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Re: [ccp4bb] Zinc or Iron binding protein, that is a question! |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: Zinc or Iron binding protein, that is a question! From: James Holton JMHolton {- at -} LBL {- dot -} GOV Date: 2009-08-06 I don't think anyone has suggested this yet, but as long as your buffer does not contain sulfur and your protein does contain a known number of S atoms, then you can get an accurate molar ratio of any metal to sulfur via PIXE (proton-induced x-ray emission). Elspeth Garman is one of the few people in the world who can do this, but last I heard she was still interested in having people send her samples. Very tiny samples (such as protein crystals) are fine for this technique. http://www.bioch.ox.ac.uk/aspsite/index.asp?pageid=578 -James Holton MAD Scientist Roger Rowlett wrote: > 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: >> 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 >> >> >> 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: YangX@moon.ibp.ac.cn >> >> >> >> We will either find a way or make one. >> >> >> >> > -- > ------------------------------------------------------------------------ > 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: rrowlett@mail.colgate.edu CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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