[RASMB] upper concentration limit AUC

[Tom Laue]

Hi all-
For equilibrium work, the upper limit is set by the optical focus and 
the concentration (refractive index) gradients. The absorbance system 
_cannot_ handle nearly as steep a concentration gradient as the 
interference optics can for the reasons pointed out by Arthur. So you 
should use interference optics.
If you use shorter pathlength cells, you must refocus the optics to 
avoid the problems Arthur pointed out. The hassle of refocusing prevents 
most people from attempting it.It can be done, though.
The bigger problem is not optics, focus or wavelength, but proper data 
interpretation. Nonideality is not dependent on the optical system, but 
is a property of the chemical system. Current data analysis programs use 
a single nonideality coefficient to describe the interactions that lead 
to nonideal solution behavior. At the concentrations where you wish to 
work, it is not likely that any of the programs will fit the data 
properly. You could use scaled particle theory to fit a single parameter 
(the effective radius) to a power series. It is not clear how to 
interpret the coefficients of the power series.
Alternatively, you could fit for the average apparent molecular weight 
as a function of cell loading concentration and determine the activity 
coefficient at several protein loading concentrations. Activity 
coefficients greater than one indicate repulsive non-ideality. Activity 
coefficients less than one indicate associative nonidealitiy. You get 
one determination per sample (unless you use a program like Biospin or 
MStar to determine the point-average apparent molecular weights).
Determining the activity coefficient is very, very quick (especially the 
data analysis) and thermodynamically rigorous. The rub is in the 
interpretation of the data. Associative behavior (e.g. under NMR 
conditions) may or may not be because of protein-protein interaction 
(e.g. dimer formation). Reduced activity coefficients may result from 
close-range favorable solute-solute weak-forces (e.g. dipole-dipole 
interactions, solute-solute solvation) or may result from less-favorable 
solute-solvent (or solvent-solvent) interactions at high concentrations.
The problem is not due to limitations of AUC, but to the model 
("cartoon") biochemists and molecular biologists use when thinking about 
high concentration solutions. A different method will provide the same 
sort of interpretative ambiguity. It may be worthwhile to interpret 
high-concentration data using a Van der Waals model that contains a 
single attractive and single repulsive coefficient.
Best wishes,
Tom




Beld, Joris wrote:
>
> 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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