[RASMB] A Complicated Equilibrium Problem

[Eric Salgado]

Jim,
    Thank you for your  input.
    The oligomers do not remain intact when I run samples down an SEC, 
and I know that EDTA, chelating the mediating Ni away, is able to 
restore all of the monomer. This leads me to believe that the 
interactions are reversible, just kinetically quick compared to the flow 
rate through the column.
    I haven't tried increasing the concentrations higher than they are 
as I am really more interested in the dimer and the trimer. It is really 
these association constants I am interested in, especially considering 
that the hexamer really is not all that prevalent. I figured it would 
have to be in the model since it is there, but it is truly the minor 
species in all of my samples.
     Thank you again,
        Eric



James Cole wrote:
> Hi Eric:
> Regardless of whatever software you use, It seems unlikely that you 
> would be able to reliably fit data to such a complicated model and I 
> agree with John Philo that it would be much better  to clean up the 
> sample before you do the SV. Do you know if all species you mentioned 
> are participating  in a  reversible equilibrium?  If you have some 
> irreversible forms, either monomer or oligomer, you might be able to 
> remove them by gel filtration. It can be useful to take the monomer 
> and oligomer fractions off the column and either rerun them on the 
> column or do SV experiments to determine whether the system is 
> completely reversible.   Alternatively, can you rule out that you 
> have  an indefinite association where the system polymerizes  to form 
> dimer, trimer, tetramer, etc. and goes on beyond hexamer at higher 
> concentrations?
>
> Jim Cole
>
> -------------
>
>
> On Jan 14, 2008, at 2:04 PM, Eric Salgado 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
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