[RASMB] Centrifugal force and protein dissociation

[Arthur Rowe]

Dear Songpan

What Jack Kornblatt says is unquestionably correct - but it is also true to
say that dissociation of oligomeric proteins at such low pressures is a very
rarely reported phenomenon.

I am not clear from what you say as to just how you have analysed your data.
If you have found a good fit to a 'mixture' - the word you use - of monomer,
dimer and tetramer, then why not believe it ?  I assume you have a good
baseline (which optics are you using ?) and that the residuals for your fit
appear to be randomly distributed, with no trend line apparent. Long-term
storage (in the cold ?) could cause slow dissociation, via weakening of
hydrophobic bonding.

How to be sure that its a mixture that you have, and not a chemical
equilibrium ? The classical way to find that out is to do multiple runs at
several concentrations. You could try for a global  fit, but equally simply
analyse all 3 runs and see if you get essentially the same estimates for
amounts of monomer etc. Bear in mind that the lower the loading
concentration, the less the precision, of course. A reversible equilibrium
must change on dilution, so this is a critical test.

If you do then find solid evidence for the presence of a reversible
equilibrium, then analysis becomes more complex, but let's leave that for
the moment !

Hope this helps

Regards

Arthur Rowe

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Arthur J Rowe
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School of Biosciences
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From: Songpon Deechongkit <songpon at scripps.edu>
Date: Tue, 23 Jul 2002 19:54:14 -0700 (PDT)
To: rasmb at rasmb-email.bbri.org
Subject: [RASMB] Centrifugal force and protein dissociation


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Dear colleagues:

Currently, our lab has tried to determine the heterogeniety of an old
protein sample.  The protein, if folded correctly and behave well, should
remain tetrameric for a long time.  However, by sed. Vel. we found that
the protein sample is the mixture of monomer, dimer, and tetramer.  The
fit was done by DCDT (by John Philo).  Here are a few questions that I
would like to ask:

1)  Can centrifugal force tear multimeric protein apart?  This protein, in
particular, has a central channel where water can go through.

2)  If there is monomer-dimer-tetramer equilibrium, can we trust
sedimentation velocity data in terms of their relative amount?

3)  What is the reliability or error from the sed. Vel. fit?

We are very confused whether the fact that we observe
monomer-dimer-tetramer is true or is just an artifact.  I would greatly
appreciate your insight, thought, or suggestion in light of these
concerns.

Sincerely yours,
Songpon Deechongkit
PhD Candidate
Jefferey W. Kelly Lab
The Scripps Research Institute
La Jolla, California


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