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Isoelectric focusing of small non‐covalent metal species from plants
Author(s) -
Köster Jessica,
Hayen Heiko,
von Wirén Nicolaus,
Weber Günther
Publication year - 2011
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201000529
Subject(s) - isoelectric focusing , chemistry , ethylenediamine , metal , covalent bond , copper , acetic acid , ferric , ligand (biochemistry) , chromatography , inorganic chemistry , organic chemistry , enzyme , biochemistry , receptor
IEF is known as a powerful electrophoretic separation technique for amphoteric molecules, in particular for proteins. The objective of the present work is to prove the suitability of IEF also for the separation of small, non‐covalent metal species. Investigations are performed with copper–glutathione complexes, with the synthetic ligand ethylenediamine‐ N , N ′‐bis( o ‐hydroxyphenyl)acetic acid (EDDHA) and respective metal complexes (Fe, Ga, Al, Ni, Zn), and with the phytosiderophore 2′‐deoxymugineic acid (DMA) and its ferric complex. It is shown that ethylenediamine‐ N , N ′‐bis( o ‐hydroxyphenyl)acetic acid and DMA species are stable during preparative scale IEF, whereas copper–glutathione dissociates considerably. It is also shown that preparative scale IEF can be applied successfully to isolate ferric DMA from real plant samples, and that multidimensional separations are possible by combining preparative scale IEF with subsequent HPLC‐MS analysis. Focusing of free ligands and respective metal complexes with di‐ and trivalent metals results in different p I s, but CIEF is usually needed for a reliable estimation of p I values. Limitations of the proposed methods (preparative IEF and CIEF) and consequences of the results with respect to metal speciation in plants are discussed.