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Dear all,
Any one please help in selecting Recombinant CYP450
Enzymes.
I want to check some of my molecules In-vitro
metabolism. How I can select Recombinant CYP450 Enzymes?
1) Based on the specific activity? (Enzyme kineticks)
2) Based on the protein content of Enzyme?
3) IC50 value?
4) Based on the are square value of enzyme vs
substrate, when i compare with the HLM?
Please suggest me in this regard.
Thanks and Regards,
chandram
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1 Specific activity is the conversion of a fixed amount of S to P in a
fixed
amount of time per unit protein
2 With most purification, specific activity increases as you purify the
enzyme from other proteins
3 IC50 and enzyme activity/substrate are fairly typical and reproducible
within reason for purified enzyme
You are essentially getting SA from the last one. The vendor should be
able to provide you with more data regarding isozymes in addition to
(3).
For examples, Isoelectrophoretic data.
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The following message was posted to: PharmPK
Dear Chandram,
Recombinant enzyme preparations are unnatural reagents where enzyme
activity is high. However, they are useful tools, namely because only
one CYP isoform is present and active.
If you want to determine whether your compound can be metabolised by a
given CYP, the best is to use the most active isoform, in order to
enhance the probability to detect the metabolite formed. Then you'll
be able to go back to HLM and investigate whether that metabolite is
formed a detectable levels in a real liver.
You may apply other criteria if you are pursuing other objectives.
If futile binding is an issue and you want to run assays with protein
content approaching that of human liver microsomes, you may add
protein to your assay, using control preparations (expressing no CYP)
which are commercially available as well (or albumin as a fallback
option).
Best wishes
Frederic MASSIERE
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