[RASMB] RE: linearity of radial scan on XL-A

[John Philo]

Seth,

Having looked at your J. Chem. Phys. paper, I see a couple of issues that
might lead to apparent changes in sedimentation coefficient at different
positions in the cell.

First, much of your data was done in a regime where there are severe optical
artifacts. If one tries to run very high concentrations with absorbance
scans by going far off the absorption maxima, and particularly when the
gradients are very steep as in your data, the strong protein concentration
gradient produces a lens effect (from the refractive index gradient) which
in severe cases like yours causes the beam to miss the detector entirely.
This gives rise to a broad spike at the boundary position, sort of like
another meniscus, as seen in your Figs. 3 and 6.

According to your paper, you dealt with this artifact by using the position
of this artifact peak to define the position of the boundary. In my opinion,
there is absolutely no reason to believe that this artifact will correctly
track the true boundary position and give the right sedimentation
coefficient!

Further, you are apparently not using any of the usual software for analysis
but instead are doing fits of ln(r) versus omega^2*t plots, where r is the
position of the boundary. However, even at low concentrations where you
don't have the optical artifact, defining exactly where the boundary is
located is not that straightforward for any real system where diffusion is
not zero. Remember, the equation defining the sedimentation coefficient (eq.
C1 in your paper) really applies only to motion of individual molecules, and
that isn't what we actually measure. It is simply not true that any
arbitrarily selected definition of where within a boundary of finite width
is "the" boundary position will give a correct sedimentation coefficient.
That is why more sophisticated mathematical approaches to defining the
boundary position, such as the second moment or transport methods, were
developed.

In your case you have strong non-ideality effects and self-sharpening on top
of that, which makes extracting the correct sedimentation coefficient even
more problematic. In such cases it is fully expected that even a homogeneous
single species (which may or may not describe your virus preps---to my eyes
your low concentration data in Fig. 5 do not look homogeneous) will show
different sedimentation coefficients in different regions of the boundary
(e.g. in a van Holde-Weischet analysis).

Overall I would summarize by saying your elegant theory probably needs to be
matched by particular care in deciding how real data can be analyzed to give
values that are directly comparable to those coming out of that theory (and
it may be necessary to directly deal with the concentration dependence of
diffusion as well as sedimentation coefficients).

Lastly, although I suspect this is not the primary effect you are seeing,
you are also working under conditions where the boundary is both quite sharp
and moving very rapidly and thus where significant boundary motion might
occur during the time required to complete a scan. This would be more
aggravated if you asked for repetitions or used step-scan mode (those
details weren't noted in the paper).

John Philo
Alliance Protein Laboratories


> -----Original Message-----
> From: rasmb-admin at rasmb-email.bbri.org
> [mailto:rasmb-admin at rasmb-email.bbri.org]On Behalf Of Seth Fraden
> Sent: Tuesday, December 18, 2001 10:38 AM
> To: rasmb at rasmb-email.bbri.org
> Subject: [RASMB] linearity of radial scan on XL-A
>
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>
> At Brandeis, we have a XL-A circa 1995 and I suspect that the radial
> scan coordinates are not being read correctly. The evidence for this
> assertion is that the sedimentation velocity depends on the radial
> position of where it is measured in the cell, with a larger Svedberg
> measured at the bottom. This is not a radial dilution effect because of
> the following two measurements. In the first measurement we evaluated S
> at the top of the cell in a dilute sample of concentration c. In the
> second measurement an initially more concentrated sample was used and S
> was determined towards the bottom of the cell, but because of radial
> dilution the concentration was reduced to the same concentration c as in
> the first measurement. We found that S was always greater at the bottom
> of the cell. The plot of ln(radius) vs. time was quadratic in time,
> instead of linear in time. This could be explained if the radial scan
> was non-linear. The coefficient of the quadratic term was independent of
> sample concentration, consistent with the effect being an instrumental
> artifact. We have published part of these results in
>
> "Concentration Dependent Sedimentation of Colloidal Rods",
> Z. Dogic et. al., J. Chem. Phys. 113, 8368 - 8380 (2000),
>
> available at the following link:
> http://www.elsie.brandeis.edu/pub/sedimentation.pdf
>
> Questions: Have any users experienced non-linear ln(radius) vs. time
> problems? How does one calibrate the linearity of the radial scan? Are
> there windows with 10 line pairs per mm (like a Moire grating) that one
> can insert in a cell?
>
> --
> Yours,
>
> Seth Fraden                      website: http://www.elsie.brandeis.edu/
> Department of Physics            phone:   (781) 736-2888
> MS-057                           fax:     (781) 736-2915
> Brandeis University              email:   fraden at brandeis.edu
> Waltham, MA 02454  USA
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--
Yours,

Seth Fraden                      website: http://www.elsie.brandeis.edu/
Department of Physics            phone:   (781) 736-2888
MS-057                           fax:     (781) 736-2915
Brandeis University              email:   fraden at brandeis.edu
Waltham, MA 02454  USA