Receptor ligand association and dissociation kinetics

Matthew Kostura mkostura at worldnet.att.net
Mon Mar 30 02:25:54 EST 1998


I have  a problem with a receptor binding assay I am setting up.  I am
trying to establish the first order dissociation constant for the receptor
and its ligand.  I am following established procedures by bringing the
labeled ligand (which is below Kd) and receptor to binding equilibrium then
diluting the reaction in the presence of an excess (1000X ) of cold
unlabeled ligand.   I then measure bound ligand by filtration.  I expected
to see a  first order rate of decay as determined by fitting the curve to y
= ae^-bx.  In addition I plotted the data as the LN(bound at time t /bound
at equilibrium) vs time.  I had hoped to see a straight line.  In neither
case did the expected fits occur.    The fit to the first order exponential
decay equation was poor and the transformed data plotted as a curved line
not unlike what one might get with a second order decay.  According to a
commercial curve fitting routine (I am using Jandel Tablecurve) the best fit
consistently comes out with the equation  y = a + b*e^-cx, a kind of
modified single exponential decay.   My suspicion is that I am observing the
displacement of the bound ligand from its binding site with its cold form.
In such an event I  would think my own data would plot out as a second order
decay.    Does anyone have any experience with allosteric models of ligand
displacement??  If so what are the accepted mathematical models and
equations for such reactions.  What are some of the accepted methodologies
for figuring this out??









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