ORIENTATION OF CYTOCHROME P450SCC IN LANGMUIR-BLODGETT MONOLAYERS

Monolayers of cytochrome P450scc and its complex with adrenodoxin were formed by Langmuir techniques and covalently immobilized on the solid substrates. The orientation of hemeprotein molecules was studied usin g polyclonal antibodies specific to the intact cytochrome P450scc mole cule and its tryptic fragments F1 and F2, representing N- and C-termin al parts of the hemeprotein molecule. Specific interactions of the Lan gmuir films of cytochrome P450scc with adrenodoxin were investigated, and the position of the ferredoxin binding site at the hemeprotein mol ecule was identified. It was shown that the molecular orientation of t he P450scc-AD (adrenodoxin) complex at the water-air interface is depe ndent on the surface density of the monolayer. The P450scc molecules d o not denature upon spreading on the water surface. The formed monolay er can be transferred from the air-water interface to the surface modi fied with siloxane polymer and covalently immobilized without damage t o the structure. A model considering the mode of orientation of cytoch rome P450scc molecules on the air-water interface in dependence on the surface pressure is discussed.