[RASMB] MW calculaiton question

[John Sumida]

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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