[RASMB] Re: RASMB digest, Vol 1 #360 - 2 msgs

Tue Nov 16 13:25:01 PST 2004

Dear Tim

If the sample is heterogeneous (i.e. there are species present that 
are not participating in the reversible equilibrium.) the larger 
species will fractionate toward the bottom of the cell independently 
of the local concentration. Remember, the composition of a purely 
heterogeneous, non-interacting system will vary purely as a function 
of the radius independently of the local concentration (cf. Yphantis, 
Biochemistry,1964). At the  lower loading concentrations one will be 
collecting data at higher radii for any given local concentration 
span, therefore, the (weight) average composition will be weighted 
toward the higher molecular weight values because the data are being 
collected at higher radii. So if you fit your data to a single 
exponential*, you will get a value for sigma that is higher at the 
lower loading concentrations (which is determined from data at higher 
radii) than at the higher loading concentrations (which is determined 
from data at lower radii). The cut-off gradient and/or maximum 
concentration observable pretty much determines where you can collect 
the data.

It sounds to me as though Satinder's system is either non-interacting 
or a mixture of interacting and non interacting components.

* By the way, fitting your data to a single exponential and calling 
that sigma a "weight average", although it seems to be a popular 
exercise, is not theoretically correct. What you get is only an 
approximation to a weight average, and if there is significant 
curvature in the ln(c) vs r**2 plot, the value you get can be quite 
far from the correct weight average.

At 1:31 PM -0500 11/15/04, Timothy Mack wrote:
>Dear  RASMBers,
>
>I have a question concerning Satinder's results. Satinder, you 
>mentioned that sigma increased with concentration and with rotor 
>speed. For a reversibly homo-associating system I thought that the 
>sigma would remain constant with speed (assuming all data between 
>the meniscus to the bottom can used in the fitting) whereas for a 
>heterogeneous system the sigma would actually decrease as a function 
>of increasing speed. What causes the increase in sigma with rotor 
>speed?   I realize that one should not put too much stock in these 
>diagnostic graphs (sigma vs speed and/or conc) but I just want to 
>make sure that I am thinking about it correctly so if someone could 
>set me straight on this issue I would greatly appreciate it.
>
>Regards,
>Tim
><mailto:rasmb-request at server1.bbri.org>rasmb-request at server1.bbri.org wrote: f
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>>Today's Topics:
>>
>>    1. question about WinNONLIN (Satinder Singh)
>>    2. Re: question about WinNONLIN (Peter Schuck)
>>
>>--__--__--
>>
>>Message: 1
>>Date: Thu, 4 Nov 2004 12:06:39 -0500 (EST)
>>From: Satinder Singh <mailto:ss2337 at columbia.edu><ss2337 at columbia.edu>
>>To: <mailto:rasmb at server1.bbri.org>rasmb at server1.bbri.org
>>Subject: [RASMB] question about WinNONLIN
>>
>>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
>>
>>
>>
>>
>>
>>--__--__--
>>
>>Message: 2
>>Date: Thu, 04 Nov 2004 13:14:01 -0500
>>To: Satinder Singh <mailto:ss2337 at columbia.edu><ss2337 at columbia.edu>
>>From: Peter Schuck <mailto:pschuck at helix.nih.gov><pschuck at helix.nih.gov>
>>Subject: Re: [RASMB] question about WinNONLIN
>>Cc: <mailto:rasmb at server1.bbri.org>rasmb at server1.bbri.org
>>
>>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
>>>
>>>
>>>
>>>
>>>_______________________________________________
>>>RASMB mailing list
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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: <mailto:Peter_Schuck at nih.gov>Peter_Schuck at nih.gov
>>***********************************************************
>>
>>
>>
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