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Doklady Biochemistry and Biophysics

, Volume 483, Issue 1, pp 302–305 | Cite as

A New Method for Quantitative Determination of Steroid Metabolites of Cytochrome P450-Dependent Reactions Using Fluorescent Spectroscopy

  • A. V. Kuzikov
  • R. A. Masamrekh
  • V. V. ShumyantsevaEmail author
  • A. I. Archakov
Biochemistry, Biophysics, and Molecular Biology
  • 15 Downloads

Abstract

A method for determination of hydroxylase activity of cytochrome P450 3A4 (CYP3A4) towards its substrate hydrocortisone using fluorescent analysis of the product was developed. 6β-hydroxycortisol, formed during CYP3A4-dependent electrocatalysis, has a characteristic fluorescent peak at λ = 427 ± 2 nm after treating with the sulfuric acid : ethanol (3 : 1) mixture and excitation at λ = 365 nm, which is different from the substrate (hydrocortisone) fluorescence (λ = 525 ± 2 nm). The limit of detection of 6β-hydroxycortisol was 0.32 μM. The developed analytical approach was used to determine the kinetic parameters of CYP3A4-dependent hydrocortisone hydroxylation.

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Copyright information

© Pleiades Publishing, Inc. 2018

Authors and Affiliations

  • A. V. Kuzikov
    • 1
    • 2
  • R. A. Masamrekh
    • 1
    • 2
  • V. V. Shumyantseva
    • 1
    • 2
    Email author
  • A. I. Archakov
    • 1
    • 2
  1. 1.Institute of Biomedical ChemistryMoscowRussia
  2. 2.Pirogov Russian National Research Medical UniversityMoscowRussia

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