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Research Collection Doctoral Thesis The relationship between membrane-water partitioning, uncoupling, and inhibitory activity of substituted phenols in chromatophores of rhodobacter sphaeroides Author(s): Escher, Beate Isabella Publication Date: 1995 Permanent Link: https://doi.org/10.3929/ethz-a-001534171 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DisslEfH QX-£> Diss. ETH Nr. 11283. The Relationship between Membrane-Water Partitioning, Uncoupling, and Inhibitory Activity of Substituted Phenols in Chromatophores of Rhodobacter Sphaeroides A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH for the degree of DOCTOR OF NATURAL SCIENCES A <'• presented by BEATE ISABELLA ESCHER Dipl. Chem. Universitat Konstanz born January 11,1965 Citizen of the Federal Republic of Germany accepted on the recommendation of Prof. Dr. Rene' Schwarzenbach, examiner Dr. Mario Snozzi, co-examiner Prof. Dr. John Westall, co-examiner Zurich, 1995 1 Acknowledgments I am deeply obliged to Prof. Rend Schwarzenbach for his engaging support and continual encouragement during the course of this work. Special thanks I also owe to Dr. Mario Snozzi for advising the biophysical part of the research. Without his excellent expertise on time resolved spectroscopy on the photosystem of Rb. sphaeroides it would have been impossible to conduct this study. I want to extend my gratitude to Dr. Renata Behra who helped me put my work into the context of environmental toxicology and QSAR studies, and to Prof. John Westall of Oregon State University for many helpful discussions. I thank all of the above for reviewing this dissertation. I am grateful to Prof. Bachofen of the University of Zurich who gave me the opportunity to perform some preliminary experiments on uncoupling in his group. I thank Prof. Venturoli and his group of the University of Bologna for inviting me to test my samples on his kinetic spectrophotometer and for interesting discussions. I would like to acknowledge those who assisted me in various parts of my research. Markus Nellen was an outstanding and independent help in the sorption study who not only produced data faster than I could evaluate it but also supported me in difficult times. D'ominik Stadler and Vagelis Baboukas also helped me on preliminary partition experiments. Beatrice Schwertfeger had her first encounter with lab work— smelly octanol. Kathrin Haberli stood nights by the bioreactor, and performed the first experiments with phenols on the spectrophotometer. Without her I would probably still be sitting in the darkness and improving the instrument. Philippe Perisset was a patient help whenever I was struggling with the electronics. Aria Amirbahman, Kathrin Haberli, Franz Gunther Kari, Thomas Poiger, and Andre Weidenhaupt reviewed parts of the manuscript. Besides the ones mentioned above, there are numerous people at EAWAG who contributed to the bits and pieces of this work, and who have made the last four years interesting, challenging, and pleasurable. I cannot name everyone but I would like to express my thanks to Werner Angst, Cedric Arnold, Michael Berg, Dieter Diem, Meg David, Claudia Fesch, Karl Fent, Guy Glod, Stefan Haderlein, Sabine Hilger, Annette Johnson, Jorg Klausen, Yael Mason, Silke Meyns, Stefan Miiller, Judith Perlinger, Tina Volker, Kenny Weissmahr. Finally, I would like to express my gratitude to my family for their love and support during all those years. ii Table of Contents Acknowledgments i Table of Contents ii List of Figures v List of Tables viii Abstract ix Zusammenfassung xi Glossary xiii 1 General Introduction 1 1.1 Substituted Phenols in the Environment 1 1.2 The Toxicity of Substituted Phenols 2 1.3 Quantitative Structure Toxicity Relationships 3 1.4 Kinetic Spectroscopy on the Photosystem of Rhodobacter Sphaeroides as Method to Determine Toxic Effects of Substituted Phenols 6 1.5 Objectives of this Work 8 2 Experimental Section 10 2.1 Chemicals 10 2.2 Culture of Rb. sphaeroides 10 2.2.1 Rb. sphaeroides 10 2.2.2 Cultures for the Chromatophore Preparation 11 2.2.3 Maintenance of the Cell Cultures 11 2.3 Chromatophores of Rb. sphaeroides 12 2.3.1 Preparation of the Chromatophores 12 2.3.2 Characterization of the Chromatophores 12 2.4 Liposomes 12 2.4.1 Preparation of Liposomes 12 2.4.2 Characterization of Liposomes 13 2.5 Partition Experiments 13 2.5.1 Determination of Octanol-Water Distribution Ratios 13 2.5.2 Determination of Membrane-Water Distribution Ratios 13 2.6 The Kinetic Spectrophotometer 16 2.6.1 Set-Up of the Instrument 16 2.6.2 Components 17 2.7 Redox-Controlled Spectroscopic Measurements 18 2.7.1 General 18 2.7.2 Concentration of the Chromatophores in Suspension 19 2.7.3 Concentration of the Redox Components 19 2.7.4 Uncoupling 20 2.7.5 Inhibition 20 The Partitioning Behavior of Substituted Phenols in Chromatophore-Water, Liposome-Water, and Octanol-Water Systems 21 3.1 Introduction 21 3.1.1 Motivation 21 3.1.2 Biological Membranes 22 3.1.3 Models for Biological Membranes 22 3.1.4 Liposomes 24 3.1.5 Sorption of Xenobiotics to Liposomes 26 3.2 Partitioning Model 28 3.2.1 The pH-Dependence of the Distribution Ratio 28 3.2.2 Dependence of the Distribution Ratio on the M+-Concentration.... 30 3.2.3 Modeling of die Sorption Isotherm of Phenoxides 31 3.3 Results and Discussion 32 3.3.1 Partitioning of Substituted Phenols in the Octanol-Water System. .32 3.3.2 Liposomes as Model Systems for Biological Membranes 35 3.3.3 Liposome-Water System 37 3.3.4 Effect of pH and K+-Concentration on the Liposome-Water Distribution Ratio Diipw 40 3.3.5 Modeling of the Sorption Isotherms in the Liposome-Water System 45 3.3.6 Comparison of Octanol- and Liposome-Water Distribution Ratios 47 3.3.7 Mechanistic Model of the Sorption of Phenols to Lipid Bilayers... 51 3.4 Conclusions 53 The Photosystem of Rhodobacter Sphaeroides as Model System for Determining Toxic Effects of Substituted Phenols 55 4.1 Chromatophores from Rhodobacter Sphaeroides 55 4.1.1 Introduction 55 4.1.2 Improvement of the Quality of the Chromatophores 56 4.2 The Photosystem of Rhodobacter Sphaeroides 57 4.2.1 The Electron Transfer Chain 57 4.2.2 The Electrochemical Proton Gradient 60 4.3 The Photosystem as Toxicity-Meter 61 4.3.1 The Carotenoid Band-Shift 61 4.3.2 The Kinetics of the Redox Components 63 4 4 Conclusions 68 5 Uncoupling of Electron Transport from ATP Synthesis 69 5 1 Introduction 69 5 1 1 Uncouplers and Their Mode of Action 69 5 12 Acidic Protonophores 70 5 13 Determination of Uncoupling Activity 72 5 2 Results and Discussion 73 5 2 1 Data Analysis 73 5 2 2 Classification of Uncouplers 77 5 2 4 First-Order Uncouplers 80 5 2 5 Second-Order Uncouplers 84 5 2 6 pH-Dependence of the Uncoupling Activity 87 5 2 7 Initial Development of a Qualitative Transport Model for the Uncoupling Activity of Phenobc Compounds m Chromatophores 91 5 2 8 Comparison with Data from Literature 94 5 3 Conclusions and Development of QSARs 97 6 Inhibition of the Electron Transport 100 6 1 Introduction 100 6 11 Quinones and Quinone-Analogue Inhibitors 100 6 12 Inhibitors of the Reaction Center Complex 102 6 13 Inhibitors of the Cyt bci-Complex 102 6 2 Results and Discussion 104 6 2 1 Non-specific Indications for Inhibition 104 6 2 2 QB-Inhibition 105 6 2 3 Qc-Inhibition 108 6 2 4 Qz-Inhibition 115 6 3 Conclusions 115 7 Summary and Outlook 117 References 120 Curriculum Vitae 127 List of Figures Fig 1 1 General scheme of the photosynthetic election transport coupled to ATP synthesis and possible target sites of substituted phenols 7 Fig 21 DeterminaUonofDmw by equilibrium dialysis 14 Fig 2 2 Diagram of the kinetic spectrophotometer 17 Fig 2 3 Cuvette for redox-controlled spectroscopic measurements 19 Fig 3 1 Fluid mosaic model of biological membranes according to Singer and Nicholson 23 Fig 3 2 Preparation and structural features of small unilamellar vesicles (SUV) 24 Fig 3 3 The composition of a selection of phospholipids abundant in the membranes of Rb sphaerotdes 26 Fig 3 4 Simple model to describe the equdibnum partitioning of phenols between a lipid and the water phase as a function of pH 28 Fig 3 5 Model to describe the liposome-water partitioning of phenoxides as a function of countenon concentration, (M+] 31 Fig 3 6 Effect of pH on the octanol-water distnbution ratio Dow of 245TnCP at 100 mMK+ 35 Fig 3 7 Comparison of the apparent distnbuUon ratios of several chlorophenols into chromatophores, Dcpi,w, and mto liposomes, Di,pW, at pH 7 and 100 mM K+ 36 Fig 3 8 Sorption isotherms of PCP on DOPC liposomes at pH 3 8 and pH 9 5 37 Fig 3 9 Sorption isotherms of some neutral chlorophenols at pH 3 8, 100 mM K+ 38 Fig 3 10 The presence of ionic PCP reduces Di,pw of the ionic 2346TeCP while the presence of non- dissociated tert -butylphenol has no significant effect on DnpW(2346TeCP) 39 Fig 3 11 Apparent hposome-water distnbution ratios E>hpw(pH) of 245TnCP as a funcnon of the fraction nondissociated species 41 Fig 3 12 Liposome-water distnbution ratios of some chlorophenols as a function of aqueous pH 42 Fig 3 13 Liposome-water distnbuUon rauos of some chloro- and nitrophenols as a function of total potassium concentration at pH 9 5 45 Fig 3 14 A Saturation isotherm of PCP at 100 mM K+, pH 9 5 B Saturation isotherm of 246TnCP at 2 5 mM K+, pH 9 5 47 Fig 3 15 Comparison of the octanol- and hposome-water distnbution ratios of DNOC and 245TnCP 48 Fig 3 16 Comparison