[RASMB] estimation of Vbar from sedimentation velocity data

[Matthew Hockin]

I am working with 25 kDa linear polymer Polyethylenimine.  What I am trying
to do is use sedimentation velocity to characterize the product obtained
upon conjugating a protein to the polyethylenimine.  There are, so far as I
can tell, no published values for the partial specific volume of this
polymer.  I have done the following experiments and would like feed back as
to what can done to strengthen my analysis.
1) SV experiments of the polymer alone in 250 mM NaCl pH 7.0 (the polymer is
a very strong buffer thus no other components)

3 concentrations were analyzed spanning 0.64-0.1 mg/ml using interference
optics at 52000 RPM.

In an attempt, perhaps feeble, to determine the vbar I began by fitting each
concentration data set using c(S) in sedfit and with known calculated buffer
density and viscosity corrected to the experimental temperature (22.5 c),
and adjusted the partial specific volume to obtain a reasonable MW estimate
after examining the peak MW distribution in the c(S) plot.

Landing on a value of 0.85 for vbar, the fits are exceptionally good as
measured by examination of the residual plot (< 0.03 variance) as well as by
examination of the graphical residual bit map, uniform grey (actually the
best uniform grey fit I have ever seen).  These fits yield a single peak at
experimentally determined S values of 0.6 at all concentrations.  Every
concentration yields a best fit experimental S value of 0.6 using frictional
ratios of 2.34, which is reasonable given that this polymer is supposed to
act as a ridgid rod in solution. Prior to the centrifuge the polymer has
been gel filtered over high resolution superdex 200 columns and
characterized with respect to the average number of titratable nitrogens.
 The gel filtration is used simply to narrow the polydispersity while the
titrations provide a lower limit to the chain length and this data is
consistent with a polymer of approximately 25 kDa.

I realize this is not the easiest way to approach this value but do not have
access to the instrumentation required to determine it.  It seems reasonable
in that my ultimate goal is to use this estimated vbar value to calculate a
weight average vbar for a protein-polymer conjugate that I am making.  The
protein vbar can of course be calculated.  I would ultimately like to
demonstrate that the actual MW of the conjugate as well as stoichiometry is
correct, and determine if the conjugate behaves as a monomer in solution.
 Initial analysis of the conjugate using the weight average vbar seem to
confirm the monomeric nature as well as support a MW addition of 25,000 more
or less.  The protein vbar is calculated to be 0.7475 and fits to the
conjuate are relatively poor at this value but improve when it is increased
to 0.78 which is the weight avg vbar.

You would be helping me quite a lot if you could provide either a criticism
of this approach (there are likely many) and a suggestion for a means to
either validate this or approach it in a different way.  Analysis of the
conjugate is somewhat complicated by the presence of free polymer and thus
two species with quite different frictional ratios so the best fits are
obtained using the c(S)ffo  model in sedfit, the fit range ffo values for
each peak in the c(s) distribution correlate with the anticipated component
represented in each peak, i.e. free polymer S experimental at 1.3 has ffo of
>2 whereas the anticipated conjugate experimental S value of 2.6 has ffo
values spanning 1.6-1.8.  The corresponding MW plots show anticipated values
of approx 28 kDa for polymer and 60-80 Kda for the presumed conjugate.
 Analysis of the free protein yields excellent fits with a single peak and
an ffo values of 1.4-1.6 and a MW of 43000 (actual 41406)

Sorry for such a long question for my first posting to this board. I hope to
have provided enough information so that a useful discussion can arrise.  I
thought I understood that I could use the buoyant MW relationship to
estimate vbar but seem not to understand how that would work in this
situation... Thanks a lot

Matthew
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20080606/1a91f1fb/attachment.htm>