Abstract
Complexation by organic ligands dominates the speciation of iron (Fe), copper (Cu), and other bioactive trace metals in seawater, controlling their bioavailability and distribution in the marine environment. Several classes of high-affinity Fe-binding ligands (siderophores) have been identified in seawater but the chemical structures of marine Cu-complexing ligands remain unknown. Immobilized metal-ion affinity chromatography (IMAC) allows Cu ligands to be isolated from bulk dissolved organic matter (DOM) in seawater and separated into fractions which can be characterized independently using electrochemical and spectroscopic techniques. Attempts have been made to combine IMAC with electrospray ionization mass spectrometry (ESI-MS) to characterize marine Cu ligands, but results have proven inconclusive due to the lack of tandem mass spectrometry (MS/MS) data to confirm ligand recovery. We used 8-hydroxyquinoline (8-HQ), a well-characterized model ligand that forms strong 1:2 metal:ligand complexes with Cu2+ at pH 8 (log β2 = 18.3), to evaluate Cu(II)-IMAC and ESI-MS/MS for recovery and identification of copper(II)-complexing ligands in seawater. One-litre samples of 0.45µm-filtered surface seawater were spiked with 8-HQ at low concentrations (up to 100 nM) and fractionated by IMAC. Fractions eluted with acidified artificial seawater were desalted and re-suspended in methanol via solid-phase extraction (SPE) to obtain extracts suitable for ESI-MS analysis. Recovery of 8-HQ by Cu(II)-IMAC was confirmed unambiguously by MS/MS and found to average 81% based upon accurate quantitation via multiple reaction monitoring (MRM). Cu(II)-IMAC fractionation of unspiked seawater using multiple UV detection wavelengths suggests an optimal fraction size of 2 mL for isolating and analyzing Cu ligands with similar properties.
Highlights
Organic complexation of dissolved trace metals like iron (Fe) and copper (Cu) exerts a major influence on their mobility, toxicity, and bioavailability in the marine environment (Florence, 1982; Sunda, 1991; Donat and Bruland, 1995; Kraemer, 2004)
UV spectrophotometric analysis of 8-HQ standards and Oceanic surface seawater (OSW) samples spiked with 8-HQ showed that the model ligand absorbs strongly at 254 nm, as noted previously (Zhang et al, 1996), and that the absorbance at this wavelength is proportional to ligand concentration
Chromatograms obtained during Cu(II)-IMAC of Artificial seawater (ASW) spiked with 8-HQ contained only the later-eluting peak, while UV absorbance spectra of collected fractions corresponding to this peak matched those of standards contained 8-HQ in artificial seawater (aASW)
Summary
Organic complexation of dissolved trace metals like iron (Fe) and copper (Cu) exerts a major influence on their mobility, toxicity, and bioavailability in the marine environment (Florence, 1982; Sunda, 1991; Donat and Bruland, 1995; Kraemer, 2004). Culturing experiments have shown that marine phytoplankton are capable of producing strong, extracellular Cu-binding ligands (McKnight and Morel, 1979, 1980; Moffett et al, 1990; Moffett and Brand, 1996; Leal et al, 1999; Croot et al, 2000; Dupont et al, 2004; Dupont and Ahner, 2005; Wiramanaden et al, 2008) Some of these exudates are thought to act like chalkophores (Semeniuk et al, 2015), promoting biological uptake and utilization of Cu for e.g., photosynthesis, radical detoxification, and iron acquisition (Maldonado et al, 2006). Numerous studies have shown that Cu in seawater exists primarily in the form of organic complexes (see Donat and Bruland, 1995) no marine copper ligands have been positively identified
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