From: Peter Schuck <pschuck@helix.nih.gov>
  To  : rasmb@bbri.org
  Date: Fri, 30 Apr 1999 10:29:46 -0700

Re: Molecular weight (Mw)

Hi Yarabe,

Although I agree with Walter and Jeff's comments, I would recommend a
slightly different strategy for examining the consitentcy of the sed
equilibrium und sed velocity: 

It can be done simply by fitting the sedimentation velocity data using a
Lamm equation fitter and constraining M to the value that was determined
from sedimentation equilibrium experiments. (For example, the software
sedfit allows transforming the fitting variables from (s,D) to (s,M) and
allows constraining M).  

The advantage of the Lamm equation analysis is that will give directly the
residuals of the fit to the raw sedimentation velocity data.  If these
residuals are acceptable within the noise of data acquisition, this
demonstrates that the model is acceptable (i.e. that the sedimentation
velocity analysis is in agreement with the equilibrium data, within the
given noise of data acquisition).  That's all there is to it, in my mind.  

If the direct analysis doesn't fit, i.e. the residuals are systematic, then
this would be an indication of a problem, such as heterogeneity, as pointed
out by Walter and Jeff. 

The reason why I would always prefer this approach is that the
determination of D from sedimentation velocity can be very problematic,
because the analysis can be an ill-conditioned problem (dependent on run
conditions, size of your sample etc.).  This can generate an apparent
inconsistency which could potentially lack any reality.  Low-speed data
contain much more information on D (and M) and therefore the use of M from
sedimentation equilibrium as a constraint in the sed-velocity analysis is a
good way of circumventing the relatively lower information content of D in
high-speed data. 

Best, 
Peter


***********************************************************
Peter Schuck, PhD
Molecular Interactions Resource
Bioengineering and Physical Science Program, ORS
National Institutes of Health
Bldg. 13 Rm. 3N17
13 South Drive 
Bethesda, MD 20892 - 5766
Tel: (301) 435-1950
Fax: (301) 496-6608
email: Peter_Schuck@nih.gov
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