Re: [ccp4bb] Zinc or Iron binding protein, that is a question!

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: Zinc or Iron binding protein, that is a question!
From: Xuan Yang pattisyang {- at -} GMAIL {- dot -} COM
Date: 2009-08-06

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From: "Moraes, Isabel" Isabel {- dot -} Moraes {- at -} EVOTEC {- dot -} COM
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Subject: Re: Zinc or Iron binding protein, that is a question!
From: Marius Schmidt marius {- dot -} schmidt {- at -} PH {- dot -} TUM {- dot -} DE
Date: 2009-08-06

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: YangX@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-schmidt@uwm.edu
http://users.physik.tu-muenchen.de/marius/

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Zinc or Iron binding protein, that is a question!
From: Xuan Yang pattisyang {- at -} GMAIL {- dot -} COM
Date: 2009-08-06

Next message:
Subject: Protein Production Senior Scientist Vacancy
From: "Moraes, Isabel" Isabel {- dot -} Moraes {- at -} EVOTEC {- dot -} COM
Date: 2009-08-06