Fig. 7. Gel filtration analysis of complexes of P450 3A4, b5, and POR. All analyses were done using a Superose 12 10/300 fast protein liquid chromatography (FPLC) column. (A) A280 profiles of individual proteins: POR (green), P450 3A4 (orange), b5 (purple), a binary mixture of b5 and P450 3A4 (blue), and a ternary mixture of all three proteins (red) are shown. Individual fractions were collected and analyzed by SDS-polyacrylamide gel electrophoresis, and densitometry was done of the Coomassie Blue-stained bands corresponding to the individual proteins. The POR preparation contained uncharacterized 280 nm-absorbing material eluting near the position of free b5 but not showing any protein after electrophoresis and staining. (B) Densitometry traces of P450 3A4 (orange) and b5 (purple) eluted in a binary equimolar mixture of the two proteins. The migration positions of the individual proteins [(P450) 3A4 and b5] are indicated. (C) Coomassie Blue staining of the proteins in a ternary complex, as eluted from the column in (A). The numbers on the left indicate Mr values of markers relevant to the three proteins of interest, which have approximate Mr values of 79 kDa (POR), 57 kDa (P450 3A4), and 17 kDa (b5). (D) Densitometry traces of P450 3A4 (orange), POR (green), and b5 (purple) eluted in a ternary equimolar mixture of the three proteins. The migration positions of proteins [POR, (P450) 3A4, and b5] are indicated in (B) and (D).