Goldstein, JA; Linko, P; Luster, MI; Sundheimer, DW
HEEP COPYRIGHT: BIOL ABS. A new form of cytochrome P-448 was isolated from livers of rats treated with 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), a compound which belongs to the 3-methylcholanthrene (MC) class of inducers. The purified cytochrome (referred to as P-448HCB) had a specific content of 18.8 nmol cytochrome/mg protein and migrated as a single band on sodium dodecyl sulfate-polyacrylamide gels. The major forms of cytochrome P-450 induced by phenobarbital and 3-MC (referred to as P-450PB and P-450MC, respectively) were also purified for comparative studies. The MW of these cytochromes on sodium dodecyl sulfate-polyacrylamide gels were 51,500 (P-450PB), 52,000 (P-448HCB) and 55,000 (P-448MC). The maxima of the CO-reduced difference spectra were at 450 (P-450PB), 448 (P-448HCB) and 447.5 nm (P-448MC). Although cytochrome P-448HCB had a MW similar to that of cytochrome P-450PB, these cytochromes differed in their spectral properties, chromatographic behavior and peptide maps after limited proteolysis. Cytochrome P-448HCB clearly differed from cytochrome P-448MC in its MW, chromatographic behavior and peptide maps. Cytochrome P-448HCB had little catalytic activity toward benzphetamine, a substrate metabolized preferentially by cytochrome P-450PB or toward benzo(a)pyrene or 7-ethoxyresorufin, substrates metabolized preferentially by cytochrome P-448MC. The oxidized spectra of cytochromes P-448MC and P-448HCB were also distinctly different. Oxidized cytochrome P-448HCB had absorbance maxima at 393 and 648 nm, indicating that this form was a high spin ferric hemoprotein. Cytochrome P-448MC had a Soret maximum at 418 nm, indicative of the low spin state. Antisera against cytochrome P-448HCB gave a single precipitin band against its antigen in Ouchterlony double diffusion plates but did not cross-react against cytochrome P-450PB. A faint cross-reaction against cytochrome P-448 was observed. This cross-reaction was removed by immunoabsorption with partially purified cytochrome P-448MC, indicating that the 3 hemoproteins had different immunological properties.