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TITLE
DEDICATION
CERTIFICATE 1
CERTIFICATE-2
DECLARATION
CONTENTS
LIST OF TABLES
LIST OF FIGURES
ABBREVIATIONS
I. INTRODUCTION
Definition and Nomenclature of Metallothioneins
Biosynthesis
Localization
Structure
Importance of Eichhornia crassipes in Metal Detoxification
Summary of the aims
II. REVIEW OF LITERATURE
Plants Heavy Metal Uptake Strategy
Heavy Metals in FVPs
Defensive Strategy of Plants
Phytochelatins
PCs Have the General Structure (γ-EC) n-G
PCs are Synthesised from GSH
PC Synthase is Activated by Heavy Metal Ions
PC Biosynthesis may be Regulated in Several Ways
PC-Cd Complexes are Sequestered in Vacuoles
Sulphide Ions Play a Role in PC Function
PC Isolation, Detection and Quantitation
III. BIOCHEMICAL INVESTIGATIONS
MATERIALS AND METHODS
Estimation of Protein - Lowrys Method (Lowry1951)
Estimation of Total Thiols (Jocelyn, 1991)
Estimation of GSH: - Ellmans Method (Ellman, 1959)
Atomic Absorption Spectroscopy (AAS)
RESULTS
GENERAL DISCUSSION
IV. PURIFICATION OF DIFFERENT PHYTOCHELATINS
4.1 PURIFICATION OF Cd EXPOSED PEPTIDES
Step-1: Extraction
Step-2: Ion-Exchange Chromatography
Step 3: Salting out and Concentration
Step 4: Gel Filtration
Estimation of Total Thiols (Jocelyn, 1991)
Atomic Absorption Spectroscopy (AAS)
RESULTS
4.2 PURIFICATION OF Cu EXPOSED PEPTIDES
Step-1: Extraction
Step-2: Ion-Exchange Chromatography
Step 3: Salting out and Concentration
Step 4: Gel Filtration Estimation of Total Thiols (Jocelyn, 1991)
Atomic Absorption Spectroscopy (AAS)
RESULTS
4.3 PURIFICATION OF Zn EXPOSED PEPTIDES
Step-1: Extraction
Step-2: Ion - Exchange Chromatography
Step 3: Salting out and Concentration
Step 4: Gel Filtration Estimation of Total Thiols (Jocelyn, 1991)
Atomic Absorption Spectroscopy (AAS)
RESULTS
GENERAL DISCUSSION
V HPLC FOR PHYTOCHELATINS
MATERIALS AND METHODS
Instrumentation
Sample Preparation
RP-HPLC Procedure
Gradient Elution
RESULTS
DISCUSSION
VI IN VITRO SYNTHESIS OF PHYTOCHELATINS
Enzyme Preparation
In Vitro Studies
RESULTS
DISCUSSION
VII STUDIES ON PURIFIED PHYTOCHELATINS
7.1 pH ANALYSIS OF PCs
MATERIALS AND METHODS
DISCUSSION
7.2 IMMOBILISATION OF PEPTIDES AND METAL ION SUBSTITUTION
MATERIALS AND METHODS
Procedure
Atomic Absorption Spectroscopy (AAS)
RESULTS
INFERENCES AND DISCUSSION
VIII SUMMARY
BIBLIOGRAPHY