What I'm looking for is the cytochrome P450 enzyme, which accepts electron from adrenodoxin (or analogue), and produces fluorescent or colorful compound. For example, I already know about CYP106A2, which produces steroids, which in order are fluorescent at strong oxidative conditions. I also know about mammalian cytochrome assay for producing luciferin-like molecules, which than accepted by luciferaze as a substrate. I'd like to learn of more examples. Thanks!
Here is another reference for you: Drug Metab Dispos. 2004 Jul;32(7):699-706. "Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes."
You can also just use UV for any dealkylation reactions using a number of derivatives such as dimedone.
Hi Sergii, Thanks to the interest of the pharmaceutical industry in CYP inhibition by drugs, there are many kits available for CYP activity assays. I found a whole suite of isozyme-specific fluoresence assays here, for example:
http://www.lifetechnologies.com/order/catalog/en/US/adirect/lt?cmd=catDisplayStyle&catKey=135009&ViewAllCatalog=135009&filterDispName=View%20All&filterType=0&OP=filter&filter=101/8201/134408/135009&_bcs_=H4sIAAAAAAAAALWRX2vCMBDAP01eViZJO2t9rIpjOJzQba8S0qsNpEaSU8m332VddWNs7GUQ7j93%0A97vcCsaLjbP1UaFPWJonFbiTVuB%2FibeIB5aVLF3SO5%2FPI6MbQFDt3hq70%2BBHynaUOnoSsCfR2g5I%0AWVeDG7XYGerC0iw%2BXqA7QvT5hJMSXFBlkXJxQ95K9%2BNlRxNnSem9DH9fISsLPowlrSRKyg5LvVQk%0AZK0dKCTLIMuWqqtZtnh4vV9T8UL7g5FhLhF21gVag4IrCFQR9%2FvM0EjjPyBivMGtmLyDNFsxnWY9%0AzDygVa2jWySbuzFPSoX6pDH8O8%2BVpcJg6Cfy6PZ2tqj0fmfgEU5gfkRN86cN2Y02GEfnvRG7rGUX%0Am1zZWDqLdKQufEP9czjEWnEJxJbDqcZfj%2FXttG8Or%2FV7qwIAAA%3D%3D
Hello Sergii,
There are plenty, as said by already by Adam.
There are microsomal mammalian P450s that oxidize susbtrates such as resorufin derivatives, fluorescein derivatives, coumarin derivatives and that yield highly fluorescent products.
I've attached two papers for you on Nile Red, which is a very interesting P450 substrate. In the papers it is described as being a substrate for CYP3A4 and for some steroid-metabolizing enzymes. I've data in my lab that show nile red is also a substrate for a number of other P450s, such as CYP1As.
Nile red is a funny substrate since it shows how deep you can go with your favorite P450s. The substrate is N-bi-alkylated. WIth CYP3A4 (and with CYP1As too) it is yielding two metabolites: one is mono N-dealkylated, the second is bi N-dealkylated. A solution of substrate is red and fluroescent at excitation 530 nm, emission 620 nm. A solution of the mono-N-delakylated product is pink and fluorescent exc = 510 nm and emm = 600 nm. A solution of bi-N-dealkylated metabolite is orange and fluorescent at exc = 490 nm and emm = 580 nm. A funny moelcule to use for looking at what's going on in the catalytic cavity and in the susbtrate access channel of any P450.
If you wish, you can go to my ResearchGate page and look at some papers of our lab available there. You will see that we heavily rely on the fluorescent properties of the P450 substrates.
Best regards,
Prof Philippe Urban
Here is another reference for you: Drug Metab Dispos. 2004 Jul;32(7):699-706. "Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes."
You can also just use UV for any dealkylation reactions using a number of derivatives such as dimedone.
Colleagues above indicated several assays for P450 enzyme kinetic assays. I would like to add to be cautious with the final solvent concentration in the assays. Majority of P450 substrates are quite hydrophobic and need to be solved in DMSO, EtOH, MeOH or ACN. However P450 isoforms are quite sensitive for these solvents which can inhibit, differentially and quite strongly P450 activity, see Busby et al., 1999 Drug Metab Dispos
http://www.ncbi.nlm.nih.gov/pubmed/9929510
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