[RASMB] MW calculaiton question

Tue Aug 13 12:04:49 PDT 2013

if 80% of mass was your major peak- what was the remaining material 
based on AUC and SEC/MALLS analyses?

At such heterogeneity, I am wondering how the DLS results look 
like?  What was Dt from DLS?

How polydisperse was the major peak from SEC/MALLS?   How well 
fractionated  was the sample during SEC/MALLS analysis?

I am a little surprised that the frictional ratio came to 1.47 or 
1.54- I would have expected the micelle to be a perfect sphere thus 
with much smaller frictional ratio.

At 08:57 PM 8/12/2013, John Sumida wrote:
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>Dear RASMB,
>
>The following was posted on the SEDFIT user list and it has been 
>suggested that I also post this message to the RASMB list.  Thus I 
>am reposting my question to RASMB and am including some edits and 
>additional information for clarity and correctness.
>
>Objective
>I am trying to reconcile results obtained using two different 
>approaches, SEDANAL and SEDFIT.
>
>Observations
>In SEDFIT, analysis of sed velocity FDS data returns an s20w of 8.5s 
>and MW=1.3 MDa for the major peak, comprising >80% of the total 
>loading concentration, determined in a c(s) fit.  Please note after 
>checking with our collaborators this value is consistent with the MW 
>returned from DLS and static light scattering experiments.  Thus the 
>SEDFIT result is consistent with the DLS and static light scattering 
>data which estimate a MW of 1.1 MDa.
>
>In SEDANAL, the simplest model necessary to fit the data well was a 
>single species model.  Using this model, analysis of the same 
>data-set returns an s20w of 8.8s, similar to the value calculated in 
>SEDFIT, but a MW of 655 kDa is calculated.
>
>My question:
>Why does the value of the MW returned for SEDANAL and SEDFIT differ 
>by a factor of ~2 when the s20w in each case are similar (8.5s 
>versus 8.8s).  My purpose here is that I believe the apparent 
>difference being returned from these parallel analyses is saying 
>something fundamentally important about the behavior of these 
>materials in buffer and our assumptions thereof.
>
>Background.
>1.     Rotor speed was 30 krpm, vbar=0.917 (measured), solution 
>density = 1.00506 g/cm3 (measured), and viscosity =0.0100281 Poise 
>(measured).  Temperature = 20oC.
>2.     The material being studies is a polymer micelle with a CMC of 
>14 micrograms/ml.
>3.     The polymer was run over a series of concentrations ranging 
>from 0.2 mgs/ml to 1.00 mgs/ml.
>a.     Thus under the conditions of the experiment I am at least 14 
>times the CMC at the lowest concentration.
>4.     A major peak is observed in the initial c(s) distribution 
>comprising >80% of the total loading and the position of this peak 
>(7.7 s-exp; s20w=8.5) does not shift with concentration.
>5.     From the analysis of raw SV data, SEDANAL returns an 
>experimental sedimentation coefficient of 7.9s and a calculated s20w of 8.8s.
>6.     A global analysis in SEDPHAT was performed over the entire 
>concentration range, transforming the initial c(s) distribution into 
>a set of 8 discrete species.
>a.     Four of these 8 species survived a critical chi square 
>analysis suggesting that these four species were important in 
>retaining the quality of the fit.
>b.     Of these four discrete species, three were grouped beneath 
>the major peak observed in the initial c(s) analysis.
>c.      The calculated weight averaged s20w for these three species was 8.1s.
>d.     The  s20w values were checked and confirmed with a manual 
>calculation, SEDNTERP verstion 1.09, as well as the calculate 
>"s(20,w) from s(xp) in SEDFIT.
>7.     Using the ratio of s/D in the Svedberg equation and values 
>for diffusion estimated by assuming 655 kDA, (the MW returned in 
>SEDANAL), I calculate a MW of 655 kDa.  Thus I believe that the 
>value for diffusion being estimated in SEDANAL is consistent with 
>the 655 kDA molecular weight.
>a.     Estimates of the diffusion coefficient in SEDFIT and SEDNTERP 
>(and thus presumable also from SEDANAL) are not very different.
>b.     The diffusion coefficient estimate in SEDFIT is 3.19E-7 
>cm2/sec and the value calculated in SEDNTERP 3.67E-7 cm2/sec (Teller 
>approximation).
>8.     Using the calculate M(s) function in SEDFIT and providing the 
>experimental sedimentation coefficient noted above I need to input 
>an ff0<1 (0.8967) in order to arrive at the 655kDA MW returned by SEDANAL.
>a.     I understand that this is nonsensical as ff0 cannot be less than 1.
>b.     Notably SEDNTERP version 1.09 also calculates a frictional 
>ratio<1; namely SEDNTERP calculate ffp=0.9449.
>c.      The c(s) analysis in SEDFIT returns an ff0=1.47.
>
>Thus to summarize:
>1.     Relatively similar s20w values are determined in both SEDFIT 
>and SEDANAL for the same data-set.
>a.     8.5s from SEDFIT
>b.     8.8s from SEDANAL
>2.     SEDFIT calculates a MW almost exactly 2 times the value of 
>the MW returned by SEDANAL for the same s20w.
>3.     The estimates of diffusion in both programs are quite similar
>4.     The frictional ratio ff0 in the SEDFIT analysis is 1.47 
>whereas the calculated ffp in SEDNTERP based on the observe MW in 
>SEDANAL is <1.
>5.     The frictional ratio calculated in SEDNTERP, assuming the 
>MW=1.3 MDa, is 1.54
>6.     There does appear to be heterogeneity in the major peak 
>observed in the c(s) analysis.
>
>Thank you in advance for your comments and suggestions.  I apologize 
>for the length of this post, but in fairness, I am attempting to 
>provide information that would enable an informed response.
>
>Best regards
>John Sumida
>University of Washington
>Analytical Biopharmacy Core
>
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