Cytochrome P450 Monooxygenase (P450) Activity

The activity of P450 was evaluated by measuring p-nitroanisole (PNOD) activities according to the method of Chang and Hodgson (1975) with minor modifications. The detection system contained 100 µL of enzyme solution, 25 mM p-nitroanisole, and 100 mM potassium phosphate buffer (pH 7.2). After the addition of 100 mM D-glucose-6-phosphate sodium salt and 100 U/mL glucose-6-phosphate dehydrogenase, the reaction was initiated by the addition of 5 mM beta-nicotinamide adenine dinucleotide phosphate. After incubation for 10 min at 30°C, the reaction was stopped by adding acetone containing 2 mM glycine and 2 U/mL sodium hydroxide. Absorbances at 405 nm were measured in the microplate reader and the amount of product formed was calculated from the p-nitrophenol standard curve. CarE, GST, P450, and MFO activities were measured in all field populations and were compared to the susceptible strain (SS).

For all the experiments of detoxification enzyme activity determination, each geographical population was as one treatment group, there are a total of 7 geographical populations or 7 treatment groups including susceptible strain (SS) (SS, AB, DB, SB, XC, XB, and ZB). The experiments were performed three determinations with three biological replicates per geographical sample (treatment group).