Assessment of electrocatalytic hydroxylase activity of cytochrome P450 3A4 (CYP3A4) by means of derivatization of 6β-hydroxycortisol by sulfuric acid for fluorimetric assay.
We used rapid one-step derivatization of 6β-hydroxylated hydrocortisone by sulfuric acid for fluorimetric determination of CYP3A4-dependent hydroxylase reaction in the electrochemical system. We have shown that CYP3A4 substrate - hydrocortisone - and its 6β-hydroxylated product have different emission wavelengths at an excitation λex = 365 nm after treatment with sulfuric acid:ethanol (3:1) mixture (λem = 525 ± 2 nm and λem = 427 ± 2 nm, respectively). The detection limit for 6β-hydroxycortisol was estimated to be 0.32 μM (corresponding to 0.095 nmol in 300 μL sample) (S/N = 3). Using the fluorimetric method of 6β-hydroxycortisol detection following the electrolysis of hydrocortisone with CYP3A4 immobilized on a screen-printed graphite electrode modified by didodecyldimethylammonium bromide we have calculated the steady-state kinetic parameters of CYP3A4 for hydrocortisone: the maximal rate of the reaction (Vmax) as 89 ± 5 pmol of product per min per pmol of electroactive enzyme and the Michaelis constant (KM) as 10 ± 2 μM. In our system, ketoconazole inhibited hydroxylase activity of CYP3A4 towards hydrocortisone with the IC50 value of 70 ± 5 nM. The approach proposed for determination of the CYP3A4 electrocatalytic activity can be used for throughput screening of different modulators of this cytochrome P450 isozyme during drug development.
['Kuzikov|Alexey|A|', 'Masamrekh|Rami|R|', 'Shkel|Tatsiana|T|', 'Strushkevich|Natallia|N|', 'Gilep|Andrei|A|', 'Usanov|Sergey|S|', 'Archakov|Alexander|A|', 'Shumyantseva|Victoria|V|']
D002384:Catalysis D051544:Cytochrome P-450 CYP3A D004572:Electrolysis D004800:Enzymes, Immobilized D005470:Fluorometry D006854:Hydrocortisone D013464:Sulfuric Acids
CYP3A4 Hydrocortisone Enzyme electrodes Fluorescence