LIPID PEROXIDATION-CYTOCHROME P450 INTERACTIONS - USE OF LINOLEIC-ACID HYDROPEROXIDE IN THE CHARACTERIZATION OF THE SPIN-STATE OF MEMBRANE-BOUND P450

1. A procedure (linolenic acid hydroperoxide (LAHP) deletion method) i s described in which LAHP is added to the reference cuvette of a pair of spectrally balanced cuvettes containing hepatic microsomes to produ ce a composite high spin (HS)-low spin (LS)-spectrum of P450. 2. The L AHP deletion method was used to determine the spin state of P450 in ra t hepatic microsomes with and without the addition of type I compounds . 3. Advantage was taken of the temperature dependency of the spin sta te of P450 to determine the overall enthalpic and entropic changes for the spin equilibrium to generate computer-derived spectra of HS and L S forms of P450, and to construct a nomogram that allows direct estima tion of the percentages of HS and LS spin forms of P450 in intact micr osomes at temperatures compatible with biochemical functions. 4. The h .p.l.c. deletion method was used to demonstrate that HS-P450 comprised 57% of the P450 in hepatic microsomes; addition of type I substrates to these microsomes raised the level of HS-P450 to 97%. 5. The percent age of HS-P450 generated by the addition of type I compounds to micros omes declined with increasing deletions of P450 until at the extrapola ted 100% level of deletion there was no HS-P450 above that of the orig inal 57% observed in the absence of added compounds. This can be expla ined if LAHP destroys part of the LS-P450 while altering the remaining LS-P450 such that it retains its LS spectral characteristics but lose s its capacity to form HS P450 when type I substrates are added. 6. Th ese studies support the concept that about 50% of hepatic microsomal P 450 is functionally in the HS state due to binding with high affinity endogenous substrates or other membrane components; the remaining P450 is LS-P450 that can bind to exogenous substrates to form HS-P450. 7. Applications of the LAHP deletion method for assessment of catalytic p roperties of menbrane-bound P450 at ambient temperatures are discussed .