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Rui Manuel Vicente Benfeitas University of Lisbon Faculty of Science DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY (in association with the Center for Neuroscience and Cell Biology, University of Coimbra) THE PHYSIOLOGICAL ROLE OF PEROXIREDOXIN 2 IN HUMAN ERYTHROCYTES: A KINETIC ANALYSIS Rui Manuel Vicente Benfeitas Biochemistry Masters 2011 University of Lisbon Faculty of Science DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY (in association with the Center for Neuroscience and Cell Biology, University of Coimbra) THE PHYSIOLOGICAL ROLE OF PEROXIREDOXIN 2 IN HUMAN ERYTHROCYTES: A KINETIC ANALYSIS Advisors Armindo Salvador, Ph.D. & Fernando Antunes Ph.D. 1Center for Neuroscience and Cell Biology, University of Coimbra 2Faculty of Science, University of Lisbon Rui Manuel Vicente Benfeitas Biochemistry Masters 2011 To my advisors Dr. Armindo Salvador and Dr. Fernando Antunes, a big thank you for all your patience, efforts and very interesting discussions. Thanks for making this so fun! To my family, friends, lab mates, thank you for pushing me forward. 3 Index Index 4 List of abbreviations 7 List of values 11 List of figures 13 List of tables 15 1. Summary 17 Resumo 19 2. Introduction 21 2.1. Reactive Oxygen Species and biological sources of H2O2 22 2.1.1. Glutathione autoxidation 23 2.1.2. Superoxide dismutation 24 2.1.3. Hemoglobin autoxidation 25 2.1.4. H2O2 influx from plasma 25 2.2. Relevance of the main defenses against H2O2 in erythrocytes 28 2.2.1. The debate around the relative importance of catalase and GPx1 28 2.2.2. Evidence of an unknown antioxidant 33 2.2.3. The relevance of peroxiredoxin 33 2.3. Peroxiredoxin 2: catalytic cycle and main features in human erythrocytes 38 2.3.1. Prx2 structure 38 2.3.2. Catalytic cycle of Prx2 39 2.3.3. Prx2 inactivation by H2O2 40 3. Problem and objective 43 4. Methods and Technical notes 45 5. Results ‐ Part I: Study of defenses against H2O2 under steady state conditions 47 5.1. What is the relative contribution of Peroxiredoxin 2 for hydrogen peroxide consumption? 48 5.1.1. Rate constant estimates 48 4 5.1.1.1. Pseudo‐first order rate constant of GPx1 with respect to H2O2 48 5.1.1.2. Pseudo‐first order rate constant of catalase with respect to H2O2 52 5.1.1.3. Pseudo‐first order rate constant of Prx2 with respect to H2O2 52 5.1.2. Relative contribution of the three enzymes under low and high oxidative loads 53 5.2. What is the maximum rate of H2O2 decomposition by Prx2? 54 5.2.1. Parameter estimates and considerations 54 5.2.1.1. Dismutase activity of catalase 54 5.2.1.2. Peroxidase activity of GPx1 61 5.2.1.3. Endogenous H2O2 production and basal intracellular H2O2 64 5.2.1.4. Kinetics of H2O2 efflux and consumption by catalase and GPx1 66 5.2.1.5. Peroxidase activity and concentration of Prx2 67 5.2.1.6. Total concentration of Trx 67 5.2.1.7. TrxR concentration 68 5.2.1.8. Estimate of the rate constants for H2O2 permeation and endogenous production in the experiments of (Low et al., 2007) 68 5.2.1.9. Pseudo‐first order rate constant for Trx reduction through TrxR 71 5.2.1.10. Estimate of the pseudo‐first order rate constant for formation of PSSP 76 5.2.2. Maximum rates of H2O2 decomposition by Prx2 at steady state 80 5.3. At what extracellular H2O2 concentration does Prx2 remain the main H2O2 scavenging process? 81 5.4. How does the redox state of Peroxiredoxin 2 vary with Trx or disulfide bond formation rate constants? 86 5.5. What is the concentration of each Prx2 form in limit situations and how does it change with system’s parameters? 88 5.5.1. Fraction of each Prx2 form under low and high oxidative loads 88 5.5.2. How H2O2 influx and Prx2 reduction affect the redox status of Prx2 91 5.6. What determines the eH2O2 at which the crossover occurs? 94 5.7. Is the sulfinylation of Prx2 physiologically relevant? 96 5.8. Is Prx2 essential for plasma H2O2 scavenging by erythrocytes? 100 5.9. Could Prx2 be replaced by catalase or GPx1? 104 Results ‐ Part II: Analysis of defenses against H2O2 in erythrocytes crossing inflammation sites 110 5.10. Physiological aspects of human circulatory system and estimates for erythrocytes facing pulses of H2O2 110 5.10.1. Human blood contents and blood flow velocities 110 5.10.2. Frequency of crossing inflammation sites 112 5 5.10.3. Basal intracellular and extracellular values of H2O2 114 5.10.4. H2O2 production in pathological conditions and cell tolerance to H2O2 115 5.11. How do different eH2O2 pulses affect the redox state of Prx2? 118 5.12. What is the role of catalase in defense against eH2O2 pulses? 120 5.13. Could there be accumulation of PSSP due to consecutive crossing of inflammatory sites? 122 5.14. Why is Prx2 so abundant in the human erythrocyte? 125 6. Discussion 127 6.1. Prx2 is the main scavenger of low/mild H2O2 128 6.1.1. Basal concentration and production of H2O2 129 6.1.2. Redox status of Prx2 under basal oxidative loads 131 6.1.3. Prx2 contributes significantly for H2O2 consumption under low/mild loads 132 6.2. Catalase becomes the main scavenger of iH2O2 under high oxidative stress 135 6.3. The contribution of Prx2 for H2O2 consumption is affected by oxidative load and NADPH supply 138 6.4. Prx2’s high concentration allows the human erythrocyte to remain protected against H2O2 when crossing inflammation sites 139 6.5. Prx2 inactivation and reaction between H2O2 and Prx2 141 6.6. Improvements and experiments needed 144 6.6.1. Basal and pathological oxidative loads 144 6.6.2. Catalytic cycle of catalase 145 6.6.3. Frequency of crossing inflammation sites and H2O2 induced by crossing inflammation sites 146 6.6.4. Peroxidase activity and relevance of Prx2 for H2O2 defense 147 6.6.5. Effects of NADPH supply on H2O2 defense 149 6.7. Relevance of the study 150 7. Conclusion 152 8. References 153 9. Appendix 168 9.1. Poster presentations 168 9.2. Oral communications 172 6 List of abbreviations Latin alphabet Abbreviation Meaning AS Erythrocyte surface area CP Peroxidatic cysteine CR Resolving cysteine DensityRBC Erythrocyte density DeoxyHb Hemoglobin (deoxyhemoglobin, Fe2+ oxidation state) Dp H2O2-induced intracellular damage during an H2O2 pulse Dr H2O2-induced intracellular damage between H2O2 pulses DTNB 5,5'-dithiobis-(2-nitrobenzoic acid) eH2O2 Extracellular hydrogen peroxide f Order of reaction of glutathione peroxidase 1 with respect to H2O2 g Order of reaction of glutathione peroxidase 1 with respect to GSH G6P Glucose-6-phosphate G6PD Glucose-6-phosphate dehydrogenase GPx1 Glutathione Peroxidase 1 GPx1ox Glutathione Peroxidase 1 (oxidized form) GPx1red Glutathione Peroxidase 1 (reduced form) GPx1-SG Glutathione Peroxidase 1 (associated with glutathione) GSH Glutathione GSR Glutathione Reductase GSSG Glutathione (oxidized form) GStot Glutathione (total concentration in the erythrocyte) H2O2 Hydrogen peroxide Hb Hemoglobin i Fraction of total body volume occupied by inflamed tissue iH2O2 Intracellular hydrogen peroxide Aggregated pseudo-first order rate constant for hydrogen peroxide consumption by catalase and glutathione kc peroxidase kcatalase Pseudo-first order rate constant for hydrogen peroxide consumption by catalase kCompoundI Reactivity between Compound I and H2O2 kCompoundII Pseudo-first order rate constant for reduction of Compound II to Ferricatalase kD Proportionality constant for the accumulation of iH2O2-induced damage to the cell kefflux Pseudo-first order rate constant for hydrogen peroxide efflux kFerricatalase Reactivity between Ferricatalase and H2O2 kGPx1 Pseudo-first order rate constant for hydrogen peroxide consumption by glutathione peroxidase 1 7 Abbreviation Meaning kH Henry’s law constant for the solubility of O2 in water kIntN Reactivity between Intermediate and NADPH kIntN’ Pseudo-first order rate constant for reduction of Intermediate to Ferricatalase, with respect to Intermediate kIntRed Pseudo-first order rate constant for reduction of Intermediate to Compound II Km Trx Michaelis-Menten constant of thioredoxin reductase for thioredoxin Km ()NADPH Michaelis-Menten constant of thioredoxin reductase for NADPH kox First-order rate constant for spontaneous glutathione oxidation kox,in vivo First-order rate constant for spontaneous glutathione oxidation in vivo, considering physiological pO2 kox,exp First-order rate constant for spontaneous glutathione oxidation experimentally determined under large pO2 kspon First-order rate constant for oxidation of glutathione by oxygen kp Pseudo-first order rate constant for H2O2 influx across the erythrocyte membrane kPrx2 Pseudo-first order rate constant for hydrogen peroxide reduction by peroxiredoxin 2 kPrxRed Reactivity between peroxiredoxin 2 and hydrogen peroxide kPSO2H Second-order rate constant for the sulfinylation of Prx2 kPSOH First-order rate constant for the formation of disulfide form of Prx2 kPSSP Reactivity between disulfide peroxiredoxin 2 and reduced thioredoxin kR Pseudo-first order rate constant for Prx2 reduction kTrxR Pseudo-first order rate constant for thioredoxin reduction by thioredoxin reductase MetHb Hemoglobin (methemoglobin, Fe3+ oxidation state) MWcatalase Molecular weight of catalase monomer MWGPx1 Molecular weight of GPx1 monomer MWPrx2 Molecular weight of each monomer of peroxiredoxin 2 MWTrx Molecular weight of thioredoxin MWTrxR Molecular weight of thioredoxin reductase NADP+ Nicotinamide adenine dinucleotide phosphate (oxidized form) NADPH Nicotinamide adenine dinucleotide phosphate (reduced form) OxyHb Hemoglobin (oxyhemoglobin, oxygen-bound state, Fe3+ oxidation state) PPP Pentose Phosphate Pathway Prx1 Peroxiredoxin 2 (singly-crosslinked monomers) Prx2
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