[RASMB] estimation of Vbar from sedimentation velocity data

[Walter Stafford]

Matthew,

    You might try an equilibrium run in both H2O and D2O to get v-bar. 
You end up with 2 equations and 2 unknowns and you can solve for both an 
average molar mass (close to weight average if you don't run at too high 
a speed) and vBar. No assumptions required except maybe an estimated 
value of H/D exchange.

See: 

*1: *Methods Enzymol. <javascript:AL_get(this, 'jour', 'Methods 
Enzymol.');> 1973;27:82-98. Links <javascript:PopUpMenu2_Set(Menu4797942);>


        Measurement of partial specific volume by sedimentation
        equilibrium in H2O-D2O solutions.

    *Edelstein SJ*
    <http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Edelstein%20SJ%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus>,
    *Schachman HK*
    <http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Schachman%20HK%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus>.



Walter

-- 
Walter F. Stafford, Ph.D.
Senior Scientist
Analytical Ultracentrifugation Research Laboratory
Boston Biomedical Research Institute
64 Grove Street
Watertown, MA 02472-2829
-------------------------
mailto:stafford at bbri.org
direct dial:   617-658-7808
receptionist:  617-658-7700
-----
Boston Biomedical Research Institute... 
Today's Research for Tomorrow's Health.	
      Please visit us at http://www.bbri.org/



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
>