Isolation of Marine Siderophores by Immobilized Metal Affinity Chromatography

• Other Authors: Farst, Carley (authoraut), Landing, William M. (professor co-directing dissertation), Cooper, William T. (professor co-directing dissertation), Salters, Vincent J. M. (university representative), Marshall, Alan G., 1944- (committee member), Stiegman, Albert E., 1953- (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Chemistry and Biochemistry (degree granting department)
• Format: Text
• Language: English
• Published: Florida State University
• Subjects: Chemistry; North Atlantic
• Online Access: http://purl.flvc.org/fsu/fd/FSU_2015fall_Farst_fsu_0071E_12828; http://fsu.digital.flvc.org/islandora/object/fsu%3A291361/datastream/TN/view/Isolation%20of%20Marine%20Siderophores%20by%20Immobilized%20Metal%20Affinity%20Chromatography.jpg

This dissertation details the isolation of marine siderophores by Immobilized Metal Affinity Chromatography (IMAC). Siderophores are biologically produced, low molecular weight (400 Da-1000Da), strong iron-binding ligands. These compounds are thought to form strong complexes with [greater than]95% of the dissolved Fe(III) in the oceans and therefore play extremely important roles in the biological uptake and biogeochemical cycling of Fe in the oceans. The challenges with isolating these ligands from seawater is they are present in very low concentrations (nM) in a matrix with a very high ionic strength (~0.7M). The most important considerations for isolating siderophores are selectivity, concentrating, and desalting. In this work, we studied the use of Immobilized Metal Affinity Chromatography (IMAC) followed by Solid-Phase Extraction (SPE) to isolate and concentrate marine siderophores from natural seawater samples. The theory behind the IMAC extraction technique is that organic compounds that form complexes with particular metal cations can be isolated and/or separated via their attraction to a cation chelating resin that has been pre-loaded with the metal cation of interest. To recover the organic compounds from the IMAC resin, elution conditions are chosen based on the analyte of interest and involves either ligand competition or stripping the column of the metal at low pH. In the case of Fe-IMAC, the best method to use was a competing ligand, in this case ethylenediaminetetraacetic acid (EDTA), since acid elution would generate an eluate with a very high ([greater than]50mM) total dissolved Fe(III) concentration that would interfere with subsequent processing and analysis. By using a high EDTA concentration (50mM) the column could be eluted effectively. The Fe-binding ligands in the EDTA eluate were further concentrated via extraction on a silica C-18 resin column (SPE) which will concentrate free ligands and ligand metal complexes that are sufficiently hydrophobic. This "purifies" the IMAC eluate by extracting the analytes of interest from the EDTA eluent. After rinsing with water, the organic compounds were eluted from the SPE column with HPLC-grade methanol, thereby converting the sample matrix to methanol which is easy to dry down for further concentration and is easy to analyze using electrospray ionization mass spectrometry (ESI-MS). Method development and method validation measurements were performed using (+)Electrospray Ionization(ESI) Time of Flight Mass Spectrometry (MS) and (+)ESI-Quattro MS. Open ocean samples were analyzed using (+)ESI Fourier Transform Ion Cyclotron MS. In addition to the internal standard (desferrioxamine B), we found evidence for the presence of three known siderophores (ferrioxamine A1/A2, agrobactin, and ferrioxamine X1) in a seawater sample from the central North Atlantic Ocean. A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Fall Semester 2015. July 30, 2015. IMAC, Immobilized Metal Affinity Chromatography, marine dissolved organic matter, Siderophores, solid phase extraction, SPE Includes bibliographical references. William M. Landing, Professor Co-Directing Dissertation; William T. Cooper, Professor Co-Directing Dissertation; Vincent Salters, University Representative; Alan G. Marshall, Committee Member; Albert Stiegman, Committee Member.