[RASMB] Why loading concentration affect main peak percentage so much?

[John Philo]

Yi-Ming,

The concentration dependence you are seeing does not seem to be consistent
with reversible self-association, and overall just doesn't make a lot of
sense to me. I suspect your experiment at 0.3 mg/ml is an outlier, and that
sample contains a few percent of irreversible aggregates for some reason.
Nonetheless keep in perspective that you are reporting peak-to-peak
variations in weight-average sedimentation coefficient of 3%, which some
labs may reasonably say is no change at all. Certainly it is clear that you
can't have very much reversible association, at least up to 2 mg/ml.

If your real interest is in the reversible association then I think your
initial focus should be on the concentration dependence of the
weight-average sedimentation coefficients, NOT on the areas of any
individual peaks. Be careful, you are saying you are measuring peaks of
"dimer" or "trimer", but for reversible associations only under certain
circumstances will those peak areas really represent the populations of
individual oligomer species, and even the sedimentation coefficients likely
don't represent the true positions for specific oligomers!

One technical comment is that I'm not sure you are calculating your
weight-average sedimentation coefficients correctly---from looking at the
graph it seems to me those numbers should be higher. I suspect you are
including the half-peak at near zero S in your calculation. Since that
half-peak clearly cannot represent your protein (since your monomer is at ~5
S), that region of the distribution near zero S should NOT be included in
calculating the weight-average sedimentation coefficient---you want an
average for the protein species only, not one that includes sedimentation of
small molecules or whatever is causing that feature at low S. Similarly,
since your buffer contains Tween 80, it would not be surprising if you
sometimes will see a peak near 2 S corresponding to Tween micelles, but if
you did detect such a peak you would not want to include it in calculating
the weight average for the protein.

On another topic, I'm not sure what you really mean by "trying to find a
good buffer system for investigating the size distribution of a protein
sample". If this protein does have a tendency to reversibly self-associate,
then the association constant(s) will definitely vary with pH, ions,
addition of stabilizers such as sucrose, etc. Thus you might find a buffer
condition where you detect no significant self-association, but that would
not imply that is true under other conditions (for example, in your
formulation buffer or in your SEC elution buffer). 

In general terms the buffer you are using seems fine to me---the pH is not
too far from the isoelectric point, and you have very high ionic strength,
so the non-ideality from electrostatic effects should not be an issue. The
Tween 80 is the only thing in that buffer that could cause some issues for
sedimentation analysis. I myself also would tend not to pick MES as the
buffer simply because we have no density or viscosity data for it. On the
other hand, your data suggests to me that the protein may be prone to making
irreversible oligomers under these conditions (and hence you are seeing
inconsistent changes with concentration).

One definition of a "good buffer system" would be one where you can get
consistent results. Have you run replicates at a single concentration to see
what kind of consistency you can get, both for peak areas and the
weight-average value? Without knowing that, it will be quite hard to judge
whether there is a small amount of reversible association. 

John Philo

-----Original Message-----
From: rasmb-admin at server1.bbri.org [mailto:rasmb-admin at server1.bbri.org] On
Behalf Of Yi-Ming_Li at hgsi.com
Sent: Friday, April 29, 2005 3:56 PM
To: rasmb at server1.bbri.org
Subject: [RASMB] Why loading concentration affect main peak percentage so
much?


Hi everyone:

I am trying to find a good buffer system for investigating the size
distribution of a protein sample (isoelectrical point 6.2, UV extinction
coefficient 0.62). In order to see whether self-association exists in the
buffer system, I diluted the samples to 2-, 1-, and 0.3 mg/ml and loaded
into 3 cells (3 mm certerpiece was used for 2 mg/ml loading) for
sedimentation velocity run. The C(S) profile of all loadings showed well
separated monomer, dimer and perhaps trimer peaks. But the percentages of
the main peak are very different between the 0.3 mg/ml and other loading
concentration (95.7% at 0.3 mg/ml, 98.9% at 1 mg/ml , 98.0 at 2mg/ml).
Moreover, the change of weight average sedimentation coefficient with
loading concentrations is complicated (4.94 s at 0.3 mg/ml, 4.80 s at 1
mg/ml and 4.84 s at 2 mg/ml). It seems to me that in 0.3-1 mg/ml
concentration range, self-association is negligible since the Sw decreases
from 4.94 s to 4.80 s when loading concentration increases from 0.3 mg/ml to
1 mg/ml. But at high concentration, it seems that weak self-association
exists, since the Sw has little change or even increase a bit when loading
concentration increases from 1 mg/ml to 2 mg/ml. Our size exclusion
chromatography showed that the main peak percentage of the sample is over
99%. Therefore the result from 1 mg/ml loading AUC data is more consistent
with the size exclusion chromatography. I would like to know why the main
peak percentage is so different at 0.3 mg/ml loading. Is it my buffer system
(10 mM MES, 500 mM NaCl, 0.01% Tween 80, pH 5.5) is not good for size
distribution analysis? The attached is the C(S) profile.

Thanks for help!

Yi-Ming

(See attached file: Comparison of C(S) at two loading concentrations.ppt)