Mechanism and Kinetics of the Hemin-catalyzed Oxidation of Linoleate in the Oil-Water Interface

Abstract The purpose of this investigation was to find out why hemin catalyzes the oxidation of linoleic acid or its esters by O2 only in the biphasic oil-in-water system but not in homogeneous solutions. We find that only those molecules of linoleic acid or methyllinoleate which form a monomolecular film at the oil-water interface react with hemin and O2. The high affinity of hemin and hemoglobin for the xylene-water interface (Haurowitz, F., Dicks, M., and Therriault, D. (1957) Nature 180, 437–438) facilitates their combination with the molecules of the interfacial oil film. Microscopic observation revealed that increase in the amount of methyllinoleate results in coalescence of small oil spherules and a concomitant decrease in the area of the oil-water interface. Consequently, increase in the amount of methyl-linoleate by a factor, n, leads to an increase in the rate of O2 uptake by a factor smaller than n⅔, close to n½. The initial rate of O2 uptake, V, in the continually shaken manometric flasks at 37° is strictly proportional to pO2 up to pO2 = 0.06 atm; further increase in pO2 does not raise V. Kinetic analysis reveals that the rate-limiting step is the transfer of linoleate molecules from the inside of oil spherules into the interfacial film. During the catalytic process hemin is degraded to non-heme iron which consists of free or loosely bound ferrous ions; their catalytic effect is lower than that of hemin. No evidence for the presence of radicals or for chain reactions in the hemin-catalyzed oxidation was found. We conclude that hemin acts in the oxidation of linoleate not only as an oxygen-oxidoreductase but also as a peroxidase which catalyzes the destruction of linoleate hydroperoxides and thus prevents the formation of free radicals. Since hemin can be replaced by hemoglobin, cytochrome c, or ferrous ions, the mechanism of the oxidation of unsaturated fatty acids in vivo is most likely identical with that proposed for the hemin-catalyzed process in vitro.