[FDS] Changes in emmission intensity

[Borries Demeler]

> 
> Bo and others, 
> How well will AUC analytical softwares deal with an experiment where a 
> fluorescent substrate is bound by a ligand, which changes the yield of the 
> fluor?  If you titrate the ligand and the  yield decreases systematically, 
> will a valid curve of fraction bound versus ligand concentration still be 
> meaningful?

Steve,

this is one of the issues that is not at all addressed so far in any
software that I am aware of. Right now, I guess you could measure
the substrate by itself - once you add the ligand, you will have a 
decrease of emission due to dilution, and an unknown change of emission
due to binding (if in fact the binding affects flo yield). If there is
a way to measure both independently, you could probably calculate some
sort of emission coefficient and plug it into the software. In velocity
experiments you would see a hydrodynamic change allowing you to tell
the bound substrate apart from the unbound (if you are lucky, two
boundaries), but the relative concentration of each is probably not
obvious at all if you have different quenching. For equilibrium, all bets
are off. But if you knew what the molar emission coefficient is, you could enter
that in place of the extinction coefficient (at least in UltraScan).

This brings up all kinds of tricky questions with FDS that are not so
obvious to address, whenever absolute knowledge of C is required.

-Borries