[RASMB] Equilibrium runs with small peptides, high offset values

Peter Schuck pschuck at helix.nih.gov
Tue Dec 10 09:33:00 PST 2002


Erin,

I recommend using a Lamm equation solver to model the whole sedimentation 
experiment, including the approach to equilibrium.  This has more 
information and can have higher precision because of the larger number of 
data points.  It implies conservation of mass, which can diminish the 
uncertainty of the baseline (but may introduce the bottom position of the 
cell as an unknown).

If you use SEDFIT, you could also do, for example, a synthetic boundary 
experiment, which will give you unambiguously the baseline offset.  A 
synthetic boundary experiment probably wouldn't work as well for the 
interference optics (because time-invariant noise decomposition is 
incompatible with using an experimental scan as initial 
condition).  However, interference optics seems to be better suited for 
work with such small peptides in conventional loading, because you can get 
closer to the bottom of the cell.  But since you're using absorbance 
anyway, this may be irrelevant.  The general approach, including the 
synthetic boundary modeling, is described in Biophys. J. (1998) 74, 466-474 
(http://www.biophysj.org/cgi/content/full/74/1/466)

This does not address directly the question about the partial-specific 
volume, but it will give you at least more confidence in the experimental 
buoyant molar mass.

Peter


>I did an equilbrium (absorbance optics) run on a water
>soluble  peptide of 1290 Daltons.  I had to spin at 60K rpm
>to get any reasonble data (data at lower speeds showed virtually no
>curvature).  My problem now is this: using Nonlin,  the best fit to the
>data gives a lower than expected sigma (.35 instead of .46 with both the
>offset and the second virial coefficent set to 0).  There doesn't appear
>to be a higher order association.  When I fix sigma to the 'known'
>value, the fit is poor, but when I then let the offset float I get a
>good fit, but the offset is .1332.  Very high!  I obviously can't do an
>overspeeding experiment to get an experimental measure of the depleted
>meniscus absorbance.  As well, I imagine that the calculated partical
>specific volume (from Durchschlag and Zipper, 1994) could be inaccurate.
>Is there something more I can be doing to get an accurate fit?  Which
>would be considered a more physically reasonble fit to the data--a fit
>using a lower than expected sigma value or a high offset?
>
>Thanks,
>
>Erin Matthews
>Graduate Student, Engelman Lab, Yale University
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