switching cations

lgbell at liverpool.ac.uk lgbell at liverpool.ac.uk
Mon Jul 28 04:20:13 EST 1997


Dirkson E. Bussiere-42T wrote:
> 
> I'm trying to swap the Zn2+ in an enzyme with other divalent
> cations, but everytime I do it, the protein precipitates.  I
> more or less dialyze the protein in 50mM Tris-HCL, pH 7.4,
> with 1-10 mM of the cation2+; the protein is stable in
> the Tris by itself.  Does anyone know of a way to stablize the
> transition?  One caveat:  the enzyme is quite concentrated
> (2.1 mg/ml), and I have thought of diluting it a bit.  But I
> will have to reconcentrate it to 12 mg/ml, so this does not
> seem to solve the problem.
> 
> Any ideas would be appreciated.
> 
> -Dirk
> 
> --
> -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-
> Dirk Bussiere                 bussierd at tvsfrank.pprd.abbott.com
> Abbott Laboratories             Office: (847)935-0916
> Department 42T                  FAX:    (847)937-2625
> 100 Abbott Park Rd.
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What happens if you first dialyse against a suitable metal chelator, say
EDTA, then switch to water and then onto your selected metal ion?

You could also try the experiment in the presence of a detergent, if you
select a suitable non-ionic detergent it shouldnt interfere with the
metal ion chelation processes and can be dialysed out at the end.

One point worth noting is that some metal ions can exhibit an ability to
produce free radicals or oxidative changes (say Cu2+ on cystienes) in th
epresence of some proteins which may also lead to aggregation.

Hope this helps.

Len Bell.
lgbell at liv.ac.uk



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