denaturing buffer for HPLC

Joe Doe None at mail.utexas.edu
Fri Jun 30 20:48:33 EST 2006


In article <1151661037.328693.317870 at h44g2000cwa.googlegroups.com>,
 "Josmar" <josmarlangner at yahoo.de> wrote:

> Hi Roland,
> I am not sure if I understand what you are saying. What comes off the
> column at the void volume must be really huge, i.e. aggregated. Can you
> explain what you mean by swept volume?
> Thanks
> Josmar
> 

Imagine your protein well folded and compact lets say a perfect sphere 
for simplicity.  Now imagine your polypeptide chain completely denatured 
and extended.  The sphere that this extended chain would occupy (the 
volume it sweeps) as it tumbles in solution is the "size" that the 
sizing column is really seeing of your unfolded protein.  So it is quite 
possible for the denatured protein to appear "bigger" than what you 
think it should be without any aggregation.  This is normally reported 
as the stokes radius of the protein.

In reality the folded structure can be anywhere from compact to quite 
extended and the same would apply to "denaturing" conditions T1 nuclease 
is active in 7M urea for example and so obviously folded.

For an example of how urea increases the stokes radius see:

 Biochemistry. 1999 Oct 5;38(40):13367-78.


Apparent radii of the native, stable intermediates and unfolded 
conformers of the alpha-subunit of tryptophan synthase from E. coli, a 
TIM barrel protein.

Gualfetti PJ, Iwakura M, Lee JC, Kihara H, Bilsel O, Zitzewitz JA, 
Matthews CR.


Roland











> > The protein may be eluting in the void volume because the swept volume
> > of the unfolded protein is much larger than the folded protein.  How do
> > you know it is aggregating?
> >
> > Analytical ultracentrifugation may be a better way to look at monomer
> > dimer equilibria and similar questions.  
> > 
> > Roland


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