[RASMB] fringe deviation

[Arthur Rowe]

Hi Tom -

Obviously we shared the same thought about 'schlieren effects' within a few
seconds of each other!

Your points are well made. Just one little thing, though, which prompts an
extra comment from me. I cannot see that you get a 'better fix' on
non-ideality in SE than you do in SV. In both cases there are ball-park
values you can use for globular systems (e.g.  ks ~ 5-6; 2BM ~ 8-10). But
for SV you can compute ks from molecular parameters (frictional ratio etc),
which you certainly cannot do for 2BM.  The latter is also more prone to
charge effects. 

I have used SV  to probe or get Kd values and published results over the
years. But right now, with software on my desktop which happily gives me
estimates for both BM and Ka from SE, I think it is a complex issue as to
which approach is best.

Regards

Arthur


Dear Barbara,
I concur with the replies of others, but wish to add a couple of quick
comments. As John Philo mentions, you working at concentrations where
both thermodynamic and hydrodynamic nonideality are significant. The two
forms of nonideality are different and have different (often opposite)
effects on the raw data, making them difficult to separate. If you stick
with equilibrium analysis, you will eliminate hydrodynamic nonideality,
and can get a better fix on the effects of thermodynamic nonideality.
Regarding the optical systems, it is important to note that it is the
*gradient* in the concentration that results in Weiner skewing, not the
concentration per se. If you can limit the steepness of your gradients
(or trim away the data from steep gradients), you will limit these effects.
Be aware that the absorbance optics are subject to the effects of
refractive gradients, too. If you have a steep concentration
(refractive) gradient, light can be deviated sufficiently that it will
not be detected by the absorbance detector. No adjustment of the
wavelength will overcome this problem. In fact, the tell-tale test for
this problem is to scan your sample at a wavelength where no absorbance
occurs- you will see a positive going peak in the data (the derivative
of the concentration profile, it turns out) where the gradient is steep.
You can help minimize the problems by using shorter pathlength cells and
reducing concentration gradients.
Best wishes,
Tom

lelj wrote:

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>
>Hello!
>
>I am trying to determine the association constant for a monomer-dimer
equilibrium. I know the association occurs because I've seen it by velocity
experiments.
>
>I run equilibrium at 3 concentrations and 3 speeds, my conc. are high and
therefore I used interference optics. I have a fringe displacement at
equilibrium of up to 50 fringes. I fit my data with WinNonlin and at the
best I can get a SQUARE ROOT OF VARIANCE=9.3317E-02.  Since I have such a
big fringe displacement when I plot deviation vs. indipendent variable, in
the worst case I have a deviation of -0.25 to 0.375 fringes, is it
reasonable?
>
>thanks 
>barbara
>
>---
>Barbara Lelj Garolla Di Bard
>Dr. Mauk's Lab
>Dept. of Biochemistry and Molecular Biology
>University of British Columbia
>2146 Health Sciences Mall
>Vancouver, B.C. V6T 1Z3
>Phone: (604) 822-2526
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-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
www.bitc.unh.edu
www.camis.unh.edu


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