From: Tom Laue <Tom.Laue@unh.edu>
  To  : Borries Demeler <demeler@bioc09.v19.uthscsa.edu>
  Date: Wed, 26 Apr 2000 15:15:42 -0400

Re: variation of local lnks

Hi Bo-
It's true that lots of people have seen it, but there are few
characterizations available for such a mixture. You stand little chance of
curve-fitting your way out of a problem that involves the sums of
exponentials, even if you have some control (i.e. rotor speed) over those
exponentials. The data from sedimentation equilibrium is ill-conditioned
with respect to deconvolution, and there is no way around it. 

If you have an incompetent species, then the sample should be fractionated
to get rid of it. In doing the fractionation, you answer two questions- who
is it that is incompetent and how much of it is present. The way to
demonstrate who is incompetent is to fractionate (say by gel filtration)
and re-run (either by gel filtration or sedimentation) the fractions at the
same loading concentrations to see which can regenerate the reversible
system. When you do the fractionation, you will learn how much of the
material was not competent.

Here is the bottom line: material trapped in an irreversible state should
be removed before analyzing the reversible states. Fractionation should be
done on the basis of the invariant characteristics of the irreversible
material (i.e. it is bigger, smaller, different charge). 
Best wishes,
Tom
At 12:39 PM 4/26/2000 -0500, you wrote:
>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
>> 
>
--------------------------------------------
Tom Laue
Professor and Director of CAMIS
University of New Hampshire
Biochemistry and Molecular Biology
Rudman-379
46 College Rd.
Durham, NH 03824-3544

Phone: 603-862-2459
FAX:   603-862-4013
---------------------------------------------