[RASMB] vbar

Wed Jun 7 00:32:03 PDT 2006

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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