Molecular Pharmacology, Vol 6, 391-401, Copyright © 1970 by the American Society for Pharmacology and Experimental Therapeutics

Ligand Interaction with Hemoprotein P-450

III. The Use of n-Octylamine and Ethyl Isocyanide Difference Spectroscopy in the Quantitative Determination of High- and Low-Spin P-450

¹ Section of Biochemistry and Molecular Biology and The Graduate School of Nutrition, Cornell University, Ithaca, New York 14850

A simple method for the determination, by ligand binding to octylamine, of the absolute amounts of high- and low-spin forms of hemoprotein P-450 of rabbit liver microsomes is described. A correlation between octylamine binding to the two forms of oxidized P-450 and the binding of ethyl isocyanide to reduced P-450 has been observed, and advantages of the amine-binding method over the use of ethyl isocyanide are pointed out. In particular, octylamine binds to each species of P-450 to produce a distinct difference spectral minimum. We have derived a relationship for the calculation of the proportion of each form of P-450.

When the amine-binding method was used to determine the amounts of high- and low-spin forms of P-450, interesting quantitative changes were observed. Variations in the contents of the two forms in rabbit liver microsomes were noted following maintenance of rabbits on different control diets; dietary oxytetracycline, commonly used in commercial feeds, further increased the proportion of the low-spin form. Microsomal P-450 of bovine adrenal cortex is almost entirely in the low-spin form, while adrenal mitochondria contain both forms in nearly equal proportions.

Application of both the octylamine and ethyl isocyanide methods provides complementary data about P-450 in either oxidations state. A distinction between the two forms of oxidized P-450 in liver microsomes is maintained quantitatively during conversion to the reduced state.