Low level chemiluminescence of liver microsomal fractions initiated by tert-butyl hydroperoxide. Relation to microsomal hemoproteins, oxygen dependence, and lipid peroxidation.

Abstract

Low‐level chemiluminescence emitted by rat liver microsomal fractions supplemented with organic hydro‐peroxides (notably tert‐butyl hydroperoxide) is resolved in a time‐course consisting of two phases. Chemiluminescence was accompanied by a loss of cytochrome P‐450 as well as by lipid peroxidation (malondialdehyde accumulation and O2 uptake). Cytochrome b5, in contrast, remained intact. With the organic hydroperoxides, lipid peroxidation is initiated without addition of electron donors such as NADPH, so that mainly the propagation reactions are investigated here (phase I1 of chemiluminescence). In fact, addition of NADPH or NADH leads to substantial restriction in chemiluminescence emission and exerts protection on cytochrome P‐450 against degradation. Optimal conditions for light emission were with cytochrome P‐450 in the oxidized state, and inhibitors of this cytochrome, such as SKF‐525A or cyanide, decreased light emission. Conversely, there was increased chemiluminescence in microsomal fractions from rats treated with phenobarbital. a condition with increased levels of cytochrome P‐450. Chemiluminescence was sensitive to a free radical trap, di‐trrt‐butylquinol, and it was enhanced by 1,4‐diazabicyclo[2.2.2]octane, an enhancer of singlet oxygen dimol emission. This and the spectral distribution of emitted light suggest that singlet molecular oxygen is involved in this chemiluminescence system. The present observations establish for cytochrome P‐450, but not for cytochrome b5, its capability to effectively catalyze homolytic scission of hydroperoxides, an activity different from the known monooxygenase and peroxidase activities. This photoemissive reaction system is similar to that previously observed for the system consisting of lipid vesicles supplemented with cytochrome c and hydroperoxides [Cadenas. E., Boveris, A., and Chance, B. (1980) Biochrm. J. 188, 577–583], The former system is 70‐fold more effective than the latter, compared on a basis of heme content.