[RASMB] Interpreting SV and SE data

[Walter Stafford]

Hi Eric,

    I don't think c(MW) is an appropriate way to analyze an interacting 
system. In the case of a self-associating system the boundary is called 
a reaction boundary and is not composed in individual peaks that can be 
resolved by any method.  Molecular weight values obtained from c(MW) 
would not have any meaning in that case. So I would be very suspicious 
of a trimer molecular weight value obtained that way.

 The boundary must be treated using one of the methods for whole 
boundary fitting to solutions of the Lamm equation for an interacting 
system. And the fit must be done globally over the widest range of 
concentrations possible to get reliable estimates of the stoichiometry 
and equilibrium constants. You could use either SEDPHAT or SEDANAL for 
global fitting to the various models you want to try.  SEDANAL has a 
Model Editor that allows you to specify a very wide variety of arbitrary 
association schemes.

SE is a good way to verify the SV results but in your case it sounds 
like you need to go to higher concentrations to populate the larger 
species in order to resolve the ambiguity between monomer-dimer and 
monomer -dimer-trimer. It is well known also that a 
monomer-dimer-tetramer system can masquerade as a monomer-trimer system 
if the concentration range is not high enough. Solubility issues may 
prevent you from increasing the concentration enough, though.


Walter Stafford

Eric Salgado wrote:
> Hello all,
>   I have a system for which I've done SV experiments at multiple 
> concentrations that, on  increasing the concentration, reveals a peak 
> that starts at an s value of about 2, shifts to a plateau between 2.4 
> and 2.6, then steadily increases at 2.6. I should point out that all 
> of these peaks are very broad.
>   The c(MW) analysis describes a monomer at low concentrations; dimer 
> at intermediate concentrations;  something between  dimer and trimer 
> at high concentrations;  and finally, as  an aggregate  starts to form 
> at very high concentrations, a trimer peak is resolved. This trimer 
> peak correlates to our crystal structure, but it appears as if, 
> because of the apparently low association constant and fast kinetics 
> involved with the trimer formation, this fact is masked in the SV data.
>   In order to hopefully rid of this ambiguity, I performed SE 
> experiments in the concentration regime that did not form an aggregate 
> (and therefore apparently only a small amount of trimer), but find 
> that, as far as a species analysis goes, both a model with 
> monomer-dimer or monomer-dimer-trimer present are statistically equal. 
> That being said, the statistics are lower for the later model, while 
> the fit and residuals for both are equally good.
>   With all of this in hand, how can I determine the best SE model to 
> use, and can I say with certainty that the trimer exists in solution 
> from these data?
>
> I know this is a lot, but I would appreciate any help I can get  with 
> this problem.
>
> Thank you in advance,
>
> Eric Salgado
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-- 
Walter F. Stafford, Ph.D.
Senior Scientist
Analytical Ultracentrifugation Research Laboratory
Boston Biomedical Research Institute
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Watertown, MA 02472-2829
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