From: John Correia <jcorreia@biochem.umsmed.edu>
  To  : rasmb@alpha.bbri.org
  Date: Tue, 16 Nov 1999 12:13:58 -0600

Re: s coeff and shape

The response's by Arthur & Bo  disagree in emphasis or bias only  - I wish
to point out my  experimental bias:

1st - Arthur has written long emails to this group on the origins of Ks
and conc dependence of primarily globular systems - this is worth
revisiting - typical globular proteins exhibit less than 1% s© at 1
mg/ml - Arthur's point - if it doesn't show up in an S vs C plot it will
not show up in a vanHolde Weischet plot either.   If a vanHolde-Weischet
plot is nonideal than plotting average S or weight average S or peak
position will also show it.  Plots of nonideal behavior by DNA were
evident long before vanHolde-Weischet plots.   I agree due to the removal
of D effects it is easier to see in a single run by vanHolde-Weischet.

2nd - this requires looking at old lit - but s(c) is typically linear over
at least one order of magnitude.  In those days a wide range of conc were
studied typically by schlieren & thus over the range 2 to 20 or 30 mg/ml. 
The Ks magnitudes observed obviously are derived from this range.

3rd - If a protein exhibits nonideal behavior by a vanHolde-Weischet plot,
than I think the experimental questions are:

  1)  how asymmetric or charged must it be to demonstrate significant
S(C)) at 1 mg/ml.   Or when is a protein like nucleic acid.   Any good
examples out there like monomeric myosin which I think is not all that
nonideal when compared to DNA???

  2)  should the best sed equil fit not actually be nonideal single
species.  This addresses the difference between a hydrodynamic Ks and a
virial coefficient in a sed equil fit.   Absorbance data is usually not
good enough to get a good B estimate - depends upon high large B is -
interference data should be precise enough to do so.  
 
  3) the result must also be evident in an average or peak position of
weight average s vs C plot - more importantly it can be verified with
greater sensitivity by a polarization experiment.  A comparison of
sedimentation & fluorescence data will highlight the fact that
interpretation of f/fo requires an assumption about hydration - the axial ratio
derived is dramatically dependent upon what value you assume - we
typically use the value predicted by amino acid composition as the best
estimate.  There is data to support this assumption.  But others then go
on to measure neutron scattering to improve the interpretation.


Finally, we have observed nonideal boundaries by vanHolde-Weischet
analysis that is artifactual due to two causes.  1)  loss of material from
the plateau due to aggregates.  Bo has documented this in his BJ paper I
believe.  2) collecting data at less than maximum point density - you must
use 0.001 cm to get a good vanHolde-Weischet plot.  I do not understand
the origin but using a spacing of 0.003 or more gives false nonideality
(in our hands - anyone else??).  With the XLA this often means running one
sample at a time, especially if you wish to do significant signal
averaging.


-------------------------------------------------------------------
 Dr. John J. "Jack" Correia
 Department of Biochemistry
 University of Mississippi Medical Center
 2500 North State Street
 Jackson, MS  39216
 (601) 984-1522                                 
 fax (601) 984-1501                             
 new email address: jcorreia@biochem.umsmed.edu     
 homepage: http://fiona.umsmed.edu/~biochem/correia.html  
 dept homepage: http://fiona.umsmed.edu/~biochem/biochem1.html
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