[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) x 10(6) M(-1) x s(-1)/0,05 +/- 0,02 s(-1), equilibrium dissociation constant (K(D)) was (0,26 +/- 0,13) x 10(-6) M. Comparison of kon, koff and K(D) for d-Fp/d-2B4 complexes in oxidation conditions with corresponding constants for the oxidized protein forms--(0,10 +/- 0,03) x 10(6) M(-1) x s(-1)/(0,14 +/- 0,06) s(-1), (0,71 +/- 0,37) x 10(-6) M--shows that the decrease in kon and K(D) 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
Iu D
IuD
Ivanov
A V
AV
Petushkova
N A
NA
Gara
O G
OG
Kuznetsov
V Iu
VIu
Podoplelov
A V
AV
Archakov
A I
AI
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D000072467:Cytochrome P450 Family 2; D015786:Cytochromes b5 / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D055620:Optical Phenomena; D055095:Optics and Photonics / Q000379:methods; D010078:Oxazines / Q000737:chemistry*
D004800:Enzymes, Immobilized; D010078:Oxazines; C044943:pentoxyresorufin; D015786:Cytochromes b5; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D009251:NADPH-Ferrihemoprotein Reductase