Properties of a Submicrosomal Particle Containing P-450 and Flavoprotein

A procedure is described for isolating submicrosomal particles from the livers of rabbits treated with phenobarbital, without the use of proteolytic or lipolytic enzymes. The preparation contains 2.5 mµmoles of P-450 per mg of protein, whereas the content of P-420 and cytochrome b5 is less than 1% of this. The preparation shows no distinct TPNH- or DPNH-cytochrome c reductase activity, but DPNH-ferricyanide reductase activity remains. P-450 in the preparation is not reduced by TPNH or by DPNH in the presence of anaerobic CO, even on addition of TPNH-cytochrome c reductase, but it is reduced by the adrenodoxin reductase system in bovine adrenal mitochondria. The physical properties of P-450 in the cytochrome b5-free preparation confirm that P-450 is a low spin protoheme protein in its oxidized state and indicate that it is a high spin ferroheme in its reduced state. The microsomal Fex signal is shown to be the electron spin resonance manifestation of oxidized P-450.