Characterizing iron complexing ligands in aquatic environments using Immobilized Metal Affinity Chromatography

Abstract

Iron plays an active role in aquatic environments, acting as a key micronutrient for micro-organisms such as phytoplankton. Iron is difficult for these organisms to access in oceanic water due to low ambient concentrations. Iron has two redox states, the II redox form is soluble in water under anoxic conditions; however, it is easily oxidized to its III form in the presence of oxygen and precipitates as Fe₂O₃(s). As a result, dissolved iron concentrations are sub-nanomolar in seawater. Microorganisms have evolved strategies to keep iron in solution, namely via the production of siderophores, specialized iron-chelating mole-cules. 99% of the dissolved iron in the ocean is bound to organic ligand complexes, which help to maintain the bioavailability of the metal. These ligands are considered part of dissolved organic matter (DOM), a complex carbon pool containing eclectic water-soluble compounds of varying chemical compositions. The vast majority of DOM is chemically uncharacterized and often referred to as humic substances (HS), which originate from the breakdown of organic matter of biological origin. A subset of HS can bind selectively to iron and retain it in its dissolved form in marine waters. Recent publications have theorized that uncharacterized humic ligands may play an essential role in the marine iron cycle, and as such, further investigation is needed.1 To further investigate the specific origin and influence of these ligands on aquatic iron cycling, the ligands need to be extracted from the ocean’s complex matrix, isolated from the rest of the DOM. A method for this extraction was developed using immobilized metal affinity chromatography (IMAC). The method works by using a column containing Sepharose, a cross-linked beaded form of agarose. This Sepharose acts as a chelator al-lowing us to charge the column with iron. By charging the column, the beads bind to the metal forming a coordination complex with available active sites. The iron binding lig-ands in our sample then bind to these available coordination sites, while other compounds pass through the column. A series of different eluants is then used to elute the retained ligands from the column based on different structural and chemical properties. The eluted ligands are then collected in fractions for further analysis. The optimized IMAC method results show three distinct regions of ligand classes of varying binding strength and structural composition. To validate the method and further explore the chemistry of binding, solutions of known ligands were used as validation samples. These specific ligands were chosen due to their likely presence in aquatic environments. By testing these ligands using the optimized method, we could further infer how the humic-iron ligands interact with our iron-charged column. The results of the model ligand experiments suggest that binding to the column was typically bidentate, with two coordination sites needed for retention to occur. After testing and validation, the method was implemented for riverine, coastal, and open ocean samples. Comparison of the behaviour of natural water samples with the model lig-ands reveals potential binding characteristics and origins of HS ligands.