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TITLE
DEDICATION
CERTIFICATE-1
CERTIFICATE-2
ACKNOWLEDGEMENT
List of Abbreviations
CONTENTS
1. FORWARD TO THE PRESENT WORK
1.1 Introduction
1.2 Lactoperoxidase
2. RETROSPECT
2.1 Review of Lactoperoxidase Functions
2.2 Review of Lactoperoxidase Structural Studies
Fig.2.1. Proposed structure of lactoperoxidase heme (Rae and Goff, 1996)
3. ISOLATION, PURIFICATION AND CHARACTERIZATION OF OVIS LACTOPEROXIDASE
3.1 Introduction
3.2 Source of the Experiment Material
3.3 Experimental Procedures
3.3.1 Activation of CM Sephadex C-50
3.3.2 Activation Procedure of Dialysis Bag
3.3.3 Ion Exchange Chromatography,
3.3.4 Enzyme Assay using ABTS
3.3.5 Definition of Enzyme Unit Activity
3.3.6 Dialysis
3.3.7 Concentration by PEG-20000
3.3.8 Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis
3.3.8.1 Preparation of stock solutions
3.3.8.2 Treatment of protein sample
3.3.8.3 Sample buffer preparation
3.3.8.4 Staining of gel
3.3.8.5 Destaining solution
3.3.9 Purification by Gel Filtration
3.3.9.1 Gel filtration
3.3.9.2 Activation of Sephadex G-100
3.3.9.3 Sephadex G-100 Chromatography
3.3.10 Native PAGE
3. 3.10.1 Preparation of sample buffer for Native PAGE
3.3 10. 2 Specific staining of peroxidase
3.3.11 Determination of Protein Concentration
3.3.11.1 Lawrys estimation of protein concentration
3 3.11. 2 Spectrophotometric method to determine protein concentration
3.3.12 Conformational study of sLP arc and metal through Electronic Spectra.
3.3.12.1 Determination of electronic spectra
3.3.12.2 Experimental procedure
3.4 Results
3.4.1 Purification Data
Table 3.1. Summary of purification of sheep lactoperoxidase
3.4.2 Enzyme Assay
3.4.3 Determination of Molecular Weight
Fig.3.1 Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) of purified sheep lactoperoxidase
Fig.3.2. Graphical representation of molecular weight dermination of sLP M1- Phosphorylase b. M2 -Human albumin and M3 - Ovalbumin
3.4.4 UV-Visible Spectral Studies
Fig.3.3. UV-Visible spectrum of sLP
3.5 Discussion
4. STUDIES ON ANTIMICROBIAL PROPERTIES
4.1 Introduction
4.2 Studies on Antibacterial Activity
4.2.1 Bacterial Strains
4.2.2 Maintenance of Bacterial Strains
4.2.2 1 Nutrient agar slants
4.2.3 Preparation of the lnoculum
4.2.4 Disc Diffusion method in medium with sLP alone_
4.2.4.1 Preparation of paper discs impregnated with protein
4.2.4.2 Mueller-Hinton agar
4.2.4.3 Experimental procedure
4.2.5 Disc Diffusion method in medium with sLP, thiocyanate and hydrogen peroxide system (sLP-S)
4.2.5.1 Medium and reagents
4.2.5.2 Experimental procedure
4.2.6 Determination of Minimum Inhibitory Concentration (MIC) of sLP
4.2.6.1 sLP in the medium without thiocyanate and hydrogen peroxide.
4.2.6.2 sLP in the medium with thocyanate and hydrogen peroxide system (sLP-S)
4.3 IR Spectral Studies
4.3.1 Experimental Procedure_
4.4 Studies on Antifungal Properties
4.4.1 Fungal Isolates and their Maintenance
4.4.2 Reagents and Medium
4.4.3 Screening of Fungi for sensitivity towards sLP system (sLP-S)
4.4.4 Experimental Procedure
4.4.5 Determination of Minimum Inhibitory Concentration
4.5 Results
4.5.1 Studies on Antibacterial Activity
4.5.1.1 Disc Diffusion studies with disc containing sLP alone
Table 4.1. Zone of bacterial Inhibition of disc diffusion studies with disc containing sLP alone
Fig.4.1 Antibacterial property of sheep lactoperoxidase without thiocyanate and hydrogen peroxide by disc diffusion method against bacterial strains (a) Escherichia coli (b) Klebsiella pneumoniae
4.5.1.2 Disc Diffusion studies with disc containing sLP-S,
Table 4.2. Zone of bacterial inhibition of disc diffusion studies with disc containing all components of sLP system
Fig.4.2 Antibacterial property of shep lactoperoxidase with thiocyante and hydrogen peroxide system by disc diffusion method against bacterial strains, (a) Escherichia Coli (b) Klesbsiella pneumoniae
4.5.2 Minimum Inhibitory Concentration studies in medium with sLP alone and with sLP-S
4.5.3 IR Spectral Studies
4.6 Studies on Antifungal Property
Table 4.4. Minimum lnhibitory Concentration of sLP in thiocyanate and hydrogen peroxide system for various fungi
4.7 Discussion
4.7.1 Studies on Antibacterial Activity
4.7.2 Studies on Antifungal Property
5. METALLOPROTEIN BEHAVIOUR
5.1 Atomic Absorption Spectrometry
5.1.1 Experimental Procedure
5.2 Inductively Coupled Plasma-Atomic Emisson Spectrometry
5.2.1 Experimental procedure
5.2.2 ICP-AES Labtam 8410 Plasma Scan
5.3 Electron Paramagnetic Resonance Spectroscopy
5.3.1 Principle
5.3.2 Experimental Procedure
5.4 Infrared Spectrophotometry
5.4.1 Experimental Procedure
5.5 General Calculation method to determine number of metal atoms
5.6 Results
5.6.1 Atomic Absorption Spectroscopic Analysis
5.6.2 Inductively Coupled Plasma-Atomic Emission Spectroscopic Analysis.
Table 5 1 ICP- AES analysis data
5.6.3 Electron Paramagnetic Resonance Spectroscopic Analysis
5.6.4 Infrared Spectrum Analysis
Fig.5.2. Infrared spectrum of sLP
5.7 Discussion
6. CIRCULAR DICHROISM AND FLUORESCENCE STUDIES
6.1 Protein Stability
6.1.1 Noncovalent Forces
6.1.2 Hydrophobic Forces
6.1.3 Hydrogen Bonds
6.1.4 Electrostatic Bonds
6.1.5 van der Waals-London Dispersion Forces
6.1.6 Disulphide Bonds
6.2 Protein Denaturation
6.3 Circular Dichroism
6.3.1 Presentation of Circular Dichroism data
6.4 Experimental Procedures
6.4.1 pH Stability
6.4.2 Thermal Stability
6.5 Fluorescence Spectroscopy
6.5.1 Three-dimensional Structure
6.5.2 Association of Proteins with Substrates and other Macromolecules
6.6 Quenching of Fluorescence
6.6.1 Theory of Collisional Quenching
6.7 Experimental Procedures
6.7.1 Urea induced Unfolding Studies
6.7.2 Guanidine Hydrochloride (GuHCI) Induced Unfolding Studies
6.7.3 Quenching of Sheep Lactoperoxidase Fluorescence
6.8 Results
6.8.1 Circular Dichroism
6. 8. 1.1 pH Stability
Fig.6.1. Native CD spectrum of sLP in 0.05 M Tris HCI buffer, pH 8
Fig.6.3. CD spectra of sLP in 0.05 M Tris HCI buffer (f) pH 7, (g) pH 6, (h) pH 5, (i) pH 4, and (j) pH 3
Fig.6.2. CD spectra of sLP in 0.05 M Tris HCI buffer (a) pH 12, (b) pH 11, (c) pH 10, (d) pH 9 (e) pH 8, and (f) pH 7
6.8.1.2 Thermal Stability
Fig.6.4. Thermal denaturation of sLP
6.8.2 Fluorescence Studies
6.8.2.1 Urea Induced Unfolding
Fig.6.5. Fluorescence spectra of sLP at varying concentrations of urea
Fig.6.6. Variation of percentage fluorescence of sLP with urea concentrations.Fluorescence intensity in the absence of urea is taken as 100%. Protein concentration was 2 μM in 0.05 M Tris HCI buffer, pH 8
Fig.6.7. Variation of emission of maxima of hptophan fluorescence of sLP with urea denaturant concentrations
6.8.2.2 GuHCl Induced Unfolding
Fig. 6.8. Fluorescence spectra of sLP at varying concentrations of GuHCl
Fig.6.9 Variation of percentage fluorescence of sLP with GuHCl concentrations.Fluorescence intensity in the absence of denaturant is taken as 100%. Protein concentration was 2 μM in 0.05 M Tris HCl buffer, pH 8
Fig.6.10. Variation of emission of maxima of tryptochan fluorescence of sLP with GuHCl denaturant concentrations
6.8.2.3 Acrylamide Quenching
Fig.6.11. Quenching of tryptophan fluorescance of sLP using acrylamide concentrations.
Fig.6.12. Stern-Volmer plot for acrylamide quenching of sLP fluorescence.
6.8.2.4 Potassium iodide Quenching
Fig.6.13. Quenching of tryptophan fluorescence of sLP at pH 8 using potassium Iodide.
Fig.6.14. Stern-Volmer plot of the quenching rate of tryptophan fluorescence for sLP at various potassium iodide concentrations.
Fig.6.15. Quenching of tryptophan fluorescence spectra of sLP at pH 5 using potassium iodide.
Fig.6.16 Stern-Volmer plot of the quenching rate of tryptophan fluorescenca for sLP at various potassium iodide concentrations.
Fig.6.17 Quenching of tryptophan fluorescence spectra of sLP at 1M GuHCl using various potassium iodide concentrations.
Fig.6.18 Stern-Volmer plot of the quenching rate of tryptophan fluorescence for sLP at various potassium iodide concentration in the presence of 1 M GuHCl
6.9 Discussion
6.9.1 Circular Dichroism
6.9.2 Fluorescence Studies
7. SUMMARY AND CONCLUSION
BIBLIOGRAPHY