Cytochrome P450 Enzyme Assays in Vesicles

Rat CYP24A1, mouse CYP27B1 and adrenodoxin and human adrenodoxin reductase were expressed in E. coli and purified as described before^{28 (link),30 (link),35 (link)}. To test metabolism of each analog, they were incorporated into phospholipid vesicles made from dioleoyl phosphatidylcholine and cardiolipin by sonication, as before^{24 (link),28 (link)}. The substrates in vesicles (510 µM phospholipid) were incubated at 37 °C with either CYP24A1 (0.14 µM) or CYP27B1 (0.8 µM) in a reconstituted system containing adrenodoxin (15 µM) and adrenodoxin reductase (0.4 µM). Samples from incubations with CYP24A1 were extracted with dichloromethane and analysed by HPLC using a 25 cm Grace Alltima C18 column, as before^{27 (link),28 (link)}. Products (except 33) were separated using an acetonitrile on water gradient (45% to 100% for 20 min then 100% acetonitrile for 40 min at a flow rate of 0.5 mL/min). For the more polar 33, the acetonitrile gradient was 30% to 100% acetonitrile for 30 min then 100% acetonitrile for 20 min, at 0.5 mL/min. Products from incubations with CYP27B1 were similarly extracted with dichloromethane and analyzed by reverse phase HPLC using a 15 cm Grace Smart C18 column and an acetonitrile in water gradient (10 min 45% to 100% acetonitrile then 20 min at 100% acetonitrile, at 0.5 mL/min).