As others have implied, quantifying metals in metalloproteins is very challenging. In my experience, the principal problems are (1) adventitious metal contamination, (2) accurate measurement of protein concentration, and (3) weak metal binding.

Zinc and iron are ubiquitous microcontaminants, and crop up in some pretty unusual places. Zinc will get into everything, and plastic bottles and microcentrifuge tubes seem to be a pretty rich source of iron contamination. In addition, we have found that some plasticware will also bind low concentrations of zinc and other metal ions from solution, making standardization of instruments very frustrating. Having said all this, ICP-OES or ICP-MS are absolutely the best methods of quantifying metals in protein solutions. ICP methods are linear over many orders of magnitude, and sample prep can be minimal. We simply dilute protein solution in high-quality deionized water to approximately 0.1-1.0 ppm in glass (acid washed and dried if possible), NOT plastic, and aspirate into an ICP-OES. The protein must be diluted enough to reduce viscosity issues in nebulization. Standards can be prepared in deionized water. We dilute our protein solutions with the same dI water we use to prepare the blank, and there is always some detectable Zn background. (Fe is usually very low in glass containers). The key to accurate measurements is Zn concentrations >0.1 ppm. This has worked very well for us for quantifying native and metal-substituted zinc-metalloproteins. If you are really up against contamination, you can extract solutions with Chelex resin, but it is possible that this treatment could also remove loosely bound metals from proteins. If your putative zinc protein has one or more sulfur ligands, this is less likely to be a problem.

Measuring protein concentrations accurately is also challenging. Every protein quantification method is subject to idiosyncrasies. The least idiosyncratic chemical methods are the microbiuret method and a variety of UV methods (I like one based on A230 in 0.1% Tween-100 or another detergent.) Measuring protein concentration will be the major source of error in metalloprotein stoichiometry measurements. Protein has to be really homogeneous for any method to be accurate.

Also keep in mind that His-tagged proteins (and some proteins with vicinial His residues or other ligands on the surface) may non-specifically bind metal ions, clouding metal stoichiometry.

Finally, if your metal is not in a tight binding site, it will be difficult to prepare solutions with the saturated stoichiometry. In these cases, it is possible that microcalorimetry might be useful.

Xuan Yang wrote:

[log in to unmask]" type="cite">
Dear Mr. Fritz,

Yes, the protein is not an E.coli protein! Instead, it was cloned from a virus. And since it was a nonstructural viral protein, I thought it might be appropiate to treat it as eukaryotic proteins.

E.coli system was quite different from eukaryotic ones, hence I was quite cautious about the ICP-ES result and trying to confirm it via alternative method. Thanks very much for mentioning the examples which suggested that Fe might be contaminants. Indeed, when I cut the protein in two parts (still with MBP) and test them via ICP-ES again, Fe became negligible in both and Zn stoichiometry increaed to 1:1 in the C-terminal part. The result lead me to focus on Zn instead of Fe. But I still want to confirm the idea.

Matallo biochemistry was exactly what I dreamed to do.

Sincerely,

Xuan Yang

2007/8/6 Guenter Fritz <[log in to unmask]>

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

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