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Picosecond Time Resolved Spectroscopy Used As a Tool To Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2004 Picosecond time resolved spectroscopy used as a tool to probe excited state photophysics of biologically and environmentally relevant systems Pramit Kumar Chowdhury Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Analytical Chemistry Commons, and the Physical Chemistry Commons Recommended Citation Chowdhury, Pramit Kumar, "Picosecond time resolved spectroscopy used as a tool to probe excited state photophysics of biologically and environmentally relevant systems " (2004). Retrospective Theses and Dissertations. 766. https://lib.dr.iastate.edu/rtd/766 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Picosecond time resolved spectroscopy used as a tool to probe excited state photophysics of biologically and environmentally relevant systems by Pramit Kumar Chowdhury A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Physical Chemistry Program of Study Committee: Jacob W. Petrich, Major Professor Mark S. Gordon Mark S. Hargrove George A. Kraus Mei Hong Iowa State University Ames, Iowa 2004 Copyright © Pramit Kumar Chowdhury, 2004. All rights reserved. UMI Number: 3136299 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 3136299 Copyright 2004 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ii Graduate College Iowa State University This is to certify that the doctoral dissertation of Pramit Kumar Chowdhury has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Major Professor Signature was redacted for privacy. Fo/the Major Program iii Dedicated to my parents for their constant support and unconditional love iv TABLE OF CONTENTS ACKNOWLEDGEMENTS viii CHAPTER L INTRODUCTION Excited state photophysics of biologically active perylene quinones 1 Heme Proteins 5 Solvation dynamics 14 Dielectric relaxation in proteins. 17 References 19 CHAPTER H. EXPERIMENTAL APPARATUS Time correlated single photon counting technique. 34 Basic principle 34 Light Source 35 Sample Chamber 38 Detector 39 Discriminators 41 Time-to-amplitude converter .42 Advantages of using TCSPC in fluorescence lifetime measurements 44 Pump-probe spectroscopy 45 Modelocking 46 Group velocity dispersion 51 The oscillator 55 The regenerative amplifier 58 The optical parametric amplifier assembly 59 The final stage 61 References 64 CHAPTER HI. EFFECT OF pH ON THE FLUORESCENT AND ABSORPTION SPECTRA OF HYPERICIN IN REVERSE MICELLES Abstract 67 Introduction 68 Materials and methods 71 Preparation of sample 72 Steady-state measurements 73 Time-resolved studies. 73 V Results and discussion 73 Conclusions 78 Acknowledgements 83 References 83 CHAPTER IV. A COMPARISON OF THE EXCITED-STATE PHOTOPHYSICS OF NEARLY SYMMETRICAL PERYLENE QUINONES: HYPOCRELLIN A AND HYPOMYCINB Abstract... 92 Introduction. 93 Materials and methods 96 Preparation of sample 97 Steady-state measurements 98 Time-resolved studies 98 Time-resolved pump-probe absorption spectroscopy ....98 Results and discussion .....98 Effects of pH on the absorption and emission spectra of hypocrellin A and hypomycin 98 Multiple H-atom transfers in the perylene quinones? 104 Summary and conclusions Ill Acknowledgements 114 References ...114 CHAPTER V. THE LEGHEMOGLOBIN PROXIMAL HEME POCKET DIRECTS OXYGEN DISSOCIATION AND STABILIZES BOUND HEME Abstract 124 Introduction 125 Material and methods 130 Mutagenesis of Lba and Mb cDNAs 130 Expression and purification of proteins 131 Spectroscopic and kinetic characterization of wild type and mutant Lba and Mb 132 Measurement of geminate recombination kinetics 133 Measurement ofhemin dissociation 134 Results 134 Removal of covalent constraints on Lba His^ 134 Effects of F-helix swapping in Lba and Mb on O] and CO binding..136 Effects of F-helix "swapping" on distal pocket mechanisms for ligand stabilization 137 Role of residue F7 in proximal regulation 138 Relationship of the proximal heme pocket to heme iron reactivity....139 vi Effects of proximal heme pocket mutations on hemin dissociation...140 Discussion 140 Differences between proximal regulation in Lba and Mb 142 Interaction of proximal and distal regulation 145 Role of the F-helix in protein stability 147 Conclusions 148 Acknowledgements 149 References 149 CHAPTER VI. EFFECTS OF DISTAL POCKET MUTATIONS ON THE GEMINATE RECOMBINATION OF NO WITH LEGHEMOGLOBIN ON THE PICOSECOND TIME SCALE Abstract 157 Introduction 158 Arguments for proximal effects in geminate recombination 159 Arguments for distal effects in geminate recombination 161 Distal and proximal effects in leghemoglobin 164 Materials and Methods 164 Preparation of NO-bound Lba samples for geminate recombination measurements 164 Measurement and analysis of geminate recombination 165 Bimolecular recombination kinetics. 166 Results and Discussion 166 Conclusions 172 Acknowledgements 175 References 175 CHAPTER VH. DYNAMIC SOLVATION IN ROOM TEMPERATURE IONIC LIQUIDS Abstract 179 Introduction 180 Materials and Methods 183 Room temperature ionic liquids 183 Spectroscopic measurements and data analysis 184 Steady-state measurements 186 Time-resolved measurements 187 Transient absorption (stimulated emission) measurements 189 vii Fluorescence upconversion measurements 191 Results 192 Discussion 197 Steady-state spectra 197 Stimulated emission, time-correlated single photon counting and single wavelength analysis 202 Analysis based on fluorescence upconversion results 206 Conclusions 206 Acknowledgements 207 References. 213 CHAPTER VHI. THE COMPLEX OF APOMYOGLOBIN WITH THE FLUORESCENT DYE, COUMARIN153 Abstract 218 Introduction. 219 Materials and methods 223 Binding constant measurement, fluorescence 224 Binding constant measurements, capillary electrophoresis 225 Steady-state spectroscopies 226 Time-resolved spectroscopies ...226 Molecular dynamics simulations. 227 Results and discussion 228 Conclusions. 235 Acknowledgements 236 References.. 237 CHAPTER IX. SUMMARY AND CONCLUSIONS 245 viii ACKNOWLEDGEMENTS I would like to take this opportunity to express my gratitude and appreciation to my research advisor, Dr. Jacob W. Petrich. I have no hesitation in saying that I have had a wonderful time working under his supervision and cherished each and every moment with him. He has always been there for me in times of need for support and guidance. I think I have been really lucky to have been given a chance to work for my advisor. I would also like to thank Dr. Mark Hargrove for collaborating with us and also allowing us to use his laser when needed. He has been very gracious and helpful in providing us with proteins and their mutants which we have used and will be using in many of our ongoing projects in collaboration with him. Thanks are also due to Dr. Suman Kundu, for working tirelessly to prepare protein samples for us. I have to admit that I have bugged him a lot over the years of my stay; being a perfect gentleman as he is, he has always provided us with samples on time, without even a single word of anger or frustration. Dr. Anindya Dutta, Dr. Kaustuv Das and Dr. Mintu Haider, post docs in our lab with whom I have had the opportunity of working with, have been so very helpful throughout my stay out here. Thanks are also due to Kyle Ashby, Jin Wen and Jaehun Park (all former graduate students of our lab), Lindsay Sanders and Prasun Mukheqee (current graduate students), Dr. Ran)an Das and Dr. Govindarajan Krishnamoorthy (present post doctoral associates) and Tessa Calhoun and Erin Campbell (current undergraduates in our group) for maintaining such a cordial work environment in the laboratory. I am also very grateful to the Chemistry Department Faculty and Staff for being of such a great help throughout my stay out here. 1 CHAPTER I: INTRODUCTION The advent of ultrafast spectroscopy and its subsequent refinements has opened up many opportunities for investigating processes taking place on timescales ranging from picoseconds to subpicoseconds and even in the femtosecond region. This thesis focuses on some of the applications of ultrafast spectroscopy we have been carrying out over the past few years, with the aid of different laser based instruments in our laboratory. 1.1 Excited state photophysics of biologically active perylene quinones Hypericin (Figure 1.1) is a photodynamic pigment found in certain members of the genus It is particularly prevalent in # (St. John's wort), which is a traditional
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