[RASMB] estimation of Vbar from sedimentation velocity data

[Ewa Folta-Stogniew]

Matthew,

if you have a good SEC conditions on Superdex 200, have you considered 
sending the samples for analysis for the SEC-LS service?

http://info.med.yale.edu/wmkeck/biophysics/Lsmemoa.htm

 From your description the system seems do be very compatible as long as 
you have at least 100 ug of the material.

Ewa

At 04:28 PM 6/6/2008, Matthew Hockin wrote:
>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
>_______________________________________________
>RASMB mailing list
>RASMB at rasmb.bbri.org
>http://rasmb.bbri.org/mailman/listinfo/rasmb