Abstract
Human cytochrome
P-450 (
P-450) 1A2 expressed in
Escherichia coli is readily converted into non-native cytochrome
P-420 (
P-420) in the presence of detergents. α-Naphthoflavone (ANF) has been used to prevent
P-450 1A2 inactivation to
P-420 during purification. However, the mechanism by which ANF modulates
P-450 1A2 is not clearly understood. We observed that recombinant human
P-450 1A2 prepared in the absence of ANF has an approx. 5 times higher maximum catalytic activity in the
O-deethylation of 7-ethoxycoumarin than that in the presence of ANF, with the same
K
m values. The results revealed that the enzyme purified with ANF is not catalytically fully active, indicating that ANF tightly binds to the enzyme, only to be dissociated by heat denaturation. Furthermore, the inactive
P-420 form of the enzyme could be reconverted to
P-450 by ANF in high concentrations of detergents. The reconversion was concentration-dependent, confirming ANF-induced regeneration of active
P-450 1A2. The reconversion coincided with the conformational change of the enzyme including increased α-helix content. The conformation of
P-450 1A2 was also stabilized by ANF, resulting in an approx. 5°C increase in thermal stability.