I am using natural inhibitor for DPP4 inhibition. Crude extract is giving results. But when i make particular conc of the extract inhibition is very less. Any changes i can make in protocol?
Yes, obviously the inhibition will be less. But let us know if you have purified the components.
One reason why the inhibition may decrease when the concentration is increased is that the solubility of the inhibitor has been exceeded, causing it to aggregate or precipitate.
Another possibility is that the extract causes interference in the measurement. For example, suppose you are using a fluorescence intensity-based assay in which the reaction causes an increase in fluorescence. If there is fluorescence in the inhibitor sample, that additional fluorescence will appear to be activity of the enzyme, making the inhibition look weaker than it is.
For a simple method to correct for interference in some types of assays, see this paper:
http://jbx.sagepub.com/content/14/8/1008.long
Hi arshad
I am working with crude extract. 100 -150 mg per ml is the maximum conc am using right now. atleast 50-60% inhibition is expected. I am using plasma to measure the activity and reading the absorbance @ 405 nm.
Thankyou
Dear Adam,
You are absolutely right . to avoid the interference of the inhibitor i take reading of inhibitor sample so that i can eliminate it. but that is also not working. i am not ableto make a visible change in the activity.
Can you please explain me the earlier point you mentioned regarding the solubility of the inhibitor.
As i am using plasma as the source of enzyme ll it make much difference?
When measuring IC50 curves for hits from compound library screens in enzyme assays, one sometimes sees that the % inhibition increases as the compound concentration increases, as expected, up to a certain concentration, then as the concentration increases further the % inhibition decreases. This phenomenon could be due to interference with the detection technology by the compound (see my original comment above), or it could be due to the compound reaching the limit of its solubility in the assay.
Although you might expect the % inhibition to level off, rather then decrease, this assumes that the soluble compound is in equilibrium with insoluble compound. What may happen instead is that the compound either "crashes out" of solution when added at a concentration above its solubility or forms aggregates that remain in suspension but don't inhibit the enzyme. The formation of precipitates or aggregates appears to be a kinetically driven, not an equilibrium phenomenon, with the result that the concentration of the monomeric compound remaining in solution is lower then the maximum soluble concentration.
The use of plasma as the source of the enzyme adds albumin and other proteins into the assay that can bind up the compound, reducing the effective (free) concentration and making the compound appear less potent than it really is. The plasma proteins, by binding up some of the compound, can also make it appear to be more soluble than it would be in an aqueous buffer.
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