[RASMB] Re: RASMB digest, Vol 1 #360 - 2 msgs
Timothy Mack
mack at cabm.rutgers.edu
Mon Nov 15 19:55:01 PST 2004
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
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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 <ss2337 at columbia.edu>
>To: 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 <ss2337 at columbia.edu>
>From: Peter Schuck <pschuck at helix.nih.gov>
>Subject: Re: [RASMB] question about WinNONLIN
>Cc: 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:
>
>
>>----------------------------------------------------------------------------------
>>The older archived RASMB emails can be found at:
>>http://rasmb-email.bbri.org/rasmb_archives
>>and current archives at
>>http://rasmb-email.bbri.org/pipermail/rasmb/
>>Search All the Archives at:
>>http://rasmb-email.bbri.org/rasmb_search.html
>>----------------------------------------------------------------------------------
>>
>>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
>>RASMB at rasmb-email.bbri.org
>>http://rasmb-email.bbri.org/mailman/listinfo/rasmb
>>
>>
>
>
>***********************************************************
>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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