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The following message was posted to: PharmPK
I have designed a mechanism based assay and have confidence in the data
obtained. The residual activity after incubation in the presence of
the inhibitor is calculated referring to the activity measured in the
presence of the solvent used to dissolve the inhibitor. Then the
natural log of the residual activity is calculated and plotted versus
the concentration of the inhibitor.
The 1/slope (from the linear plot) is plotted versus the
1/concentration of the inhibitor and the Kitz-Wilson plot is used to
estimate the Kinact and KI.
When interpreting the results I have noticed that a very slight
difference in the residual activity in the presence of no inhibitor can
give a significant difference in the results obtained. Has anyone else
noticed this or have I made a flaw in my calculations?
Regards Dave
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The following message was posted to: PharmPK
Dave
I have seen similar results and prefer a different method for
calculating Ki and Kinact because of this and potential saturation of
the inhibitor. The two methods are described below.
Method 1: e.g. Palamanda et al (2001) DMD 29(6) pg 863
Step 1. Plot Log of remaining activity vs. time and determine the slope
for all inhibitor concentration levels
Step 2. Plot 1/-slope vs. 1/inhibitor concentration. Y intercept
=1/kinact and X intercept = -1/Ki (Kitz-Wilson plot using half-life
can also be used - gives same answer)
Method 2: e.g. Heydariet al (2004) DMD 32(11) pg 1213
Step 1. Plot Log of remaining activity vs. time and determine the slope
for all inhibitor concentration levels
Step 2. -slope vs. inhibitor concentration. Curve fit using standard V
vs S M-M kinetics --- Km=Ki and Vmax=kinact
The calculated values are almost identical in most cases. As stated
previously, I have a preference for method 2, because it allows for
saturation of the inhibitor where Method 1 loses linearity if the
inhibitor is saturated. Method two also accommodates this "background"
inactivation that you were describing. Method 1 does not allow for
consideration of "background" inactivation because the zero inhibitor
concentration can not be plotted for the Kitz-Wilson plot because you
have to divide by inhibitor concentration = zero.
Hope this Helps
Mike Cameron
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)