CONTENTS
1. BACKGROUND OF THE WORK
1.1 Introduction
1.1.1 L-asparaginase
1.1.2 Antineoplastic mechanism
1.1.3 Antigenically modified L-asparaginase
1.1.4 Crystal structure of L-asparaginase
1.1.5 Scope for more research
1.2 Forces Determining the Globular Structure of Proteins
1.2.1 Local interactions and hydrogen bonding
1.2.2 Non-local forces and hydrophobic interactions
1.2.3 Side chain interactions of amino acids
1.2.4 Electrostatic interactions
1.2.5 Disulphide bonds
1.2.6 Quaternary association of proteins
1.3 Denaturation of Proteins
1.4 Aggregation
1.5 Molten Globule
1.6 Protein Stability
1.6.1 Physical denaturing conditions
1.6.2 Chemical denaturing conditions
1.7 Classical Strategies for Enhancing Enzyme Stability
1.7.1 Stabilizing compounds
1.7.1.1 Substrates, inhibitors and cofactors
1.7.1.2 Polyols and sugars
1.71.3 Salmi
1.7.2 Immobilization and chemical modification
1.7.3 Protein engineering
1.7.4 Enzymes of thermophilic organisms
1.8 General Methodology
1.8.1 Matrix assisted laser desorption and ionization (MALDI) spectroscopy
1.8.2 Fluorescence spectroscopy
1.8.2.1 Introduction
1.8.2.2 Three-dimensional structure
1.8.2.3 Association of proteins with substrates and other macromolecules
1.8.2.4 Quenching of protein fluorescence
1.8.3 Circular dichroic spectroscopy
1.8.3.1 Introduction
1.8.3.2 Classes of proteins
1.8.3.3 Methods of estimation of protein secondary structures
2. ISOLATION, PURIFICATION AND CHARACTERIZATION OF AEROMONAS L-ASPARAGINASE 2.1 Introduction
2.1 Introduction
2.2 Experimental Procedures
2.2.1 Maintenance of bacterial culture
2.2.2 Growth and harvesting of bacterial cells
2.2.3 Preparation of cell free extract
2.2.4 Determination of protein
2.2.5 Lowrys estimation of protein concentration
2.2.6 Spectro photometric method to determine protein concentration
2.2.7 Enzyme assay
2.2.8 Definition of enzyme unit
2.2.9 Purification of enzyme
2.2.9.1 Ammonium sulphate precipitation
2.2.9.2 DEAE- cellulose chromatography
2.2.9.3 Sephadex G-200 chromatography
2.2.9.4 DEAE-Sephadex A-50 chromatography
2.2.10 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
2.2.11 Native PAGE
2.2.12 Determination of molecular weight
2.2.12.1 MALDI time of flight mass spectrometry
2.2.12.2 Electrophoresis
2.2.12.3 Gel filtration chromatography
2.2.13 Circular dichroism (CD) spectroscopy
2.3 Results
2.3.1 Purification and enzymatic activity
2.3.2 Molecular weight
2.3.3 effect of pH on Aeromonas L-asparaginase
2.3.4 Effect of temperature on Aeromonas L-asparaginase
2.3.5 Effect of modifiers on Aeromonas L-asparaginase
2.3.6 Circular dichroism
2.4 Discussion
3. UNFOLDING STUDIES OF AEROMONAS L-ASPARAGINASE
3.1 Introduction
3.2 Experimental Procedures
3.2.1 Thermal denaturation
3.2.1.1 Circular dichroism
3.2.1.2 Rayleigh scattering
3.2.2 Guanidine hydrochloride induced unfolding
3.2.2.1 Circular dichroism
3.2.2.2 Huorescence studies
3.2.3 Urea induced unfolding
3.2.3.1 Circular dichroism
3.2 3.2 Fluorescence studies
3.2.3 3 Quenching of fluorescence
3.3 Results
3.3.1 Thermal denaturation
3.3.2 GuHCI induced unfolding
3.3.2.1 Circular dichroism
3.3.2.2. Intrinsic tryptophan fluorescence
3.3.2.3 Activity studies
3.3.3 Urea induced unfolding
3.3.3.1 Circular dichroism
3.3.3.2 Fluorescence study
3.3.4 Quenching of fluorescence
3.3.4.1 Acrylamide quenching
3.3.4.2 Iodide quenching
3.3.4.3 pH dependence and quenching
3.4 Discussion
4. THERMODYNAMIC ANALYSIS OF THE STABILIZATION OF AEROMONAS L-ASPARAGINASE
4.1 Stability Studies: The Effect of Cosolvents
4.1.1 Introduction
4.1.2 Experimental procedures
4.1.2.1 Enzyme and reagents
4.1.2.2 Activity assays
4.1 2.3 Analysis of enzyme stabilization
4.1.2.4 Incubation and assay
4.1.2.5 Derivation of the thermodynamic parameters
4.1.3 Results
4.1.3.1 Measurement of stability curves
4.1.3.2 Determination of thermodynamic parameters
4.1.4 Discussion
4.2 Freeze-drying
4.2.1 Introduction
4.2.2 Experimental Procedures
4.2.3 Results
4.2.4 Discussion
5. SUMMARY AND CONCLUSIONS
BIBILIOGRAPHY