Molecular Pharmacology, Vol 19, 162-167, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Use of Linoleic Acid Hydroperoxide in the Determination of Absolute Spectra of Membrane-Bound Cytochrome P-450

¹ Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

Linoleic acid hydroperoxide (LAHP) destroys cytochrome P-450 in microsomes without destroying cytochrome b₅. This is the basis for a simple and direct method of determining the absolute spectra of membrane-bound cytochrome P-450. The method consists of adding LAHP to the reference cuvette of a spectrally balanced pair of cuvettes containing the same microsomal suspension and observing the resulting difference spectrum. The absolute spectra of oxidized, reduced, or reduced carbon monoxide-complexed cytochrome P-450 in microsomes from untreated or phenobarbital- or 3-methylcholanthrene-treated rats were very similar to corresponding spectra that have been reported for a highly purified preparation of P-450LM₂ with the exception that the oxidized spectrum of membrane-bound cytochrome P-450 represented a mixture of high-and low-spin forms of cytochrome P-450, whereas P-450LM₂ gives a typical low-spin spectrum.