From: E.braswell <BRASS@UConnVM.UConn.Edu>
  To  : rasmb@alpha.bbri.org
  Date: Fri, 27 Jul 2001 09:43:34 EDT

small molecules

Ms Morris,  We have done a number of sed. equil. studies of the association of
small molecule with MWs as low as a few hundred (methylene blue and
other dyes) many years ago.  These studies had some aspects that were special,
therefore I think that our more recent work is more to your interest. These
involved the trimerization of collagen peptides (monomer MWT = 3k) with
Brodsky and more recently, like Richard Thomas (mentioned by Correia)
coiled coils with Holtzer (Biophysical J. 78, 2037 [2000]). The latter involved
thermodynamic studies of the association of
monomers of ca.4 kD associating to dimers and tetramers. In order to get enough
curvature in your data, you must spin as fast as possible and use as long a
solution column as possible.  The trade off will be that the time to reach
equil. increase with the square of the column height.  For the collagen
and coiled coil studies we used a column height of 5mm and 48-50kRPM.  BUT
it took 2-3 days to reach equil.  For your studies you will need a
proportionately higher column height-- I don't like running at 60k for a long
time for a number of reasons, which are probably over conservative.
Velocity studies are a different beast. you should run as fast as you can
but more importantly, the analysis method is critical.  A mass transport
method (currently not used much) will work well.
Peter Schuck's approach has been mentioned and is very good--but even John
Philo's program "Svedberg" which returns S & D, and is NOT designed for
experiments which have not cleared the meniscus gave fairly good values
for the mol.wts. of model cmpds.in the low thousands and therefore presumably
returned even better values of S.  Synthetic boundary methods would
prbably work well--Even the S value of sucrose was determined!
BUT if the material is also self-associating--I believe
that you would have tremendous difficulties getting much out of any of these
except perhaps the Schuck approach (any comments Peter?).  Good luck! I think
you'll need it.

Emory H. Braswell
Professor of Biophysics and Head,
National Analytical Ultracentrifugation Facility
U-149
University of Connecticut
STORRS, CT 06269-3149
TEL 860 486 5032
FAX         5005
EMORY.BRASWELL@UCONN.EDU