Florida State University Libraries 2015 Isolation of Marine Siderophores by Immobilized Metal Affinity Chromatography Carley Farst Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES ISOLATION OF MARINE SIDEROPHORES BY IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY By CARLEY FARST A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2015 Carley Farst defended this dissertation on July 30, 2015. The members of the supervisory committee were: William M. Landing Co-Professor Directing Dissertation William T. Cooper Co-Professor Directing Dissertation Vincent Salters University Representative Alan G. Marshall Committee Member Albert Stiegman Committee Member The Graduate School has verified and approved the above-named committee members, and certifies that the dissertation has been approved in accordance with university requirements. ii I would like to dedicate this dissertation to my father, Troy Timothy Farst (b.1956-d.2011). iii ACKNOWLEDGMENTS Alexandra Stenson for assisting in my understanding of mass spectrometry. Umesh Goli for teaching me how to use the time of flight mass spectrometer and quadrupole mass spectrometer. Huan Chen for running my samples on the Fourier Transform Ion Cyclotron Resonance mass spectrometer. My friends and family for providing me the necessary emotional support to finish my degree. iv TABLE OF CONTENTS List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii Abstract ......................................................................................................................................... xii 1. BACKGROUND ........................................................................................................................1 1.1 Introduction ..........................................................................................................................1 1.2 IMAC ...................................................................................................................................6 2. METHOD DEVELOPMENT ...................................................................................................14 2.1 Method Concept .................................................................................................................14 2.2 Method Outline ..................................................................................................................15 2.3 Sample Collection ..............................................................................................................15 2.4 IMAC .................................................................................................................................16 2.5 SPE .....................................................................................................................................20 2.6 Gauging Instrumental Response ........................................................................................20 2.7 Final Method ......................................................................................................................21 3. METHOD APPLICATION ......................................................................................................36 3.1 Outline................................................................................................................................36 3.2 Sample Collection ..............................................................................................................37 3.3 Sample Processing .............................................................................................................37 3.4 Sample Analysis.................................................................................................................38 3.5 Results ................................................................................................................................39 4. DISCUSSION ............................................................................................................................58 4.1 Isolation of Marine Siderophores ........................................................................................58 4.2 Characterization of Marine Siderophores ...........................................................................60 4.3 Implications of Our Research Project .................................................................................63 APPENDIX A. LIST OF SIDEROPHORES ................................................................................71 BIBLIOGRAPHY ..........................................................................................................................78 BIOGRAPHICAL SKETCH .........................................................................................................81 v LIST OF TABLES Table 1.1 Proteins Requiring Iron And Their Associated Functions (Twining And Baines) ...8 Table 1.2 Typical Siderophore Functional Groups and Their pKa Values (Hider and Kong) ..9 Table 2.1 ESI-TOF MS Settings ............................................................................................24 Table 2.2 ESI Quattro MS Settings .........................................................................................25 Table 2.3 Measuring SPE Extraction Efficiency ....................................................................26 Table 2.4 Measuring Flow Rate ..............................................................................................26 Table 2.5 Iron Addition Experiment .......................................................................................26 Table 2.6 Testing Different Volumes of Sample ....................................................................26 Table 2.7 Gauging Instrument Response Reproducibility Over Time ....................................27 Table 3.1 Masses of Compounds in the HP Matrix in Positive Mode ....................................44 Table 3.2 Isotopic Masses and Relative Abundance for Ferrioxamine A1 and A2 ................47 Table 3.3 Isotopic Masses and Relative Abundance for Ferrioxamine B (DFOB +Fe(III)) ...49 Table 3.4 Isotopic Masses and Relative Abundance for Ferrioxamine X1 .............................51 Table 3.5 Isotopic Masses and Relative Abundance for Agrobactin ......................................53 Table 4.1 Species to Consider in IMAC Elution .....................................................................65 vi LIST OF FIGURES Figure 1.1 A. Nitrate concentrations in the surface ocean ranging from very low in the central gyres to >35µM nitrate in the Southern Ocean. B. Chlorophyll data for the surface . ocean ranging from 0.01 to >10µg/L. Elevated nitrate concentrations in the Southern Ocean, along the equator, and in the sub-Arctic northeastern Pacific are associated with relatively low chlorophyll concentrations: these regions are referred to as HNLC regions (Martin and Fitzwater) ...........................................................................................................................10 Figure 1.2 Iron is required for these steps in the marine nitrogen cycle: oxidation (red), nitrification (blue), denitrification (yellow), and nitrogen fixation (green) (Morel and Price) ...............................................................................................................11 Figure 1.3 Vertical profiles for Nitrate and iron from three different curse initiatives: USJGOFS (NABE), VERTEX (VII), and FeLine (FeL) (Johnson, Gordon, and Coale). These profiles demonstrate nutrient behavior with the most obvious seen with the VERTEX cruise data .....................................................................................................................................12 Figure 1.4 The iron(III) cellular uptake strategy for both low and high-affinity iron acquisition mechanisms. Exocellular Fe(III) reduction (1) is followed by Fe(II) transport through the cell wall (2), re-oxidation to Fe(III) (3) and transport of Fe(III) into the cytoplasm (4) ..........13 Figure 1.5 A. a tricatechol structure 1,3,5-N,N', N"-tris-(2,3- dihydroxybenzoyl)triaminomethylbenzene (MECAM). B. a hydroxyamate structure desferrioxamine B (Hider, 1984) .......................................................................................13 Figure 2.1 This box diagram shows the processes that occur on the Chelex-100 resin. The first panel shows that Chelex-100 resin is derivatized with imidodiacetate (IDA) groups. The second panel refers to when the Chelex-100 resin is loaded with Fe(III). The next panel illustrates the goal of capturing the ligands that have high affinity for Fe(III) on the resin. The final panel shows the EDTA competing with the natural ligands bound to Fe(III) on the Chelex-100 resin and subsequently eluting the ligands. ....................................................28 Figure 2.2 This box diagram shows the process used to separate iron binding organics from seawater..............................................................................................................................28 Figure 2.3 (+) ESI-TOFMS spectrum of a standard solution extracted