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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: Roger Rowlett rrowlett {- at -} COLGATE {- dot -} EDU Date: 2009-08-06 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: 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 < style="border-left: 1px solid rgb(204, 204, 204); margin: 0px 0px 0px 0.8ex; padding-left: 1ex;">Hi -- 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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