I have come accross this tricky question and could not find a suitable explanation anywhere. Since both abovementioned types of inhibition show the same pattern, how can I make sure which one I am dealing with? Does anyone know how to measure the ''lost'' enzymes in irreversible inhibition?
Washing? Precipitate the enzyme with ammonium sulfate or concentrate on centricon or similar, resuspend in fresh buffer (if you used ammonium sulfate you might need to dialize) and check activity. If it came back - noncompetitive. If not - either irreversible or your process 'killed' the enzyme.
Yoram
In addition to washing you may also investigate time-dependence of the inhibition process. Irreversible inhibitors often act as time-dependent inhibitors, while reversible inhibitors do not show time-dependence.
Alexei
You also can do Enzyme vs Activity curve, increasing the concentration of enzyme with the same concentration of irreversible inhibitor wil start at the point where no enzyme is bound to inhibitor, while the noncompetitive inhibitor the curve will start at zero but the slope is lower. Look for it in Irwin Segel "Enzyme Kinetics"
Yes, what Ana has explained is the method to distinguish between noncompetitive & Irreversible inhibitions. See "Biochemical Calculations" by Irwin H. Segel.
To make it more clear, determine the maximal velocity (Vmax) using different enzyme concentrations in different experiments. Plot a graph between Vmax and total enzyme concentration [Et].
Do this exercise with (1) control (without inhibitor) and in the presence of a (2) noncompetitive inhibitor (NCI) and (3) irreversible inhibitor.
All plots thus obtained would be linear plots. Both plots obtained with control and in the presence of NCI will start from the origin (zero), however, plot obtained with NCI will show lower slope than that obtained with the control.
On the other hand, the plot obtained in the presence of irreversible inhibitor will start at a certain distance from zero on 'X' axis and it would be parallel to the control curve. The distance from zero to the start of this plot on 'X' axis will yield the concentration of the irreversible inhibitor.
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