Assessment of electrocatalytic hydroxylase activity of cytochrome P450 3A4 (CYP3A4) by means of derivatization of 6β-hydroxycortisol by sulfuric acid for fluorimetric assay

Alexey Kuzikov, Rami Masamrekh, Tatsiana Shkel, Natallia Strushkevich, Andrei Gilep, Sergey Usanov, Alexander Archakov, Victoria Shumyantseva

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7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)231-236
Number of pages6
JournalTalanta
Volume196
DOIs
Publication statusPublished - 1 May 2019
Externally publishedYes

Keywords

  • CYP3A4
  • Enzyme electrodes
  • Fluorescence
  • Hydrocortisone

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