The determination of ferric iron in plants by HPLC using the microbial iron chelator desferrioxamine E |
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Authors: | Victoria?Fernández Email author" target="_blank">Günther?WinkelmannEmail author |
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Institution: | (1) Fruit Science Department, Humboldt University of Berlin, Albrecht-Thaer-Weg 3, 14195 Berlin, Germany;(2) Department of Microbiology & Biotechnology, University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany |
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Abstract: | Common methods for plant iron determination are based on atomic absorption spectroscopy, radioactive measurements or extraction
with subsequent spectrophotometry. However, accuracy is often a problem due to background, contamination and interfering compounds.
We here describe a novel method for the easy determination of ferric iron in plants by chelation with a highly effective microbial
siderophore and separation by high performance liquid chromatography (HPLC). After addition of colourless desferrioxamine
E (DFE) to plant fluids, the soluble iron is trapped as a brown-red ferrioxamine E (FoxE) complex which is subsequently separated
by HPLC on a reversed phase column. The formed FoxE complex can be identified due to its ligand-to-metal charge transfer band
at 435 nm. Alternatively, elution of both, DFE and FoxE can be followed as separate peaks at 220 nm wavelength with characteristic
retention times. The extraordinarily high stability constant of DFE with ferric iron of K=1032 enables extraction of iron from a variety of ferrous and ferric iron compounds and allows quantitation after separation by
HPLC without interference by coloured by-products. Thus, iron bound to protein, amino acids, citrate and other organic acid
ligands and even insoluble ferric hydroxides and phosphates can be solubilized in the presence desferrioxamine E. The “Ferrioxamine
E method” can be applied to all kinds of plant fluids (apoplasmic, xylem, phloem, intracellular) either at physiological pH
or even at acid pH values. The FoxE complex is stable down to pH 1 allowing protein removal by perchloric acid treatment and
HPLC separation in the presence of trifluoroacetic acid containing eluents.
Published online December 2004 |
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Keywords: | iron iron deficiency ferrioxamines siderophores |
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