[RASMB] A Complicated Equilibrium Problem

[Eric Salgado]

Peter,
    As far as the c(s) distributions go, it looks as if the s-value of 
both the dimer and trimer are close to each other; so close that I 
thought there was only 1 broad peak at the lower concentrations. I can 
now definitely see that there is a trend of the higher s-value shoulder 
of that broad peak becoming sharper and more abundant as concentration 
increases. In short, it does appear as if the s-values only change in 
height and not value for these two species. With all of this in mind, I 
will try your suggestions for the ball park estimates.
     Thank you very much for your time and advice,
        Eric

Peter Schuck wrote:
> Hi Eric,
>
> if you're able to determine the species concentrations in SV through 
> c(s) in SEDFIT or by discrete species analysis in SEDPHAT, it must 
> mean that the interconversion is sufficiently slow such that the 
> oligomers to not fall apart on the time-scale of sedimentation.  If 
> this is true, then the signals from each oligomer still approximately 
> reflect their equilibrium populations.  In that case, you can simply 
> use the signals of each of the oligomers, convert into concentration, 
> and plug that into any mass action law to get the binding constants.
>
> With a system as complicated as yours, even if it is pretty clean, I'm 
> not sure if there is a good chance to do much better.  For reasons 
> I've outlined previously, I don't think that kinetic rate constants 
> will be sufficiently well determined not to have enormous parameter 
> correlations and be extremely susceptible from systematic errors 
> arising from slight inaccuracies in the assumptions regarding buoyancy 
> and purity.
>
> On the other hand, even if there's slight dissociation during SV, the 
> numbers you may be able to get from species analysis may still be 
> pretty good ball-park estimates.  You can find out if that 
> approximation works well by running your sample at different 
> concentrations. If the s-value of the peaks for each species appears 
> independent of loading concentration (i.e. only the relative height 
> changes, but not peak position), then this should be pretty good.
>
> Peter
>
>
>
> At 02:04 PM 1/14/2008, you wrote:
>> Hello,
>>   I have a system that contains four different molecular weight 
>> species of my protein ( monomer, dimer, trimer, hexamer), all 
>> mediated by Nickel coordination. I can see this through previous SV 
>> experiments, as well as the species analysis model in SEDPHAT. I 
>> cannot, however, determine any dissociation constants using any of 
>> the pre-existing models in that program. I'm thinking that there 
>> might be competing monomer-dimer and monomer-trimer-hexamer 
>> equilibriums, or some combination thereof.
>>   With all of this in mind, I was wondering if there was a program 
>> with a model such as this that I might employ, or if it would be 
>> possible to add my own model to such programs as Heteroanaylsis, 
>> Sedphat, etc.
>>   Thank you for your time and advice in advance,
>>
>>      Eric Salgado
>>      Univ. Cal. San Diego
>>      Dept. Chemistry and Biochemistry
>>
>> _______________________________________________
>> RASMB mailing list
>> RASMB at rasmb.bbri.org
>> http://rasmb.bbri.org/mailman/listinfo/rasmb
>