The optical biosensor study of the redox partner interaction with the cytochrome P450 2B4-containing monooxygenase system under hydroxylation conditions

  • Yu. D. Ivanov
  • A. V. Ivanov
  • N. A. Petushkova
  • O. G. Gara
  • V. Yu. Kuznetsov
  • A. V. Podoplelov
  • A. I. Archakov
Experimental Studies


The interactions between cytochrome P450 2B4 (d-2B4), NADPH:cytochrome P450 reductase and cytochrome b5 have been investigated in the monomeric reconstituted P450 2B4-containing monooxygenase system in the presence of a substrate (7-pentoxyresorufin) and an electron donor, NADPH. Each partner was immobilized via its amino groups on the carboxymethyldextran biochip surface of the optical biosensor IAsys+. Such mode immobilization was not accompanied by any loss of activities of the immobilized proteins. The formation of binary d-Fp/d-2B4 complexes was registered. The association/dissociation rate constants (kon/koff) were (0.013 ± 0.005) × 106 M−1 s−1/0.05 ± 0.02 s−1, and dissociation constant (KD) was (0.26 ± 0.13) × 10−6 M. Comparison of kon, koff and KD values for d-Fp/d-2B4 complexes formed under hydroxylation (O-dealkylation) with corresponding constants obtained for the oxidized proteins of (0.10 ± 0.03) × 106 M−1 s−1/(0.14 ± 0.06) s−1, and (0.71 ± 0.37) × 10−6 M, respectively shows that the decrease in kon and an insignificant decrease in KD are associated with the increase of complex lifetime during transition from the oxidized to hydroxylation conditions. Complex formation between d-Fp and d-b5 was not registered in both hydroxylation conditions and in the case of oxidized forms of these proteins. In both cases formation of the ternary d-Fp/d-2B4/d-b5 complexes occurred.

Key words

cytochrome P450 2B4 cytochrome b5 NADPH:cytochrome P450 reductase optical biosensor protein-protein interactions 


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Copyright information

© MAIK Nauka 2008

Authors and Affiliations

  • Yu. D. Ivanov
    • 1
  • A. V. Ivanov
    • 1
  • N. A. Petushkova
    • 1
  • O. G. Gara
    • 1
  • V. Yu. Kuznetsov
    • 1
  • A. V. Podoplelov
    • 1
  • A. I. Archakov
    • 1
  1. 1.Orekhovich Institute of Biomedical ChemistryRussian Academy of Medical SciencesMoscowRussia

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