From: UMJ <xuyu@UMDNJ.EDU>
  To  : Borries Demeler <demeler@bioc09.v19.uthscsa.edu>
  Date: Thu, 27 Apr 2000 10:35:48 -0400

Re: variation of local lnks

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

----- Original Message -----
From: Borries Demeler <demeler@bioc09.v19.uthscsa.edu>
To: <rasmb@alpha.bbri.org>
Sent: Wednesday, April 26, 2000 1:39 PM
Subject: Re: variation of local lnks


> More to the point:
>
> The protein I am investigating is heme containing, although a small
> population lost the heme group. When I track the association at around
> 400nm (where heme absorbs) I get a perfect dimer-tetramer-octamer system,
> when looking at it at 280, 230 and 207 where also the non-heme containing
> subpopulation absorbs, I see this presence of incompetent material.
> Clearly, Nonlin is not capable of extracting this information, so I need
> to know what models people have used to describe such (apparently
frequent)
> phenomena. In my case it looks like the heme-containing subpopulation
> doesn't produce incompetent species, only the non-heme containing stuff.
> This in itself is very interesting biologically in my case.
> However I would like to quantify this effect for the monomer-dimer
> assoc. constant. However, only at 207 is the conc. low enough for me to
> see monomer, but both populations absorb so I can't separate them.
> The proper model/software should be able to extract the k2 for the
> "competent" species. Has anybody written something like this?
>
> First the question of which assoc. state is incompetent? Does anybody
> have experience in telling which one is (OK, I can simulate, but I'd
> rather not)? PAGE is not conclusive.  Next, on first glance it seems
> that it should be possible to formulate a global model. Any suggestions?
>
> Thanks so much for any pointers, -Borries
>
> >
> > Borries and others; We have frequently run into this situation.  Do you
> > notice that there is a trend in the values of lnK, in that they increase
> > in a positive direction as loading concentration decreases? If so, that
> > is probably due to the presence of incompetent species, not only
monomer.
> > Irreversible species can be any size--but usually we have found that
> > they are at the high end.  Of course it could also be an impurity with a
> > MW not related to any of the species that you are trying to fit.
> > The best fitting procedure is to solve your
> > model so that you are fitting globally all but the last step of the
> > association sequence.  Then let that step be fit globally followed by
> > letting that step be fit locally.  Then compare rms and systematic
errors.
> > There are attempts being made to get info re: the species that is
incompetent
> > and what fractional concentration is present.  Yujia Xu, Dave's last
student
> > has a dissertation devoted to this which I hope will be published soon.
> > You could play around with it in a similar way by simulating various
mixtures
> > of incompetent species, and seeing what Nonlin will do. Hope this helps.
> >                                                         Emory
> >