HYDROPEROXIDE-MEDIATED CYTOCHROME P450-DEPENDENT 8-ANILINO-1-NAPHTHALENESULFONIC ACID DESTRUCTION, PRODUCT FORMATION AND P450 MODIFICATION

The interaction between hydroperoxides, cytochrome P450 and 8-anilino- 1-naphthalenesulfonic acid (ANS) has been investigated. The addition o f ANS to the cytochrome P450 solution did not effect the P450 Soret ab sorption peak or the reduced CO difference spectrum, suggesting that A NS may not bind to P450 heme directly. H2O2 or CuOOH alone did not eff ect ANS fluorescence and absorption spectra indicating that no detecta ble reaction occurs between hydroperoxide and ANS in the absence of P4 50. The reconstituted system of cytochrome P450, P450 reductase, lipid and NADPH did not mediate ANS metabolism. In the presence of P450, th e addition of either H2O2 or CuOOH, however, leads to a decrease in AN S absorption around 258 nm and 350 nm indicating possible destruction of ANS. ANS destruction was confirmed with the disappearance of the AN S elution peak in the reverse phase HPLC profiles and with the changes in P450-bound ANS fluorescence intensity and the shift of lambda(max) of ANS. Moreover, a very sensitive method to detect trace fluorescent products of ANS by thin layer chromatography has been developed based on the fact that ANS fluorescence is enhanced more than 1000-fold by the organic solvent butanol. A UV-sensitive fluorescent product was de tected on thin layer chromatography profiles of the reaction mixtures. P450 was also observed to be modified by a fluorescent derivative of ANS, when the fluorescence was enhanced by butanol. These results also show that an organic compound which can not be metabolized by the rec onstituted system of cytochrome P450 and NADPH-P450 reductase is metab olized by the reconstituted system of P450 and hydroperoxide, suggesti ng the activities of these two systems may not be completely comparabl e.