From: Peter Schuck <pschuck@helix.nih.gov>
  To  : judithk@vax2.concordia.ca
  Date: Mon, 01 Oct 2001 09:02:29 -0700

Re: Keq from sedimentatin velocity

Hi Judith,

I think how to do this best depends a bit on your system.  In any case it
is very helpful to know the sedimentation coefficients of the monomer and
dimer.  

If the self-association is rapid relative to the time-scale of
sedimentation, you could use direct boundary modeling with the
monomer-dimer self-association model in sedfit.  This will simulate the
sedimentation with instantaneous monomer-dimer equilibrium at all points in
the cell, and the analysis will directly give you the equilibrium constant
in signal units (i.e. it will ultimately require the extinction coefficient
or the refractive index increment; a more detailed description of that can
be found at
http://www.analyticalultracentrifugation.com/sedfit_help_selfassociation.htm
).  Knowing the monomer and dimer sedimentation coefficients (and I presume
also the molar masses) should make this a well-conditioned problem. 

If, on the other hand, the self-association is slow relative to the
sedimentation time, no redistribution of monomer and dimer will occur
during the sedimentation velocity run, and one can use the loading
concentration as the total (at equilibrium before the run).  The fractional
concentrations of monomer and dimer could be obtained by direct boundary
modeling with discrete species, or by integrating the monomer and dimer
peaks under c(s).  This approach has been used recently by Christine Ebel,
published in Buisson et al, JMB (2001) 311:217-228.  

Of course, there are other ways of analyzing such a system, for example
using weight-average sedimentation coefficients from dcdt, as Jack Correia
has pointed out. 

Best, 
Peter







At 05:22 PM 9/27/01 -0400, you wrote:
>Dear RASMBers, I am trying to measure the equilibrium constant for a
>monomer/dimer system using sedimentation velocity. I feel reasonably sure
>of the s20,w values for the monomer and dimer and reasonably sure that
>there are no higher order mers. However, when calculating Keq, what
>protein concentration do I use? Is it the concentration of the solution
>that I loaded? Or is it the concentration at some point in the cell? If
>the later - at what point in the cell do I measure it and when during the
>run? Many thanks.
>
>Judith Kornblatt
>Dept of Chemistry and Biochemistry
>Concordia University
>1455 de Maisonneuve Ouest
>Montreal, Qc H3G 1M8
>Tel: 1 514 848 3384   FAX: 1 514 848 2868
>