[RASMB] vbar

Ewa Folta-Stogniew stogniew at yahoo.com
Wed Jun 7 09:07:14 PDT 2006 

Hi all,

my point was that for such big oligomers (i.e.
composed of many units) one should go after molar mass
rather than size to distinguish 21-mer vs. 24-mer. 
The 24-mer differs by ~14.3% from 21-mer in mass while
it differs only by ~5.8% in size.  Thus, unless one
can measure Rh with accuracy better than 2% (it this
doable?), it seems virtually impossible to assign the
stoichiometry with confidence.... 

Am I missing something?

Ewa 




--- "Schoenfeld, Hans-J."
<hans-j.schoenfeld at roche.com> wrote:

> Static light scattering may be simpler as it saves
> the sedimentation experiment.
> However, to feel confident I would do both. Both
> methods depend exclusively on absolute measurements
> of parameters (M vers. D and s), should therefore
> result in similar numbers for M and would in the
> best case confirm each other...
> 
> Hans-Joachim Schönfeld.
> 
> -----Original Message-----
> From: rasmb-bounces at rasmb.bbri.org
> [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of
> Allen Minton
> Sent: Wednesday, June 07, 2006 3:21 PM
> To: rasmb at server1.bbri.org
> Subject: RE: [RASMB] vbar
> 
> 
> I second Ewa's suggestion that static, not 
> dynamic, light scattering seems to be the method 
> of choice here, provided that you have an 
> accurate determination of concentration and 
> refractive increment.  The ratio of scattering 
> intensity to concentration is proportional to 
> molar mass and can easily be determined to within 
> a few percent in less than 15 min.  We do this 
> routinely.  The only caveat is that special 
> attention must be paid to sample preparation: for 
> best results the sample must be filtered through 
> a 0.02 micron filter and degassed by centrifugation
> just prior to measurement.
> 
> Allen Minton
> 
> At 03:32 AM 6/7/2006, you wrote:
> >Ewa and Peter,
> >I agree with both of you, that DLS is not
> >precise in the characterization of single 
> >components of polydisperse samples. However, I 
> >also agree with Peter that the method is far 
> >better doing in the analysis of monodisperse 
> >samples. To my experience, diffusion 
> >coefficients as obtained by DLS are precise and 
> >highly reproducible (determination is absolute 
> >and is mainly based on measurement of time which 
> >can be done with very high precision...).
> >Our experience was that the strategy to combine 
> >s from AUC with D from DLS resulted in molecular 
> >masses that were very close to values as 
> >obtained from sedimentation equilibrium runs and 
> >I therefore recommend to give it a trial (see 
> >table in: Schoenfeld, Poeschl, Mueller: 
> >"Quasi-elastic light scattering and analytical 
> >ultracentrifugation are indispensable tools...", 
> >Biochemical Society Transactions, 26, pp.753-758
> (1998)).
> >Best regards,
> >Hans-Joachim.
> > >============================================
> > >Dr. Hans-Joachim Schönfeld
> > >F. Hoffmann-La Roche Inc.
> > >PRBD-E, B93/5.44
> > >CH-4070 Basel
> > >Switzerland
> > >
> > >Tel. (+41) 61 688 28 95
> > >Fax. (+41) 61 688 90 60
> >mailto:hans-j.schoenfeld at roche.com
> >
> >
> >-----Original Message-----
> >From: rasmb-bounces at rasmb.bbri.org
> >[mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of
> Peter Schuck
> >Sent: Tuesday, June 06, 2006 11:08 PM
> >To: Ewa Folta-Stogniew; Chad Brautigam
> >Cc: rasmb at server1.bbri.org
> >Subject: Re: [RASMB] vbar
> >
> >
> >Ewa,
> >
> >I agree with you that one maybe should not use a
> size-distribution 
> >model in that case.  However, this limitation is
> not one of DLS, but 
> >the analysis method used.
> >
> >  From the sedimentation velocity, it should be
> possible to assess very 
> >well how monodisperse the peak is.  If it appears
> to be pretty much a 
> >single species (without very large aggregates, or
> those could perhaps 
> >be filtered out), then one can do the DLS analysis
> nicely with a single 
> >species model.  For example, both SEDFIT and
> SEDPHAT allow you to use 
> >those discrete models and fit DLS autocorrelation
> functions, and I 
> >believe some instrument makers also supply software
> that can give a 
> >single diffusion coefficient, rather than a
> distribution.  That's what 
> >I would try to combine with s.
> >
> >Peter
> >
> >
> >At 04:32 PM 6/6/2006, Ewa Folta-Stogniew wrote:
> > >Peter and Chad,
> > >
> > >I maybe too pessimistic, but I do not believe
> that DLS
> > >has enough precision to distinguish 21 vs. 24
> > >configuration. The standard fitting protocol for
> Rh determination is 
> > >for Gaussian distribution of sizes with 15% SD,
> which would encompass 
> > >sizes for both configurations making them
> virtually identical.
> > >
> > >Static LS for MW determination maybe an option
> given
> > >the experiment is done with accuracy at least +/-
> 3%.
> > >
> > >
> > >Ewa
> > >
> > >--- Peter Schuck <pschuck at helix.nih.gov> wrote:
> > >
> > > > Hi Chad,
> > > > if the oligomer is relatively pure, would it
> be
> > > > possible to do
> > > > DLS? Perhaps taken together with s, that would
> give another quick 
> > > > way of confirming M, instead of equilibrium
> sedimentation.
> > > > I don't think enclosed
> > > > solvent adds to the buoyant molar mass,
> therefore it
> > > > would not contribute
> > > > an error to the vbar. However, due to error
> > > > propagation from vbar to the
> > > > (1-vbar*rho) term, small errors in vbar are
> > > > amplified; my feeling is that
> > > > this usually could account for a few percent
> > > > uncertainty (i.e. in the
> > > > ballpark of 1/24).
> > > > Peter
> > > >
> > > > At 02:45 PM 6/6/2006, you wrote:
> > > > >Hello, All,
> > > > >
> > > > >Sorry if this is a rudimentary question. I
> have
> > > > been using velocity
> > > > >sedimentation to examine the oligomeric
> states of a
> > > > protein and
> > > > >mutants thereof. Some mutants are trimers,
> and the
> > > > molecular weight
> > > > >estimates given by sedfit (either a c(M)
> > > > distribution or a discrete
> > > > >species model) are very reasonable. However,
> based
> > > > on a crystal
> > > > >structure, we expect the wild-type to be a
> 24-mer.
> > > > Sedfit
> > > > >consistently underestimates the molecular
> weight (
> > > > I get something
> > > > >more akin to 21-mer).
> > > > >
> > > > >I assume that there are at least to
> possibilites
> > > > here:
> > > > >
> > > > >1. The crystal structure is wrong, and the
> thing
> > > > really is a 21-mer
> > > > >in solution.
> > > > >
> > > > >2. The vbar calculated by Sednterp is
> inaccurate-
> > > > it is not
> > > > >accounting for the fact that some of the
> volume of
> > > > the 24-mer is not
> > > > >taken up by protein, but by solvent. The vbar
> is
> > > > therefore
> > > > >significantly too low, with adverse effects
> on the
> > > > MW calculation.
> > > > >
> > > > >Does anyone know if there is a more accurate
> way to
> > > > estimate vbar in
> > > > >cases of large macromolecular assemblies? Can
> our
> > > > crystal structure
> > > > >help us out in any way?
> > > > >
> > > > >BTW, yes, I know that sed. equilibrium might
> be the
> > > > preferred
> > > > >approach in this case, but instrument time is
> > > > limited at the moment.
> > > > >
> > > > >Thanks,
> > > > >Chad
> > > > >
> > > > >
> > > > >==================================
> > > > >Chad A. Brautigam, Ph.D.
> > > > >Research Scientist
> > > > >The University of Texas
> > > > >Southwestern Medical Center at Dallas
> > > > >5323 Harry Hines Blvd.
> > > > >Dallas, TX 75390
> > > > >Office:  (214) 645-6384
> > > > >Fax:      (214) 645-5383
> > > > >
> > > > >
> > > > >
> > > > >
> > > >
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