Progress 09/01/00 to 08/31/05
Outputs
In seawater, copper (Cu) is present in concentrations that are potentially toxic to many species of algae. However, most of the Cu is complexed by organic chemicals called ligands that dramatically reduce its bioavailability. The first goal of our project was to examine known sulfhydryl-containing ligands to determine whether they are directly important in the field with respect to Cu complexation and to use them as experimental surrogates for natural ligands in the field. We began with phytochelatin, a small peptide that is synthesized by eukaryotic algae in response to trace metal stress. We made significant progress with respect to our understanding of the production, exudation and degradation rates of phytochelatin in the natural environment and have a fairly complete model that may be able to predict phytochelatin concentrations in seawater under some conditions. This is the work of Liping Wei who has received partial support from this grant and recently finished
her PhD. We have recently published a paper on the effects of metal interactions on the intracellular concentrations of thiols in eucaryotic algae and have another in preparation that includes the model. Our second goal was to determine whether some algae produce novel compounds to chelate Cu. We successfully developed a method to couple our chemical separation technique (using high pressure liquid chromatography or HPLC) to electrospray mass spectroscopy (ESMS) in order to identify specific chemical structures. We also developed a pre-concentration strategy that has enabled us to measure very low concentrations of important thiols in field samples. This has been the work of Christopher Dupont, funded in full by this grant, who finished his MS during the summer of 2003. Using these techniques we discovered two previously unknown compounds in the culture medium of the species Emiliania huxleyi following Cu addition and also found that this organism constitutively contains these two
compounds (arginine-cysteine and glutamine-cysteine) in high intracellular concentrations. We found that E. huxleyi exudes these thiols, along with cysteine, stoichiometrically in response to increased copper concentrations in the growth media. Furthermore, stable Cu (I) complexes with the exuded thiols were observed in the growth media using matrix assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry following size exclusion chromatography. Additionally, E. huxleyi appears to utilize these novel thiols in nitrogen storage and assimilation as they are rapidly synthesized upon nitrogen addition to a nitrogen-depleted culture. We examined several strains of E. huxleyi, all of which contained these compound; they were not present in the handful of other algae that we examined. We currently have two publications in review on this work. This grant also partially funded a student to participate in a cruise to the north Pacific to measure thiols. All of the thiols we
measure in the laboratory cultures were also measured in the field. We have a paper in preparation on this work.
Impacts
A better understanding of trace metal chemistry in seawater will enable us to better model the cycling and bioavailability of toxic and nutrient trace metals. These metals can exert significant control over primary production in seawater and hence can affect the cycling and sequestration of numerous other elements such as carbon.
Publications
- Wei, L., Donat, J. R., Fones G. and Ahner, B. A. 2003. Interactions between Cd, Cu, and Zn influence particulate phytochelatin concentrations in marine phytoplankton: Laboratory results and preliminary field data. Environmental Science and Technology 37:3609-3618.
- Wei, L. 2003. Phytoplankton-metal interactions: Phytochelatin production, exudation and degradation in natural seawater, and molecular studies of phytochelatin synthesis in marine microalgae. Cornell University PhD Thesis.
- Dupont, C.L. 2003. Production and exudation of thiols by Emiliania huxleyi: Establishing links between marine phytoplankton and metal speciation in surface seawater. Cornell University MS Thesis.
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Progress 01/01/02 to 12/31/02
Outputs
In seawater, biologically active trace metals, such as Cu, are complexed by organic ligands; it is our goal in this project to determine the sources, sinks and chemical identities of these ligands. We have taken a multi-pronged approached to this work to specifically characterize thiol ligands that would be likely Cu ligands. We have been working to characterize known sulfhydryl-containing metal chelators (primarily phytochelatin and glutathione) as a model system for unknown ligands. We have made significant progress with respect to our understanding of the production, exudation and degradation rates of phytochelatin in the natural environment and have the beginnings of a comprehensive model of phytochelatin in seawater. This is the PhD work of Liping Wei who has received partial support from this grant and has just submitted a paper on the effects of metal interactions on the intracellular concentrations of thiols in eucaryotic algae. We have also been working on
both the analytical challenges of coupling an HPLC separation with electrospray mass spectroscopy (ESMS) as well as developing a pre-concentration strategy that will enable us to obtain enough material to perform the former technique on field samples. Graduate student, Christopher Dupont, funded in full by this grant, successfully continued our efforts to optimize an HPLC separation coupled to ESMS with Moffett and another chemist (R. Nelson) this last summer. One approach to finding novel Cu ligands is to investigate compounds made by pure cultures of algae. We recently discovered two particular compounds (eluting as unique retention times) in the culture medium of the species Emiliania huxleyi following Cu addition. Concentrations were too low in the medium for the ESMS method so we used concentrated culture extracts to get structural information about these two compounds. We were able to determine a structure and then we had these compounds synthesized. MS fragmentation patterns of
the synthesized compounds matched those of our putative compounds and likewise retention times were identical. We have surveyed a large group of algae and found that these compounds appear to be specific to this species, though small amounts of one of the compounds may have been found in a closely related species. We are continuing this investigation. To identify specific thiol-Cu complexes, we fractionated copper-treated E. huxleyi culture filtrate using size exclusion chromatography. The thiol and copper co-eluted in one fraction, which was then analyzed using matrix assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS). This was done in collaboration with post-doctoral associate Saj Bashir who works in the laboratory of Jocelyn Rose (Plant Biology, Cornell University). Peaks corresponding to exact masses of the cysteine-Cu(I) complex as well as a mixed-ligand Cu(I)-complex of cysteine along with one our new compounds was also found. This is the first
time that biologically-derived specific Cu complexes have been identified in a seawater medium. We are preparing a manuscript for submission to the journal Nature.
Impacts
A better understanding of trace metal chemistry in seawater will enable us to better model the cycling and bioavailability of toxic and nutrient trace metals. These metals can exert significant control over primary production in seawater and hence can affect the cycling of numerous other elements such as carbon.
Publications
- Ahner, B. A., J. R. Oleson, N. Ogura, and L. Wei. 2002. Glutathione and other low molecular weight thiols in marine phytoplankton under metal stress. Marine Ecology Progress Series. 232:93-103.
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Progress 01/01/01 to 12/31/01
Outputs
Almost all biologically active trace metals in seawater are complexed by organic ligands; it is our goal in this project to determine the sources, sinks and chemical identities of these ligands. We have taken a multi-pronged approached to this work to specifically characterize Cu ligands. We have been working to characterize known sulfhydryl-containing metal chelators (primarily phytochelatin and glutathione) as a model system for unknown ligands. We have made significant progress with respect to our understanding of the production, exudation and degradation rates of phytochelatin in the natural environment and have the beginnings of a comprehensive model of phytochelatin in seawater. This is the PhD work of Liping Wei who has received partial support from this grant. Second, we have been developing tools to chromatographically separate and characterize ligands that are as of yet unknown. We have been working on both the analytical challenges of coupling an HPLC
separation with electrospray mass spectroscopy (ESMS) as well as developing a pre-concentration strategy that will enable us to obtain enough material to perform the former technique. While at WHOI this last summer, Ahner worked with Moffett and another chemist (R. Nelson) to optimize an HPLC separation coupled to ESMS. A good separation scheme was developed (this primarily involved removing sodium and ion pairing reagents from our buffer) and spectral analyses were performed on a suite of standard thiol-containin compounds. We use a pre-column derivatization technique that labels the cysteine sulfhydryl with the fluorescent marker monobromobimane (Molecular Probes). Currently we have a nice method for separation and detection, now the primary goal is to obtain enough material to use our methods. Several techniques are currently being explored to pre-concentrate thiols from seawater. We have been working with a commercial product Thiolpropyl-Sepharose 6B (Pharmacia) that specifically
targets thiol containing compounds. Thus far we have confirmed that it is possible to selectively remove known thiols (cysteine, cys-gly, glutathione) from seawater with this material and, with a strong reductant, it is possible to retrieve them. Unfortunately impurities from the material interfere with the chromatographic separation of our target biochemicals. Complete regeneration of the material (following a published Pharmacia protocol) prior to its use reduces but does not eliminate these interferences. Extraction techniques to remove this interference are being actively sought. We are also continuing to experiment with a variety of reductants (DTT, TCEP, hydrazine, sodium borohydride) to optimize the recovery and derivatization of known thiols. We have begun testing our protocol with growth medium from Cu-stressed E. huxleyi cultures (in which we have detected unknown thiol exudates). This part of the project is the work of Christopher Dupont, a first year graduate student,
funded in full by this grant.
Impacts
A better understanding of trace metal chemistry in seawater will enable us to better model the cycling and bioavailability of toxic and nutrient trace metals. These metals can exert significant control over primary production in seawater and hence can affect the cycling of numerous other elements such as carbon.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
Almost all biologically active trace metals in seawater are complexed by organic ligands; it is our goal in this project to determine the sources, sinks and chemical identities of these ligands. Though this is a relatively new project we have made some significant progress. First we have been performing studies on a known intracellular metal chelator called phytochelatin. This sulfhydryl-containing polypeptide is synthesized by eukaryotic phytoplankton in response to metals and has binding characteristics similar to those of the unknown ligands. Phytochelatins are released to the surrounding seawater by exudation and cell breakage, however little work has been done to determine their turnover rates in seawater. Both biological and chemical degradation are possible. As peptides, they are probably degraded readily by bacteria, serving as a source of necessary carbon, nitrogen and sulfur. Using a synthesized phytochelatin standard, chemical degradation rates were
measured in artificial seawater in the presence of sodium azide, a bacterial inhibitor. First order kinetics were used to estimate rates and half-lives. Biological degradation experiments were performed in fresh seawater. By comparing degradation rates, it is apparent that bacteria play an important role in degrading phytochelatin in natural waters. We found that rates were lower when the phytochelatin was added as a metal complex but we have not yet to determine whether this was due to bacterial inhibition by the metals. These newly obtained rates can then be used to model dissolved phytochelatin concentrations that can then be compared to measured values. Measurements of dissolved phytochelatin can be compared to predictions of organic ligand concentrations (based on electrochemical measurements) to determine their potential contribution to this pool. We have also begun developing HPLC techniques that are complementary to electrospray mass spectroscopy (ES-MS) methods. Our
established technique for measuring small sulfhydryl compounds relies upon buffers that contain reagents that interfere with ES-MS. Ultimately characterization of unknown ligands will require ES-MS analyses. We have used some of these new separation schemes to measure sulfhydryl containing compounds in spent culture medium of copper stressed Synechococcus. Thus far we have observed significant concentrations of several common sulfhydryls as well as some unknown peaks that we are exploring further.
Impacts
A better understanding of trace metal chemistry in seawater will enable us to better model the cycling and bioavailability of toxic and nutrient trace metals. These metals can exert significant control over primary production in seawater and hence can affect the cycling of numerous other elements such as carbon.
Publications
- No publications reported this period
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