Long time ago I had a superoxide dismutase that was active with iron as well as manganese. No matter what functional metal (Fe or Mn) was bound a substantial fraction up to 1/3 of the molecules had the (non-functional) Zinc in their metal binding site (found by AAS and EXAFS). Zinc is everywhere, even in plastic bottles for your distilled water, it is extremely hard to get rid of. And it seems to fit to many iron binding sites. When I recall correctly a fairly stable source of Fe(II) is Mohr's salt. However, Fe binds also unspecifically to the protein, so it is very hard to quantify iron binding to their specific site because eg. EPR spectra change with each washing step.
Best Marius > Hi Xuan, > I guess your protein is not an E.coli protein. There are several > examples that eukaryotic Zn-proteins expressed in E.coli contain Fe > instead of Zn. I am sceptic whether IMAC with different metal ions > will > give the solution of the problem. If you really want to get > information > on the metal ion binding properties you will have to do some matallo > biochemistry: preparing apo protein, reconstitution with metal ions, > UV-Vis spectroscopy, EPR would be great, ... > >> Dear Sir or Madam, >> >> The ICP-ES results indicated that 1 molar my protein purified from >> E.coli Origami(DE3) contained about a half molar Zinc and nearly a >> quarter molar Iron (whether II or III was not available). The protein >> carried a MBP tag on the N-terminal and the situation was similar with >> or without His tag at the C terminal. I want to determine whether my >> protein really bind Zinc or Iron. Does anyone have any experience >> about such problems? >> >> Specifically, now I want to compare the binding efficiency on various >> IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or >> CuSO4(control). However, considering the instability of Fe(II) in >> solution, the design still seemed problematic. >> >> Sincerely, >> >> Xuan Yang >> >> National Laboratory of Biomacromolecules and >> Center for Infection and Immunity, >> Institute of Biophysics, >> Chinese Academy of Sciences, >> Room 1617, 15 DaTun Road,Chaoyang District, >> Beijing, China, 100101 >> Tel: 86-10-64884329 >> Academic email: ya...@moon.ibp.ac.cn <mailto:ya...@moon.ibp.ac.cn> >> We will either find a way or make one. >> Dr.habil. Marius Schmidt Asst. Professor University of Wisconsin-Milwaukee Department of Physics Room 454 1900 E. Kenwood Blvd. Milwaukee, WI 53211 phone: +1-414-229-4338 email: m-schm...@uwm.edu http://users.physik.tu-muenchen.de/marius/
