[RASMB] upper concentration limit AUC

[Arthur Rowe]

Hi Christine (and everyone)

I think this discussion is already in the RASMB Archives.

To summarise very briefly, k(D) is indeed lower than k(s), but not hugely
so. For simple, spherical particles, defining all coefficients in volume
fraction terms and at limiting infinite dilution, we can write

k(D) = k(P) - k(s) where k(P), the concentration dependence of the chemical
potential which is the force driving the flux in translational diffusion,
and is given for s single component by (2BM + higher terms) where B is the
2nd virial coefficient. For spheres, 2BM = 8 ml/g; k(s) = 4* ml/g:  so k(D)
= 4 ml/g. I am afraid that neglecting k(D) is not a good approximation.

Kind regards - will be seeing you (Christine) and lots of others in
Newcastle this week.

Arthur

*see "The sedimentation rate of disordered suspensions"  Brady, John F.;
Durlofsky, Louis J.
Physics of Fluids 1988 31 717-727 for the last word from the fluid mechanics
people. You can also look up my Chapter in the 1992 Book (the 'black book')
ed s Harding, Rowe & Horton) for details of my own derivation. Which happens
to be in total numerical agreement with the Brady/Durlovsky treatment, right
p to 64% volume fraction . . . . .
Note: the value given above for k(s) = 4 ml/g is for DYNAMIC k(s). The usual
measured value which gets reported is the KINEMATIC k(s), which for spheres
has the value 5.0 ml/g. You can get either value out of theory, depending
how you play it. Brady & Durlovsky report the kinematic value. All of which
was first noted by Burgers in 1939/40, and has been much ignored ever since!
As no density 'correction' is called for in the estimation of k(D) or of
k(P), it seems reasonable to use the dynamic k(s) in the equation above.


Dear Joris, dear all,

This reference may be perhaps useful.

 

Solovyova A., Schuck P., Costenaro L., Ebel C,

Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in
complex solvents, 

(2001) Biophys. J, 81 1868-1880.

 

In this work, we analyzed sedimentation velocity profiles considering
hydrodynamic and thermodynamic non ideality. (i.e. concentration dependency
of s and D) in the case of an homogeneous  solution of our protein of
interest. The modified Lamm equation was implemented in a model of analysis
in Sedfit (note that, unless the programme was recently modified, the kS and
kD are expressed in signal unit in sedfit)

From my experience, the sedimentation velocity profiles are essentially
modified by the concentration dependency of the sedimentation coefficient.
Thus the concentration dependency of D can be neglected in a first
approximation. Also from a theoretical point of view, kD is much lower that
ks. 

All the best 

Christine 

 
Christine EBEL 

Institut de Biologie Structurale CEA-CNRS-UJF

41 rue Jules Horowitz, F-38027 Grenoble France

Tel (33) (0) 4 38 78 95 70; Fax (33) (0) 4 38 78 54 94

christine.ebel at ibs.fr

http://www.ibs.fr/content/ibs_eng/presentation/lab/lbm/ebel.htm
-----Message d'origine-----
De : rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] De
la part de Beld, Joris
Envoyé : lundi 1 septembre 2008 15:24
À : RASMB at rasmb.bbri.org
Objet : [RASMB] upper concentration limit AUC

 

Dear all, 

 

A colleague asked me whether analytical ultracentrifugation has an upper
limit with regard to the concentration of the protein. They want to measure
the protein at the same concentration as the NMR experiments (> 1 mM). I am
not entirely sure but I thought this should be no problem. One could easily
measure off-peak at another wavelength than 230nm, e.g. 235nm or 280nm,
right?! Or does one run into non-ideality phenomena when doing sedimentation
equilibrium at these high protein concentrations?!

Thanks a lot in advance for any feedback.

Best wishes, 

 

Joris Beld 

 

Hilvert Group 

ETH Zürich 

Switzerland 

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