[RASMB] fringe deviation

Allen Minton minton at helix.nih.gov
Tue Feb 17 07:26:00 PST 2004


Bo's reply regarding Wiener skewing is on the mark for interference 
optics.  However, Wiener skewing doesn't have a significant effect on 
absorbance gradients [Gonzalez et al, Analytical Biochem 313: 133-136 
(2003)].  I suggest that Barbara redo the equilibrium experiments with 
absorption optics, using a wavelength where the solute has a smaller 
extinction coefficient, so that she can go to higher concentrations without 
maxing out the absorbance detector.  Of course a model incorporating 
nonideality is necessary if you go to protein concentrations exceeding ca 
10 mg/ml, but I have to warn you that the nonideality contribution 
implemented in NONLIN is incorrect and may lead in certain cases to 
incorrect values of association constants.  There are correct treatments 
out there if anyone is interested.

Allen Minton


At 07:21 PM 2/16/2004 -0600, Borries Demeler 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
>
>Hi Barbara!
>
>Whenever you get very big interference gradients (50 fringes over a 2-3
>mm equilibrium column is a lot!) you need to be aware of systematic
>errors introduced into the equilibrium experiment. In my experience
>there are two sources: 1. Wiener skewing and aperture related problems,
>which can be significant with such large concentration gradients, and
>as John Philo mentioned, concentration dependent nonideality. There was
>a good explanation about Wiener skewing posted to this forum by Arthur
>Rowe, you can find it at:
>
>http://server1.bbri.org/rasmb_archives/1997/113.html
>
>If you have a good idea of your Kd, my recommendation would be to measure
>1 order of magnitude above and below of this concentration, so you get a good
>signal for both the monomer and the dimer.
>
>Good luck, -Borries
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