[RASMB] minimum and maximum

[Peter Schuck]

Hi Chin and Joris,

my experience with large and small particles is the following:

1) as for the maximum, there is in principle no upper limit in 
c(s).  However, there is a limitation due to the experimental data that are 
loaded in sedfit.  For example, if you want to observe a speices with an 
s-value so large that for the first scan it would already be at the bottom 
of the cell, or outside your fitting range, then it doesn't make sense and 
sedfit will prevent you from using such an s-max.  An error message should 
appear with the maximal s-value that you can possibly observe for the given 
data.  But if you run slow enough, that's not limiting.  I've looked at 
size-distributions of bacteria with 10,000  - 100,000 S.  For very large 
particles, you may want to consider switching to the ls-g*(s), since 
diffusion can likely be neglected.

2) for the smallest species, we can routinely see the sedimentation of 
buffer salts (and measure their s-value...), and to characterize things in 
the couple hundred Dalton range is absolutely feasible.  I would definitely 
recommend to go to 60,000 rpm and a long (maybe 10 mm) solution 
column.  The question of equilibrium versus velocity is simple to answer, 
since they will be essentially the same - except that if you regard it as a 
velocity you would acquire all the data towards the approach of 
equilibrium, which is extremely valuable information.  Of course, it will 
be best to also let the solution actually come to equilibrium, which 
shouldn't take too long.  Including the approach to equilibrium in the 
analysis is helpful, in particular, for resolving different species, which 
may be difficult to unravel from the equilibrium alone.   You can find some 
examples for the shapes of sedimentation profiles in Biophys J (1998) 
74:466, but the c(s) can work well in that range, too.  Examples for 
approach to equilibrium analyses with c(s) and c(M) can be found in 
Biophys. J. (2001) 81:371

The problem with velocity that Borries referred to is probably the 
expectation to pull a traditional boundary, clear the meniscus and get 
plateaus, which is required, for example, for an analysis with the van 
Holde-Weischet method.  That would not work.  But if you regard a velocity 
run as simply observing the time-course of sedimentation, it should work 
perfectly fine if you use Lamm equation solutions in the analysis and you 
can get perfectly good s and D values.  If by any chance your two molecules 
have significantly different absorption spectra, I would try to take 
advantage of that in multi-signal detection.  For interacting systems with 
known reaction scheme, the Lamm equation solutions incorporating reaction 
terms should be applicable.

One issue that in my experience makes more difficulties with very small 
species is the v-bar problem, maybe due to the larger surface-to-volume 
ratio and relative contributions from solvation.  But if the problem is 
such that the 500 Da and the 1500 Da can be characterized separately, 
first, then you could take the buoyant molar mass of the individual species 
(together with the known mass from mass spec) to calculate an effective 
v-bar which is correct for the experimental conditions.

Best,
Peter


At 11:22 AM 1/26/2005 +0100, you wrote:
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>Dear all,
>
>Some time ago I asked around for the highest measurable molecular weight 
>by analytical ultracentrifugation. This time a colleague asked me whether 
>it is possible to measure host-guest complexes by analytical 
>ultracentrifugation in which the host is around 1500 Da and the guest 
>around 500 Da. I checked literature on this more supramolecular chemistry 
>related research (in contrast to the more biochemical research normally 
>done by AU) and so far without any luck. Could someone help me with a 
>lead? To me it seems to be possible, although one needs to spin the sample 
>pretty fast. Apart from this question: what would be the experiment of 
>choice? Sedimentation velocity or sedimentation equilibrium?
>Thanks!
>Kind regards,
>
>Joris Beld
>Swiss Federal Institute of Technology
>Hilvert Group
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