[RASMB] time to reach equilibrium

[D.Schubert]

Dear colleagues,

based on a long experience with "difficult" samples I would like to contribute to the
discussion.

I fully agree with Peter Schuck and Michael Morris that what prolongs the time
required to reach sedimentation equilibrium are chemical reactions of different types.
The question is, however, whether the "final" state corresponding to the "true"
sedimentation equilibrium is what one is interested in or whether it represents some
type of artefact, e.g., due to slow dimerization via S-S bonds or changes induced by
oxidizing degradation products in detergents. In the latter cases the information of
interest would be in the pre-equilibrium A(r) profiles. Unfortunately, Peter is right
that fitting these data in many if not most cases will support incorrect models,
despite a seemingly high quality of the fits. One way to overcome this problem may be
to work with smaller sample volumes, which will bring A(r) at different times closer
to "quasi-sedimentation equilibrium" states. This may reveal what is actually
happening. The reduced number of data points often makes it necessary to restrict
oneself to determining Mw(r) instead of calculating the contributions of, e.g.,
monomers and dimers; nevertheless the results may be helpful. In addition, we found it
very helpful to compare results from a series of sedimentation velocity runs on
samples from the same stock solution, performed at time intervals which together cover
the time range required to reach sedimentation equilibrium. In many cases the changes
occurring will be slow enough to consider the components as non-interacting; in others
the s-values (determined, e.g., by sedfit) may be slightly shifted. In any case, there
is a good chance to find out what is happening to the sample and whether that state of
it which is really of  interest  will be accessible to sedimentation equilibrium
analysis.

Best regards,
Dieter


--
Prof. Dr. Dieter Schubert
Institut fuer Biophysik der JWG-Universitaet
Theodor-Stern-Kai 7, Haus 74
D-60590 Frankfurt am Main
Germany
Tel. +49 (0)69 63017073
Fax  +49 (0)69 63015838