Brigham Young University BYU ScholarsArchive Theses and Dissertations 2011-08-04 Ferritin Diversity: Mechanistic Studies, Disease Implications, and Materials Chemistry Robert Joseph Hilton Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Biochemistry Commons, and the Chemistry Commons BYU ScholarsArchive Citation Hilton, Robert Joseph, "Ferritin Diversity: Mechanistic Studies, Disease Implications, and Materials Chemistry" (2011). Theses and Dissertations. 3070. https://scholarsarchive.byu.edu/etd/3070 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Ferritin Diversity: Mechanistic Studies, Disease Implications, and Materials Chemistry Robert J. Hilton A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Richard K. Watt, Chair Steven W. Graves Roger G. Harrison Barry M. Willardson Dixon J. Woodbury Department of Chemistry and Biochemistry Brigham Young University December 2011 Copyright © 2011 Robert J. Hilton All Rights Reserved ABSTRACT Ferritin Diversity: Mechanistic Studies, Disease Implications, and Materials Chemistry Robert J. Hilton Department of Chemistry and Biochemistry Doctor of Philosophy The study of ferritin includes a rich history of discoveries and scientific progress. Initially, the composition of ferritin was determined. Soon, it was shown that ferritin is a spherical, hollow protein. Eventually, over several decades of research, the structure and some function of this interesting protein was elucidated. However, the ferritin field was not completely satisfied. Today, for example, researchers are interested in refining the details of ferritin function, in discovering the role of ferritin in a variety of diseases, and in using ferritin for materials chemistry applications. The work presented in this dissertation highlights the progress that we have made in each of these three areas: 1) Mechanistic studies: The buffer used during horse spleen ferritin iron loading significantly influences the mineralization process and the quantity of iron deposited in ferritin. The ferrihydrite core of ferritin is crystalline and ordered when iron is loaded into ferritin in the presence of imidazole buffer. On the other hand, when iron is loaded into ferritin in the presence of MOPS buffer, the ferrihydrite core is less crystalline and less ordered, and a smaller amount of total iron is loaded in ferritin. We also show that iron can be released from the ferritin core in a non-reductive manner. The rate of Fe3+ release from horse spleen ferritin was measured using the Fe3+-specific chelator desferoxamine. We show that iron release occurs by three kinetic events. 2) Disease studies: In order to better understand iron disruption during disease states, we performed in vitro assays that mimicked chronic kidney disease. We tested the hypothesis that elevated levels of serum phosphate interrupted normal iron binding by transferrin and ferritin. Results show that phosphate competes for iron, forming an iron(III)-phosphate complex that is inaccessible to either transferrin or ferritin. Ferritin samples separated from the iron(III)- phosphate complex shows that as the phosphate concentration increases, iron loading into ferritin decreases. 3) Materials chemistry studies: Anion sequestration during ferritin core reduction was studied. When the core of horse spleen ferritin is fully reduced using formamidine sulfinic acid, a variety of anions, including halides and oxoanions, cross the protein shell and enter the ferritin interior. Efforts have been made to use ferritin to control the concentration of anions for reactions. In addition, the native ferrihydrite mineral core of ferritin is a semi-conductor capable − of catalyzing oxidation/reduction reactions. Light can photo-reduce AuCl4 to form gold nanoparticles (AuNPs) with ferritin as a photocatalyst. The mechanism of AuNP formation using ferritin as a photocatalyst was examined. From this work, we propose that the ferrihydrite core of ferritin photo-reduces; the mineral core dissolves into a soluble iron(II) mineral. The iron(II) then re-oxidizes, and a new mineral forms that appears to be the new photocatalyst, as the lag phase is significantly decreased with this new mineral form of ferritin. Keywords: ferritin, chronic kidney disease, nanocage, anion, gold nanoparticles, photochemistry ACKNOWLEDGEMENTS The success of the work contained in this dissertation was possible thanks to significant contributions from many individuals. First, I wish to thank Richard Watt for his incredible patience, advisement, and direction with the work. The research would not have progressed without his intellectual contributions, energy, and persevering attitude. Richard has had a great impact in the way that I approach problems, scientific or otherwise. His mentorship has taught me how to be a better person. I would also like to thank several undergraduate and graduate students, post-doctoral researchers, and collaborators; Dr. Gary Watt, Dr. Alejandro Yevenes, Matthew Graff, Jeremiah Keyes, David Andros, Zach Kenealey, Curtis Seare, Naomi Martineau, Cata Matias, Dr. Kwang Min Shin, and Dr. Jeffery Farrer. Without their assistance, this work would not have been possible. Finally, I would like to thank my unwavering family. Because of the tremendous support system that they have constantly offered through encouragement and praise, I have been able to persevere. Most especially, thanks to my beloved wife, Lacy. Her attitude, patience, and persistence has lifted me up and strengthened me. Our late-night conversions that centered on my research generated many ideas and thoughts, and her contributions are as significant as any. Ultimately, I wish to thank my Father in Heaven for creating the world in which we live, full of complexities and mysteries. Discovery of these complexities turn out to be beautiful and profoundly spiritual. I am grateful for the opportunity I have had to catch but a glimpse of the creations of God. TABLE OF CONTENTS LIST OF TABLES ............................................................................................................................. vii LIST OF FIGURES ........................................................................................................................... viii LIST OF SCHEMES ............................................................................................................................ xi ABBREVIATIONS ............................................................................................................................ xii CHAPTER 1: INTRODUCTION TO FERRITIN ........................................................................................ 1 Single Ferritin Subunit Structure ................................................................................................ 2 Nanocage Structure ..................................................................................................................... 3 The Role of H and L Chain Ferritin ............................................................................................ 6 Mechanism of Ferritin Function ................................................................................................. 8 Disease ...................................................................................................................................... 10 Ferritin in Materials Chemistry ................................................................................................ 12 Conclusion ................................................................................................................................. 13 References ................................................................................................................................. 13 CHAPTER 2: CRYSTALLINE FERRIHYDRITE FORMATION IN FERRITIN ............................................. 19 Abstract ..................................................................................................................................... 19 Introduction ............................................................................................................................... 20 Materials and Methods .............................................................................................................. 22 Results ....................................................................................................................................... 25 Discussion ................................................................................................................................. 36 References ................................................................................................................................. 36 CHAPTER 3: FERRIC IRON RELEASE FROM FERRITIN USING DESFEROXAMINE .............................. 40 Abstract ..................................................................................................................................... 40 Introduction ............................................................................................................................... 40 iv Materials and Methods .............................................................................................................