[RASMB] question about WinNONLIN

[Peter Schuck]

Hi Satinder,
there's probably several possibilities, but it reminds me of a protein we 
studied when there was a bit of low molecular weight impurities in the 
prep.  Sometimes, these can go undetected on SDS PAGE, but would skew the 
sedimentation profiles in a way consistent to what you describe.  I would 
try a model fixing the molecular weight (and v-bar - I assume that's 
known), floating the binding constant, and adding an unrelated species of 
fixed molar mass (low Mw, perhaps a few kDa) into the global fit to see if 
that can explain the data.  (I'm not sure if WinNONLIN let's you do this, 
I'm using SEDPHAT.)  In order to verify this, if you still have the sample, 
it might be useful to do a velocity run (after shaking up the sample, that 
should work well enough even with a few mm column).  In our case, we saw 
the same species in a very slow boundary in a velocity experiment done in 
parallel.
Hope that helps,
Peter



At 12:06 PM 11/4/2004 -0500, you wrote:
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>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
>
>
>
>
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***********************************************************
Peter Schuck, PhD
Division of Bioengineering & Physical Science
National Institutes of Health
Bldg. 13 Rm. 3N17
13 South Drive
Bethesda, MD 20892 - 5766
Tel: (301) 435-1950
Fax: (301) 480-1242
email: Peter_Schuck at nih.gov
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