[RASMB] Aggregation AND m-d Kd

[David Hayes]

Hi Mitra,

Regardless of the type of analysis you do (whole boundary fitting or 
weight average S points) an aggregate species will affect the 
analysis.  How it affects the analysis depends on the S value and 
kinetics of the aggregate.

We have had aggregates that are so large that they are essentially 
pelleted by the time the rotor reaches final speed before the first 
scan and no new aggregates form during the time span of the 
experiment.  These have very little effect on the analysis except you 
must use the concentration from the centrifuge's absorbance or 
refractive increment measurement instead of the believed loading 
concentration.  At times it seems that trying to remove such 
aggregates before loading the cells (by pre-spinning and pelleting 
them) disturbs the dialysis equilibrium, and so for interference 
optics experiments, it is best to just let them pellet during the run 
rather than trying to get rid of them before loading the analytical cells.

In all other cases, the best way to deal with irreversible aggregates 
is to fit only the data that does not include the aggregates.  The 
early scans near the meniscus always include the aggregates and 
should not be used in the fit or the determination of weight average 
S.  For best separation, choose the data scans where the smaller 
species of interest are close to the bottom of the cell and the 
aggregate is pelleted.    If you use Sedanal, you simply choose a 
late range of scans.  We jokingly call this 'purifying' our 
sample.  It is a valid strategy when the aggregate boundary does not 
overlap the boundary of the samples of interest.  DCDT+ actually lets 
you set an S value range to fit, though you still have to avoid early 
scans where diffusion dominates and the boundaries almost always 
overlap on the S scale.  In the recent AUC workshop, John Philo 
demonstrated on some data that fitting the whole range resulted in an 
incorrect determination of Molecular Weight while fitting the 
restricted range ignoring the aggregates gave the right 
answer.  Presumably, if you get the wrong Molecular Weight you would 
also get the wrong weight average S.

In one case, we successfully modeled a monomer, dimer, tetramer, 
octomer equilibrium in which one sample had aggregate going faster 
than octomer.  In this case it was necessary to hold the S value of 
the octomer fixed (to a value from the sample when it was fresher and 
had no aggregate) or the S value for the octomer went too high to try 
to best fit the aggregate.  There seemed to be too much overlap 
between the octomer and the aggregate to just choose the right scans, 
but when we held the octomer S value fixed, then it seemed to ignore 
the aggregate and you saw the bump in the residuals at higher S.

If the aggregates are forming during the experiment in a measurable 
amount, you can never choose a set of scans that do not include them 
and in that case have to add them to your model.  In this case, I 
have heard of people modeling the 'aggregate' equilibrium like Jack 
Correia models Tubulin polymerization to tease out tubulin to sample 
binding constants.  I am not familiar with anyone doing weight 
average S isotherms with a background of aggregate formation, though 
it does not sound impossible it probably requires that some one of 
the theorists have worked out the equations for this kind of fit (or 
maybe you would be brave enough to write out the equations yourself).

With any of the major AUC software packages (SEDANAL, DCDT, SEDFIT) 
you can simulate a set of experiments.  I know that with the model 
editor in SEDANAL you can model any of the situations I described 
(reversible or irreversible aggregation) with the model editor, you 
can then investigate yourself what effect an aggregate like what you 
see has on the analysis:  just simulate data sets and then try out 
your analysis strategy and see if it returns the values set in the simulation.

Hope this helps.

Dr. David B Hayes
Analytical Ultracentrifugation Research Laboratory
Boston Biomedical Research Institute
64 Grove St.
Watertown, MA  02472
617-658-7738

Boston Biomedical Research Institute... Today's Research for 
Tomorrow's Health.
Please visit us at www.bbri.org



At 04:04 PM 5/29/2007, you wrote:
>Hi Folks
>I have a question (s) - how does the aggregation (and maybe the 
>subsequent loss)
>of a protein sample during SV affect the Kd of the monomer-dimer equilibrium?
>Either by direct fitting of the boundary or considering the average
>sedimentation coefficient. Is there any way around this?
>
>Mitra
>--
>Mitra S. Rana
>Graduate Student
>Institute for Cellular and Molecular Biology
>2500 Speedway, UT-Austin
>Austin, TX-78712
>_______________________________________________
>RASMB mailing list
>RASMB at rasmb.bbri.org
>http://rasmb.bbri.org/mailman/listinfo/rasmb
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