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Stability Characteristics and Applications of Native and Chemically-Modified Horseradish Peroxidases

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Stability Characteristics and Applications of Native and Chemically-Modified Horseradish Peroxidases Stability Characteristics and Applications of Native and Chemically-Modified Horseradish Peroxidases By Enda Miland B.Sc. A thesis submitted for the degree of Doctor of Philosophy Supervised by Prof. Malcolm R. Smyth and Dr. Ciarán Ó Fágáin Dublin City University January 1996 DECLARATION I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Doctor of Philosophy, is entirely my own work and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed: TyufloAc l Enda Miland Date: 2 o / 2 / 7<?6 II ACKNOWLEDGEMENTS I would like to thank most sincerely Prof. Malcolm R. Smyth and Dr. Ciarán Ó Fágáin for giving me the opportunity to carry out this research. I greatly appreciate their help, guidance and enthusiasm over the last three and a half years. I wish to thank Prof. Paulino Tunon-Blanco and his research group for allowing me to work in their laboratories at the University of Oviedo, Asturias, Spain. I would like to thank the members of Malcolm’s research group: Siobhán, Gemma, Michaela, Mick, Caroline, Brian, Declan, Bumni, Brendan and Bin; and Ciarán’s group: Ann, Fiona, Noel and Anne-Marie. Thank you to the Biology technical staff. To all the chemistry postgraduates, especially, Rod, Kevin, Karen, Lorraine and Frances, all of whom were a great help, and everybody here from the AS4 class of 1992. Thanks to the Chemistry technical staff, especially Niall, Damien, Veronica and Shane. To all the staff in the house: Rod, Nobby, 2 Nialls, Noel, Cailiosa, John and Emma. A big thank you to some other people, Marie, Nuala, all the boys at home, and a large number of other people who have been there over the years. Finally, I would like to thank Mam, Dad and Ronan for their support and encouragement all the way through college. Ill ABBREVIATIONS A electrode area AA Amino acid A Angstrom AA-NHS Acetic acid N-hydroxysuccinimide ester Abs Absorbance Ag/AgCl Silver/Silver Chloride AH2 Hydrogen acceptors Arg Arginine Asp Asparagine C bulk concentration of substrate CD Circular dichroism C6H5OH Phenol CO2 Carbon dioxide CP Chlorophenol CPE Carbon paste electrode CSTR Continuous stirred tank reator Cys Cysteine CV Cyclic voltammetry Da Dalton DEAE Diethylaminoethyl DH Hydrogen donor DMF Dimethylformamide DMSO Dimethylsulphoxide DNA Deoxyribonucleic acid DSC Differential Scanning Calorimetry E Applied potential Ey2 Half-wave potential in voltammetry EDTA Ethylenediaminetetra-acetic acid IV EG-NHS Ethylene glycol bis-succinic acid ester of N-hydroxysuccinimide EIA Enzymeimmunosassay ELISA Enzyme-linked immunosorbent assay ESR Electron spin resonance FIA Flow injection analysis GA Glutaraldehyde GnCl Guanidine hydrochloride h 2o 2 Hydrogen peroxide His Histidine HPLC High performance liquid chromatography HQ Hydroquinone HRP Horseradish peroxidase HS-(CH2)„-SH Dithiol h 2so 4 Sulphuric acid i,I Current Inactive state of protein IAA Indole acetic acid ISE Ion selective electrode K Equilibrium constant Km Michaelis-Menten constant k Rate constant for protein inactivation kDa kilodaltons Lys Lysine M Molar mM millimolar Mr Molecular weight N Native state of protein nA Nanoampere Na2B407 Sodium tetraborate NaH2P0 4 Sodium dihydrogen orthophosphate NaOH Sodium hydroxide V -n h 2 Amino group n h 3 Ammonia NH4+ Ammonium NHS N-hydroxysuccinimide NMR Nuclear magnetic resonance o 2 Oxygen o-AP o-aminophenol OPD o-phenylenediamine Ox Oxidised form of enzyme PAP Poly(o-aminophenol) PEG Polyethylene glycol Phe Phenylalanine POPHA p-hydroxy-phenylacetic acid Red Reduced form of enzyme rpm Revolutions per minute SA-NHS Suberic acid N-hydroxysuccinimide ester SCE Saturated calomel electrode TMB 3, 3 \ 5, 5’-tetramethylbenzidine TNBS T rinitrobenzenesulphonate Tris Hcl T ris(hydroxymethyl)aminomethane hydrochloride Trp Tryptophan Tyr Tyrosine U Unfolded form of enzyme U.V./Vis. Ultra violet/Visible spectroscopy (v/v) Volume per volume Vinax Maximum rate of enzyme reaction (w/v) Weight per volume VI TABLE OF CONTENTS Declaration II Acknowledgements III Abbreviations IV Abstract XII CHAPTER ONE INTRODUCTION 1 1.1 Foreword 2 1.2 Biochemistry and Stabilisation of HRP 3 1.2.1 Introduction 3 1.2.2 Structure of HRP molecule 3 1.2.3 General peroxidase isoenzymes 5 1.2.4 Functions and catalysis of peroxidases 6 1.2.5 Stability of HRP 8 1.2.6 Peroxidase activity assays 11 1.2.7 Other functions of HRP 13 1.2.8 Protein stabilisation 14 1.2.9 Naturally existing stable enzymes 14 1.2.10 Denaturation 15 1.2.11 Enzyme immobilisation 16 1.2.12 Chemical modification of proteins 17 1.2.13 Enzyme stabilisation in organic solvents 20 1.2.14 Stabilisation through the use of additives 22 1.2.15 Other stabilisation techniques 23 1.2.16 Aplications of HRP 24 1.3 The use of peroxidase in wastewater treatment 26 1.3.1 Introduction 26 1.3.2 General peroxidase catalysed reactions 27 VII 1.3.3 Methods for treating phenolic wastes 28 1.3.3.1 Microbial 28 1.3.3.2 Incineration 29 1.3.3.3 Activated carbon 29 1.3.3.4 Chemical oxidation methods 30 1.3.3.5 Aeration 31 1.3.4 Use of immobilised peroxidases in phenol removal 31 1.3.5 Use of soluble peroxidases in phenol removal 33 1.3.6 Strategies to enhance phenol removal by peroxidases 34 1.3.7 Catalytic cycle of HRP 34 1.3 .8 Resistance of aromatic compounds to biodégradation 36 1.3.9 Reactor configurations 37 1.3.10 Oxidation of phenolics in non-aqueous media 38 1.3.11 General treatment of industrial effluents 39 1.4 Biosensors 40 1.4.1 Introduction 40 1.4.2 Transduction 40 1.4.3 Working electrodes 41 1.4.4 Immobilisation of biocomponents onto electrode surfaces 45 1.4.4.1 Adsorption 46 1.4.4.2 Covalent binding 47 1.4.4.3 Entrapment in a matrix 48 1.4.4.4 Electropolymerisation 49 1.4.5 Kinetics of amperometric electrodes 51 1.4.6 Applications 54 1.4.6.1 Medical 54 1.4.6.2 Food industry 56 1.4.6.3 Environmental monitoring 56 1.4.6.4 Industrial 57 1.5 Bibliography 58 VIII CHAPTER TWO CHEMICAL MODIFICATION OF HORSERADISH PEROXIDASE 65 2.1 Introduction 66 2.2 Experimental 69 2.2.1 Materials 69 2.2.2 Equipment 70 2.3 Methods 71 2.3.1 Horseradish peroxidase microassay 71 2.3.2 Thermal inactivation of HRP 72 2.3.3 Determination of protein concentration 72 2.3.4 Accelerated storage studies 73 2.3.5 Chemical modification of HRP 73 2.3.6 Comparison of fresh and aged succinimides 74 2.3.7 Organic solvent profiles 74 2.3.8 pH activity profiles 75 2.3.9 Determination of free amino groups 75 2.3.10 Stability towards denaturing and reducing agents 76 2.3.11 Fluorescence studies 77 2.3.12 UV/Visible spectrophotometric analysis 77 2.4 Results and Discussion 78 2.4.1 TMB assay 78 2.4.2 Optimisation of HRP modification protocol 82 2.4.3 Effect of pH on enzyme modification and activity 84 2.4.4 Thermal studies on Native HRP 86 2.4.5 Thermal studies on HRP derivatives 89 2.4.6 Exposure to water-miscible organic solvents 95 2.4.7 Long and short term stability studies 103 2.4.8 Determination of free amino groups 104 2.4.9 Effects of denaturing and reducing agents 107 2.4.10 UV/Visible spectrophotometric analysis 111 2.4.11 Fluorescence Studies 113 2.4.12 Conclusion 119 IX 2.5.1 Bibliography 121 CHAPTER THREE PHENOL REMOVAL FROM AQUEOUS SYSTEMS AT HIGH TEMPERATURES BY CHEMICALLY MODIFIED HORSERADISH PEROXIDASES 123 3.1 Introduction 124 3.2 Experimental 128 3.2.1 Materials 128 3.2.2 Equipment 129 3.3 Methods 130 3.3.1 Chemical modification of Horseradish peroxidase 130 3.3.2 Determination of protein concentration 130 3.3.3 Determination of HRP activity 130 3.3.4 Phenol precipitation experiments 131 3.3.5 HPLC analysis of residual phenols 132 3.4 Results and Discussion 133 3.4.1 Optimisation of HPLC analysis 133 3.4.2 HRP-catalysed oxidation of phenol 136 3.4.3 Effect of pH 138 3.4.4 Temperature studies 140 3.4.5 Effect of succinimides on phenol removal 142 3.4.6 Effect of reaction time on 4-CP removal 147 3.4.7 Effect of hydrogen peroxide on polymerisation 149 3.4.8 Effect of HRP concentration on 4-CP removal 152 3.4.9 Simultaneous biodégradation of phenol and 4-CP 156 3.4.10 Removal of a range of phenolics 160 3.4.11 Conclusion 163 3.5 Bibliography 165 X CHAPTER FOUR THE USE OF HORSERADISH PEROXIDASE IN A POLYMER-BASED SENSOR FOR DETECTING URIC ACID 167 4.1 Introduction 168 4.2 Experimental 173 4.2.1 Materials 173 4.2.2 Equipment 174 4.3 Methods 175 4.3.1 Carbon paste preparation 175 4.3.2 Electrode preparation 175 4.3.3 Polymer film preparation 176 4.4 Results and Discussion 177 4.4.1 Cyclic voltammetry studies on poly(o-aminophenol) 177 4.4.2 Mediator studies 180 4.4.3 Effect of enzyme loading on sensor response 182 4.4.4 Amperometric studies on poly(o-aminophenol) 186 4.4.5 Hydrodynamic studies 191 4.4.6 Kinetic studies 194 4.4.7 pH dependence of biosensor 199 4.4.8 Interference studies 202 4.4.9 Calibration characteristics in static systems 205 4.4.10 Application of sensor in a flow injection system 207 4.4.11 Analysis of real samples 213 4.4.12 Conclusion 215 4.5 Bibliography 217 CHAPTER FIVE CONCLUSIONS 219 Appendix 225 XI ABSTRACT Due to its inherent stability, ease of handling and availability, horseradish peroxidase (HRP) will continue to be used more extensively in analytical and industrial situations.
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