From: Cole, James L <jim_cole@merck.com>
  To  : 'Borries Demeler' <demeler@bioc09.v19.uthscsa.edu>
  Date: Thu, 27 Apr 2000 17:22:14 -0400

RE: variation of local lnks

Borries
It does seem reasonable that your system contains incompetent monomer due to
loss of heme.  In this case, the apparent  association constant  ought to
decrease  with increasing protein concentration, since some of  the monomer
is not equilibrating with the dimer, as you showed in the graph.  However, I
expect that the apparent association constants  would also go down as you
increase the rotor speed, since the effect of increasing rotor speed is to
increase the concentration of the sample towards the bottom of the cell.
Tom Laue  doesn't expect to see a systematic effect of speed on lnK. Why ?
In any case, rather than trying to fit  a heterogeneous system  to account
for the presence of a non-equilibrating monomer,  I would put the problem
back in the hands of the biochemists to  try to make a  more  homogeneous
sample. Do you know how much apo-enzyme is present ?   If the heme can come
out, maybe it can be put back in. Reversible extraction of  porphyrins is
feasible in a number of proteins.

Jim
---------------------------------------
>  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".
>