Purification and immunochemical detection of β-naphthoflavone-and phenobarbital-induced avian cytochrome P450 enzymes

Livers from mallards (Anas platyrhynchos) were treated with either β-naphthoflavone (50 mg/kg) or phenobarbital (70 mg/kg). Purification of induced hepatic cytochrome P450 was accomplished using both DEAE and hydroxyapatite columns, as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis separation. Polyclonal antibodies to these proteins were then produced in young male New Zealand White rabbits. β-Naphthoflavone (βNF)- and phenobarbital (PB)-treated red-winged blackbird, screech owl, European starling, and lesser scaup liver microsomes were analyzed in western blots for species cross-reactivity. Although all four of these avian species exhibited cross-reactivity with antibodies to βNF-induced mallard P450, all but the lesser scaup revealed a protein of higher molecular weight than that of the βNF-induced mallard. In addition, only the lesser scaup exhibited cross-reactivity with the anti-PB-induced mallard P450 antibodies.