Since you have only Asp and His ligands coordinating the Fe ion, dialysis against, say, 0.1 M Citrate at pH 5 will do the trick. Citrate will chelate the Fe(III) (if the protein has Fe(II) it will be oxidized during dialysis due to air oxygen) avoiding precipitation of Fe(OH)3 and the acid will protonate the protein ligands. If the affinity is really high, adding also some guanidinium chloride (0.5-1M) in the first dialysis step to loosen the protein a little bit will also help.
Best, Javier M. Gonzalez, PhD. University of Maryland Baltimore Department of Pharmaceutical Sciences X-Ray Crystallography Shared Service (UMXSS) 20 Penn St., HSFII, Rm 514 Phone/Fax: 410-7061124/410-7060886 21201 Baltimore, MD http://www2.pharmacy.umaryland.edu/psc/xray/ On Tue, May 11, 2010 at 12:26 PM, Roger Rowlett <[email protected]>wrote: > I would try dialyzing against a solution with 1,10-phenanthroline, > 1,10-phenanthroline-2-carboxylate, > or pyridine-2,6-dicarboxylate. Removal of metals from proteins is often not > just dissociative, but requires the associative interaction of a chelating > agent. Which one works is often empirical. > > Cheers. > > > On 5/11/2010 11:11 AM, Wenguang LIANG wrote: > > Dear all, > > I have a protein which binds iron with two D and two H as active site. I > have tried to extract the iron by dialysis. First, 20mM tris, pH7.5, 150mM > NaCl, 20EDTA, 10mM Na2S2O3, O/N. Then, followed by dialysis with 20mM tris, > pH7.5, 150mM NaCl, 1EDTA O/N to remove the EDTA and Na2S2O3. However, after > dialysis, the density of the Iron is still in the protein. > > Could anybody please give me some suggestion on how to extract Iron out of > the protein? Or I can just use Chelex-100 to extract it instead of Iron. > > Thank you very much for help, > > Best wishes, > > Vinson Liang > > > > -- > ------------------------------ > Roger S. Rowlett > Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: [email protected] >
