These are the per minute fluorescence observed. Please guide me how can I determine Vi, Vo and IC50 ? By slope determination or any other method?
Control: 28.11 40.30 40.78 37.61 45.34 42.56 44.76 44.51 44.57
Inhibitor: 12.81 12.78 12.84 10.83 10.64 11.24 11.42 11.67 11.31
Vo is the initial rate of fluorescence increase when there is no inhibitor added. If the inhibitor is dissolved in a solvent other than the assay buffer, the same amount of solvent should be added to this control. We will call this rate MAX.
Vi is the initial rate of fluorescence increase in the presence of the inhibitor at a given concentration. We will call this rate X.
You should also have a negative control rate, in which there is no activity of the enzyme, either because the enzyme was omitted or because the enzyme was fully inhibited by a potent inhibitor. We will call this rate MIN.
For each inhibitor concentration, calculate % inhibition as follows:
% inhibition = 100 x [(X - MIN)/(MAX - MIN)].
Calculate the IC50 by nonlinear regression using the full set of % inhibition measurements versus inhibitor concentration [I]. The equation for this appears in many forms. My preference is this form of the Hill equation:
% inhibition = 100 [I]n/(IC50n + [I]n).
n is the Hill coefficient. It is usually close to 1, but not always.
The value 100 assumes that the enzyme can be fully inhibited by the inhibitor. If this is not the case, you can replace this number with a variable parameter that can be part of the nonlinear regression.
Hi there,
Can you be more accurate about the way your data are expressed? Are they instant velocities or fluorescence intensities measured at different times. If the former, velocity in the control is stable from 1 minute on and is around 40-45/min whereas in the inhibition assay velocity is around 10-12/min which means the inhibition is around 75%, provided the reaction mix are strictly identical in terms of contents (except for the inhibitor). It would be easier to work with purely kinetics data: fluorescence intensity versus time and estimate the slope during the linear phase.
Dear Dominique these values are the measurement of fluorescence intensities at different time intervals for about 30 minutes.
Dominique Liger
So you should repeat the experiments on a much shorter time scale as from the second point of your control nothing much happened (reaction is finished) and nothing seems to happen in the second experiment... As well as running negative control experiment (without enzyme) to determine fluorescence background.
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