[RASMB] vbar

Ewa Folta-Stogniew stogniew at yahoo.com
Wed Jun 7 11:28:45 PDT 2006


John, 

--- John Philo <jphilo at mailway.com> wrote:

> Ewa,
> 
> Yes I think you have missed a key point. The point
> was NOT to estimate
> stoichiometry based on the actual Rh values and some
> calibration curve of Rh
> versus M. Rather, Peter's proposal was to get the
> molar mass by taking the
> ratio of the sedimentation coefficient (from SV of
> course) to the diffusion
> coefficient (measured directly by DLS), using the
> Svedberg equation. 
> 
thank you for the clarification. 

> For that approach in order to distinguish 21-mer
> from 24-mer you need only
> measure the diffusion coefficient to ~8% accuracy.
> (The sedimentation
> coefficient will easily be accurate to 0.5% or
> better.) The drawback to this
> approach is that it still requires an accurate vbar
> value to get an accurate
> true molar mass.
> 

wouldn't this be the starting point of the difficulty
that Chad described? 

> But actually yes I do find that the precision of the
> diffusion coefficients
> from DLS (and therefore Rh values too) can be about
> 2%. And as Hans
> Schoenfeld said, for homogeneous samples the
> diffusion coefficients from DLS
> do generally agree quite well with those from
> sedimentation velocity.
> 
> But overall I would say static light scattering
> (after SEC or using Allen
> Minton's approach) is probably the easiest and most
> reliable approach to
> resolving this discrepancy.
> 

thank you, Ewa

> John
> 
> -----Original Message-----
> From: rasmb-bounces at rasmb.bbri.org
> [mailto:rasmb-bounces at rasmb.bbri.org] On
> Behalf Of Ewa Folta-Stogniew
> Sent: Wednesday, June 07, 2006 9:07 AM
> To: Schoenfeld, Hans-J.; Allen Minton;
> rasmb at server1.bbri.org
> Subject: RE: [RASMB] vbar
> 
> 
> 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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