From: John Philo <jphilo@mailway.com>
  To  : Rasmb <rasmb@alpha.bbri.org>
  Date: Fri, 27 Jul 2001 09:14:16 -0700

RE: small peptides

I wanted to add one clarification in response to several comments by others
suggesting that the lack of strong curvature in equilibrium data for low
mass species is a major limitation.

That is certainly true IF you are fitting baseline offsets (as you always
must for interference data), but NOT for absorbance data if you fix the
offset. With fixed offsets, you really don't need ANY curvature (the data
can look like a straight line, and the total change in concentration across
the cell could be only 30% or so) to get the correct mass.  For those of you
who have been at the Beckman training workshops, you may recall that we show
an example for the peptide hormone LHRH (buoyant mass 330 Dalton) at 50K rpm
(Fig. 2-12), where the data are nearly linear and concentration varies
<2-fold across the cell.

The problem, of course, is that with these low mass things you can't go to
high speed at the end of the equilibrium run to clear the meniscus and get
an experimental value for the baseline offset, as one normally would for
higher mass species. Usually, though, one can be pretty confident the
offsets at 280 or 230 nm will be <0.02 AU with quartz windows (and usually
less than that), so you can simply vary the fixed offset as part of
evaluating the errors in the estimated mass. Another approach sometimes used
for getting an experimental offset when you can't overspeed is to take a
scan at a wavelength where you expect the absorbance from the sample to be
zero (perhaps 360 nm for peptides) and then assume that offset applies at
other wavelengths.

John Philo
Alliance Protein Laboratories