6M Guanidine HCl and IEC

Andrew Wallace a.wallace at qub.ac.uk
Wed May 28 13:51:06 EST 1997


Peter Hains wrote:
> 
> Hello all,
> 
> I was wondering if anybody knows for sure if you can deionise 6M
> guanidine HCl for use in ion-exchange chromatography the way you can with
> 8M urea. I have tried 8M urea but I am not convinced that it fully
> denatures the proteins and I know that 6M Gdn HCl does.
> 
> Thanks in advance, Peter.

Not in the sense that you mean since guanidine is a base and will become
protonated to form guanidinium ions at any pH below the pKa. These
denature the protein by interfering with ionic interactions and are
therefore certain to interfere with whatever kind of IEC that you do.
Even if you could deionise GdnHCl (e.g. by high pH) there would be no
point as it wouldn't denature your protein anymore. Urea works in IEC
because pure urea is a neutral compound, denaturing proteins by
disturbing structure-maintaining non-ionic interactions. Urea is
unstable in solution and decomposes to charged species therefore it can
be deionised before use in IEC to remove these contaminants.

It may be worthwhile to add some other non-ionic denaturant to your 
8M urea, such as low concentrations of organic solvents like methanol or
ethanol and try the IEC with that. You could also try denaturing your
protein with 6M GdnHCl then exchanging this with 8M urea by gel
filtration or dialysis before IEC.

Andrew
-- 
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==================================================================
Andrew Wallace,Ph.D., Queens University Belfast,  N. Ireland (UK)   
a.wallace at qub.ac.uk   http://web.qub.ac.uk/bb/awpage/wallace.html   
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