Comparative Immunochemical Studies of Cytochrome P‐450 CAM of Pseudomonas putida and of Cytochrome P‐450 SCC , of Bovine Adrenocortical Mitochondria

1 Antibodies raised in rabbits against highly purified preparations of two cytochromes (cytochrome P‐450 CAM from Pseudomonas putida and cytochrome P‐450 scc from bovine adrenocortical mitochondria) were found to be specific for their respective antigens in Ouchterlony doublediffusion assays. In each case a single sharp curve was observed and no response was seen with other P‐450 hemeproteins. 2 In enzyme inhibition studies with reconstituted monooxygenase systems both the precipitating antibodies and soluble, monovalent Fab −1 fragments obtained from them by peptic digestion were effective inhibitors. In contrast to the Ouchterlony assay, anti(P‐450 CAM ) not only stopped camphor hydroxylation completely but also caused measurable inhibition of the cholesterol side‐chain cleavage (desmolase) reaction while anti(P‐450 scc ) inhibited camphor hydrolxylation significantly in addition to abolishing desmolase activity. 3 Iodination of cytochromes P‐450 CAM and P‐450 scc with the help of chloramine‐T produced soluble, radiolabeled preparations with specific activities ranging from 3 × 10 8 to 1 × 10 9 counts × min −1 ×μmol −1 which showed undiminished enzymatic activities and gave a single precipitin curve each in the Ouchterlony assay with anti(P‐450 CAM ) and anti(P‐450 scc ), respectively. In the iodinated preparations 60–70% of the tyrosine residues were found to be present as monoiodotyrosines while no other amino acid was labeled. 4 A sensitive radioimmunoassay was developed, measuring inhibition of binding between 125 I‐labeled cytochrome P‐450 CAM and its antibodies by cross‐reacting material, which revealed substantial cross‐reactivity of cytochromes P‐450 11β (88%), P‐450 scc (64%) and P‐450 LM‐2 (60%). Conversely, competitive binding o. 125 I‐labeled cytochrome P‐450 scc and unlabeled cross‐reacting antigens to anti(P‐450 scc demonstrated cross‐reactivities for cytochromes P‐450 scc β (60%), P‐450CAM (75%) and P‐450 LM2 (90%). Incubation of cytochrome P‐450 scc with adrenodoxin, and of cytochrome P‐450 CAM with putidaredoxin, prior to interaction with anti(P‐450 scc ) and anti(P‐450 CAM ), respectively, caused marked decreases in the ability of the corresponding P‐450 hemeproteins to bind to their antibodies. 5 Even the BrCN‐derived hemepeptides of cytochromes P‐450 CAM and P‐450 ssc were still able to recognize the antibodies elicited by their parent proteins as evidenced by significant crossreactivities in the radioimmunoassay. It is concluded that the four P‐450 hemeproteins tested in this investigation, cytochromes P‐450 CAM , P‐450 ssc , P‐450 11β , and P‐450 LM‐2 , have one or more antigenic determinants in common and that a major antibody binding site is associated with the hemepeptide.