From: John Correia <jcorreia@biochem.umsmed.edu>
  To  : jphilo@mailway.com
  Date: Fri, 27 Jul 2001 16:07:03 -0500

RE: small peptides

John - let me rephrase your comment to clarify - if there is no
association and using absorbance optics, assuming a small offset will accurately
determine MW from a single channel fit - or in a LnC vs r2/2 plot the
slope of the linear line will correctly give back the correct MW.

On my machine it is not unusual to have offsets of 0.002 up to 0.05 in
global fits of self associating data sets.  I typically do 9 loading
concentrations at one speed (lately all my data for a monomer-dimer-trimer
system I am working on is also two wavelengths).  

So in practise how dependable is this offset assumption?

My understanding of globally fitting many channels of data, conc or speed,
is in part to help constrain the offsets and the Ao or meniscus
concentrations, thus giving the best global MW and/or association constants.  If the
system is (reversible and) associating, then assuming offsets or
neglecting offsets sets up an error propagation between offset and Ao and thus K. 
When fiitting associating systems more curvature helps to determine all
parameters in a global sense, especially the Nmer values, although I must
admit I do not compare wide ranges of curvature, wide ranges of sigma, to
see the effect on Ks.  (A caveat in this is the fractionation of monomer
from polymer - too much curvature may mean the polymer is pelleting - I
usually try to spin at speeds where the largest Nmer has a sigma less than
4 to 5) 

An additional point worth making, although not necessarily for the small
peptide case, if one uses conservation of concentration fitting methods,
then one must in fact spin al lower speeds and thus less curvature to
avoid loss of material.  This may be especially critical for analysis of
hetero-associating cases.  The conservation of mass in part compensates
for the information lost in the low curvature plus the complexity of
having two thermodynamic species.