INABILITY OF SUBSTRATES TO ALTER THE CARBON MONOXIDE AND ETHYL ISOCYANIDE DIFFERENCE SPECTRA OF MICROSOMAL HEMOPROTEIN

¹ The Wellcome Research Laboratories, Burroughs Wellcome and Company (U.S.A.) Inc., Tuckahoe, New York

Previous studies have shown that treatment of rats with polycyclic hydrocarbons alters the CO and ethyl isocyanide difference spectra of microsomal hemoproteins. The purpose of the present studies was to determine if spectral alterations were due to synthesis of hemoprotein or to substrate binding. Rats were killed 3 or 24 hours after administration of 25 mg/kg of H²-3-methylcholanthrene (3-MC). At 3 or 24 hours, respectively, the maximum absorption of the CO difference spectra was at 450 or 448 mµ, the ratio of the 455: 430-mµ peaks of the ethyl isocyanide difference spectra at pH 7.4 was 0.65 or 1.20 and the content of 3-MC and its metabolites in microsomes was 0.69 or 1.11 µg/g of liver. The spectral characteristics of the microsomal hemoproteins at 3 hours were identical to those obtained with untreated rats. Calculations showed that 1 mµmol of 3-MC and its metabolites was present for every 24 mµmoles of cytochrome P-450 induced between 3 and 24 hours. When liver homogenates were incubated with 667 µg of H³3-MC per g of liver and reduced nicotinamide adenine dinucleotide phosphate, the isolated microsomes contained 95 µg of 3-MC and 10 µg of metabolites per g of liver but no spectral changes occurred. Addition of hexobarbital, benzpyrene or aniline to reduced microsomes had no effect on the CO difference spectra. Addition of hexobarbital or benzpyrene to reduced microsomes did not affect the ethyl isocyanide difference spectra. Aniline additions to reduced microsomes had an effect on the ethyl isocyanide difference spectra of microsomes from 3-MC-induced rats. These studies are in accord with other observations indicating that polycyclic hydrocarbons induce formation of a new hemoprotein.