[RASMB] question about WinNONLIN

[Satinder Singh]

Hello.

I am trying to determine the dissociation constant of a homodimer. The 
monomer molecular weight (by both sequence & molecular weight) is 33,400 
Da. At 15,000 rpm, the sigma is 0.95. I ran the experiment at 15,000, 
20,000, 27,000, and 35,000 rpm with 4 different protein concentrations 
(0.2, 0.4, 0.8, & 1.2 mg/ml) using A280 detection.

Before concluding that I indeed had a monomer-dimer equilibrium, I used 
the "sigma test" to see if there was evidence of association. Indeed, the 
sigma increased as a function of protein concentration (for a single
rotor speed). It also increased as a function of rpm (for a single protein
concentration).

The data are best fit to a monomer-dimer equilibrium, although the 
residuals with this model (sigma fixed) still look a bit skewed. The SRV, 
however, is pretty good at 5.6384x10-3. The question I have is 
this: When I FIX sigma at the monomer molecular weight, I get a 
dissociation constant of 12 uM. However, if I let sigma float, sigma falls 
to 0.8276, which corresponds to a molecular weight of 28,000 Da,  and I 
get a dissociation constant of 2.3 uM.

If the data are fitted correctly and nothing has happened to the protein
sample during the experiment, i.e., aggregation, precipitation, shouldn't
the 2 dissociation constants be relatively close? Could anyone tell me 
what I may be doing wrong?

Thanks in advance for your help.

Satinder