Interactions of 8-anilino-1-napthalenesulfonic acid (ANS) and cytochrome P450 2B1: Role of ANS as an effector as well as a reporter group

Interactions of cytochrome P450 2B1 were probed using 8-anilino-1-naphthalenesulfonic acid (ANS), a well known and frequently used reporter group for hydrophobic interactions. Titration of cytochrome P450 2B1 with ANS revealed 6.6 ± 0.2 ANS binding sites per molecule of P450 2B1 with a K_d value of 42 ± 2 M. In our evaluation of the consequences of the binding of ANS to cytochrome P450 2B1, we found that the binding of ANS to P450 2B1 increased benzphetamine demethylation activity by 1.5-fold, indicating a role for ANS as an effector in addition to its role as a reporter group. Kinetic analysis of the effects of ANS on P450 2B1-dependent demethylation activity revealed that ANS increased both the V_max and K_m of the benzphetamine demethylation reaction. ANS stimulation of activity appears not to be due to the replacement or augmentation of the role of lipid since studies of binding and catalytic activities in the presence and absence of added lipid gave the same array of effects. These results demonstrate that ANS can bind to cytochrome P450 2B1 as would be expected of a reporter group probe but show in addition that this probe also acts as an effector molecule stimulating catalytic activity. Thus, results of ANS studies should be viewed with caution since the molecule may play more than one role in its reaction with a protein.Abbreviations ANS 8-anilino-1-naphthalenesulfonic acid - bis-ANS 1,1-bis(4-anilino-5-naphthalenesulfonic acid - DTT dithiothreitol - P450 cytochrome P450