Hi
I'm currently working on proteases inhibition in the blood. Being fairly new to the field I was wondering how would one characterize a new protease inhibitor.
I'm especially interested in the constant one would try to determine, which method to choose to determine the Ki for instance?
Thanks in advance for any help one could provide!
A great source of information on characterization of enzyme inhibitors is a book by Robert Copeland called "Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists"
Do you have the enzyme, substrate, and inhibitor in a purified form? Do you have a convenient assay for the activity of the enzyme?
Is the inhibition time-dependent or -independent? This makes a big difference on how best to characterize the inhibition.
Dear Henri
Is it necessary to know the nature of your protease.
I recommend an efficient source: BIOTECHNOLOGY - Volume IV: Fundamentals in Biotechnology publié par Horst W. Doelle,J. Stefan Rokem,Marin Berovic.
Regards
Marius
Hi all, thank you for your answers I started to take a look in the copeland book, but it might take a while.
Basically my PI gave me a plasma protease inhibitor and a plasma protease and asked me to check whether or not the inhibitor would affect the protease. So currently at my disposal I have human plasma, the purified inhibitor, a chromogenic substrate for the enzyme. I'm still waiting for the purified enzyme to arrive but I will have it soon.
The inhibitor is present free at 11µg/ml and in protein complex with other proteins between 200 and 500µg/ml in the blood. The enzyme is around 40µg/ml. What I did so far is to activate the plasma (and thus the enzyme), add 200µg of the purified free inhibitor to the plasma and then add the chromogenic substrate and mesure the absorbance. The goal was to ensure that the inhibitor indeed had an effect on the enzyme, but so far I don't see any effect. Might be more relevant to check in the purified system first...
How do I determine what kind of inhibitor I'm dealing with here? doesn't that depend on the inhibition curve I would observe when incubated with the target protease? From a structural point of view, I know that this inhibitor has some kunitz domains, which interacts by electrostatic and hydrophobic bonds (BTW if somebody knows anything about how these domains work, I am VERY interested, because so far I found more about Mr Kunitz himself than about the domain he described...).
Obtaining reaction progress curves (product formation versus time) with the inhibitor at various concentrations is a good way to start. This will show whether the inhibition is time-dependent or not. It will also allow you to measure the potency of the inhibitor, once you have measured the kinetic parameters of the enzyme (Vmax, Km).
Chose a substrate concentration and an enzyme concentration for this experiment that give a linear progress curve for about 1 hour with no inhibitor present.
Kunitz domains are non-covalent protease inhibitors that are small proteins. They are held together by disulfide bonds, so it is important that no reducing agent be included in the assay. The classic one is bovine pancreatic trypsin inhibitor (BPTI), a competitive inhibitor of trypsin.
You may be dealing with a very potent inhibitor of your enzyme, so you may have to treat it as a slow-tight binder (see Copeland).
Hi Sir
yo can see this book title Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists .
Dear Henri
You can see the attached files, they could be useful for what you looking about
Good luck
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