Dear all,
Anybody could explain to me what is the difference between crude type and purified enzyme? Why the IC50 value for both enzyme would be different when tested on particular inhibitor? Your kind answer is highly appreciated
Thanks in advance.
Here are a few possible reasons.
1. With a crude enzyme mixture, there may be more than one enzyme with the same catalytic activity, but which are not inhibited equally by the inhibitor.
2. The enzyme concentrations may be different when the measurement is made with the crude mixture versus the purified protein. This can affect the IC50 in one of two ways.
a. If the inhibitor has very high affinity for the enzyme (tight binding), then the IC50 can depend on the enzyme concentration.
b. If the inhibitor is non-specific, such as an aggregator, its potency can be affected by the enzyme concentration (see papers on non-specific inhibitors from Brian Shoichet's lab).
3. The inhibitor may bind to other components in the crude mixture, or be chemically modified by them, reducing the effective inhibitor concentration.
4. If the inhibitor is of the substrate-competitive type, and the substrate is present in the crude enzyme mixture at some unknown concentration in addition to the amount that is deliberately added, then the substrate concentration will be different from the concentration used for the ourified enzyme. The IC50 of a competitive inhibitor depends on the concentration of the substrate.
5. There may be an allosteric regulator of the enzyme present in the crude enzyme mixture that is not present in the purified enzyme.
I totally agree with Dr Adams. His explanations are very valuable and succinctly explained.
I agree with Dr Adams!
It is a very clear and convincing explanation!
Dear Rachal Cmp
Pure enzyme is normally a single homogenous protein which shows the highest specific activity expressed, let us say, micro moles/mg protein/hour. So the specific activity will be less for an impure (heterogeneous) enzyme mixed with other junk proteins. The other partial is generally lost during the enrichment procedure of the pure form involving many steps from contaminated proteins.
Kindly follow link below for some suggestions
http://www.pnas.org/content/pnas/50/5/900.full.pdf
There is a wonderful book by Trevor and Palmer on enzymology. It explains things in a lucid manner I suggest U read that book.
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