The optical biosensor study of the redox partners interactions within the cytochrome P450 2B4 - containing monooxygenase system in hydroxylation conditions
Interactions between cytochrome P450 2B4, NADPH:cytochrome P450 reductase and cytochrome b5 have been investigated in the presence of a substrate (7-pentoxyresorufin) and an electron donor, NADPH, in the monomeric reconstituted P450 2B4-contained monooxygenase system. Each partner was immobilized via its amino groups on the carboxymethyldextran biochip surface of an optical biosensor IAsys+. It was shown that, despite immobilization of any of the partners (via their respective amino groups) onto the carboxymethyldextran surface of the IAsys+ optical biosensor, its activity didn't loss. 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, equilibrium dissociation constant (KD) was (0,26±0,13)⋅10-6 M. Comparison of kon, koff and KD for d-Fp/d-2B4 complexes in oxidation conditions with corresponding constants for the oxidized protein forms - (0,10±0,03)⋅106 M-1⋅s-1/ (0,14±0,06) s-1, (0,71±0,37)⋅10-6 M - shows that the decrease in kon and KD occurs due to the increase in lifetime during transition from oxidized to hydroxylation conditions. Complex formation between d-Fp and d-b5 was not registered in oxidation and hydroxylation conditions. The ternary d-Fp/d-2B4/d-b5 complexes formation was shown in hydroxylation and oxidation conditions.
Ivanov Yu.D., Ivanov A.V., Petushkova N.A., Gara O.G., Kuznetsov V.Yu., Podoplelov A.V., Archakov A.I. (2008) The optical biosensor study of the redox partners interactions within the cytochrome P450 2B4 - containing monooxygenase system in hydroxylation conditions. Biomeditsinskaya Khimiya, 54(4), 435-444.
Ivanov Yu.D. et al. The optical biosensor study of the redox partners interactions within the cytochrome P450 2B4 - containing monooxygenase system in hydroxylation conditions // Biomeditsinskaya Khimiya. - 2008. - V. 54. -N 4. - P. 435-444.
Ivanov Yu.D. et al., "The optical biosensor study of the redox partners interactions within the cytochrome P450 2B4 - containing monooxygenase system in hydroxylation conditions." Biomeditsinskaya Khimiya 54.4 (2008): 435-444.
Ivanov, Yu. D., Ivanov, A. V., Petushkova, N. A., Gara, O. G., Kuznetsov, V. Yu., Podoplelov, A. V., Archakov, A. I. (2008). The optical biosensor study of the redox partners interactions within the cytochrome P450 2B4 - containing monooxygenase system in hydroxylation conditions. Biomeditsinskaya Khimiya, 54(4), 435-444.
Yeung D., Gill A., C.H. Maule, R.J. Davies (1995) Trends in Anal. Chem., 14, 49-56. Scholar google search
Jonsson U., Fagerstam L., Ivarsson B., Johnsson B., Karlsson R., Lundh K., Lofas S., Persson B., Roos H., Ronnberg I. (1991) BioTechniques, 11, 620-627. Scholar google search
Way S., Hill B. (1996) Methods Guide for IAsys plus and IAsys Auto+. Affinity Sensors. Human-Computer Interface Limited, Cambridge, England. Scholar google search
