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<TITLE>Re: [RASMB] Centrifugal force and protein dissociation</TITLE>
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Dear Songpan<BR>
<BR>
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.<BR>
<BR>
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 <FONT COLOR="#800000"><B>'mixture'</B></FONT> - 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.<BR>
<BR>
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 <U>must</U> change on dilution, so this is a critical test. <BR>
<BR>
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 !<BR>
<BR>
Hope this helps<BR>
<BR>
Regards<BR>
<BR>
Arthur Rowe<BR>
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<BLOCKQUOTE><B>From: </B>Songpon Deechongkit <songpon@scripps.edu><BR>
<B>Date: </B>Tue, 23 Jul 2002 19:54:14 -0700 (PDT)<BR>
<B>To: </B>rasmb@rasmb-email.bbri.org<BR>
<B>Subject: </B>[RASMB] Centrifugal force and protein dissociation<BR>
<BR>
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<BR>
Dear colleagues:<BR>
<BR>
Currently, our lab has tried to determine the heterogeniety of an old<BR>
protein sample. The protein, if folded correctly and behave well, should<BR>
remain tetrameric for a long time. However, by sed. Vel. we found that<BR>
the protein sample is the mixture of monomer, dimer, and tetramer. The<BR>
fit was done by DCDT (by John Philo). Here are a few questions that I<BR>
would like to ask:<BR>
<BR>
1) Can centrifugal force tear multimeric protein apart? This protein, in<BR>
particular, has a central channel where water can go through.<BR>
<BR>
2) If there is monomer-dimer-tetramer equilibrium, can we trust<BR>
sedimentation velocity data in terms of their relative amount?<BR>
<BR>
3) What is the reliability or error from the sed. Vel. fit?<BR>
<BR>
We are very confused whether the fact that we observe<BR>
monomer-dimer-tetramer is true or is just an artifact. I would greatly<BR>
appreciate your insight, thought, or suggestion in light of these<BR>
concerns.<BR>
<BR>
Sincerely yours,<BR>
Songpon Deechongkit<BR>
PhD Candidate<BR>
Jefferey W. Kelly Lab<BR>
The Scripps Research Institute<BR>
La Jolla, California<BR>
<BR>
<BR>
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