Dear Marius, I checked out the references, and it was mentioned that " The zinc EXAFS spectra of P. shermanii superoxide dismutase have shown that zinc can be incorporated in the active center instead of the iron." (Eur. Biophys. J. * 24* , 243-250). And this example further aroused my concern about the possibility of non-specific metal binding. Especially when the SOD showed full activity regardless of Mg or Fe being incorporated (J. biol. inorg. chem. 1 , 532-541). I think the best solution available was in-cell NMR if in vitro experiments could not tell the difference.
Thanks for sharing your experience. Sometimes it just seemed that anything was possible in protein science. Sincerely, Xuan 2009/8/6 Marius Schmidt <marius.schm...@ph.tum.de> > 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/ >
