Abstract
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.
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