I will extract and purify elastase from the pancreatic tissues, and then calculate the optimum pH, temperature, and substrate concentration. After, I will apply different plant extracts to the enzyme to identify the inhibition effect.
my question is:
1. what is the chemical inhibitor of serine elastase?
2. what is the perfect method for extraction?
3. what is the substrate
4. what is the better method to add the plant extracts ?
Serine proteases are often inhibited by phenylmethylsulfonyl fluoride (PMSF) and 3,4-dichloroisocoumarin.
Chromogenic and fluorogenic artificial substrates for neutrophil elastase are available, consisting of short peptides with a C-terminal nitroaniline or aminomethylcoumarin group. An example is:
Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide
I don't know whether these will work with a plant enzyme.
Adding plant extracts may be tricky if you are measuring a color or fluorescence change because the extracts may interfere with detection. In that case, you may want to use a protein substrate and follow the cleavage by SDS-PAGE.
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