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

  • Biochemistry, Biophysics, and Molecular Biology
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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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Authors and Affiliations

  1. Institute of Biomedical Chemistry, ul. Pogodinskaya 10, str. 8, Moscow, 119121, Russia

    A. V. Kuzikov, R. A. Masamrekh, V. V. Shumyantseva & A. I. Archakov

  2. Pirogov Russian National Research Medical University, ul. Ostrovityanova 1, Moscow, 117997, Russia

    A. V. Kuzikov, R. A. Masamrekh, V. V. Shumyantseva & A. I. Archakov

Authors
  1. A. V. Kuzikov
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  2. R. A. Masamrekh
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  3. V. V. Shumyantseva
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  4. A. I. Archakov
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Corresponding author

Correspondence to V. V. Shumyantseva.

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Original Russian Text © A.V. Kuzikov, R.A. Masamrekh, V.V. Shumyantseva, A.I. Archakov, 2018, published in Doklady Akademii Nauk, 2018, Vol. 483, No. 1.

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Kuzikov, A.V., Masamrekh, R.A., Shumyantseva, V.V. et al. A New Method for Quantitative Determination of Steroid Metabolites of Cytochrome P450-Dependent Reactions Using Fluorescent Spectroscopy. Dokl Biochem Biophys 483, 302–305 (2018). https://doi.org/10.1134/S1607672918060054

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  • DOI: https://doi.org/10.1134/S1607672918060054

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