From: Joachim Behlke <behlke@mdc-berlin.de>
  To  : Borries Demeler <demeler@bioc09.v19.uthscsa.edu>
  Date: Thu, 27 Apr 2000 20:03:56 +0200

Re: variation of local lnks

Lieber Herr Demeler,
Ihr Problem scheint ja schon fast geloest zu sein. Ich wollte Ihnen nur mitteilen,
dass ich vor 30 Jahren schon einmal ein fast aehnliches Problem mit dem
Lamprey-MetHb hatte, das unligandiert monomer und ligandiert (z. B. mit Azid)
dimerisieren kann. Das ligandierte MetHb bildete also ein Mo-Di-Gleichgewicht, das
aber durch das unligandierte MetHb gestoert wird. Damals hatte ich die Chance
genutzt auf spektroskopischem Wege den Grad der Ligandierung zu testen und den
Konzentration-
anteil unligandierten Proteins abgezogen. So konnte ich eine echte
Gleichgewichtskonstante bestimmen (publiziert 1970 in Eur. J. Biochem.)
Austen Riggs kannte sogar das Zitat als wir uns in Galveston trafen.
Herzliche Gruesse.
Joachim Behlke

Borries Demeler schrieb:

> >
> > If you are sure the monomer-dimer is the correct model for your system I
> > have a way to estimate the correct K2 from samples contaminated by either
> > incompetent monomer or incompetent dimer, not by direct fitting though.
> > Rather, the method requires NONLIN fitting of a series of data sets with
> > different loading concentration and then, the K2 can be calculated using the
> > estimated NONLIN parameters - I call it the secondary calculation. If you
> > are interested, I can provide you more info on that.
> > John is right, simply including more fitting parameters can always improve
> > the RMS of a fit, but it does not guarantee the fitting is correct. To be
> > sure that incompetent species is what causes the problem, one should follow
> > Emory's suggestion - to observe the trend of variations of estimated lnK
> > with loading concentration and/or speed. If it was incompetent large
> > species, you can probably get rid of it at a higher speed. To deal with
> > incompetent monomer is more challenging.
> >
> > Regards
> > Yujia
> >
>
> Yes, I would be interested, I have sent a copy of a plot to Tom Laue
> yesterday that shows the K2's plotted against loading concentration and
> against speed, and these plots seem to indicate that I do infact have the
> presence of incompetent monomers, which is what I suspected all along,
> and it matches well with the biochemical hypothesis (how often can you say
> that!). For those that are interested, I put the plot on the web at:
>
> http://demeler.uthscsa.edu/data/k2.gif
>
> It shows a clear decrease of k2 with increasing loading concentration,
> but an uncorrelated effect with rotor speed. Tom indicated that this
> means "incompetent monomer".
>
> If Yujia or someone else has some software to do some secondary
> analysis on this data, please let me know. If there is a way to
> extract the k2 for the competent part I would be happy.
>
> Regards, -Borries