Three different methods to evaluate antioxidative effect using assay systems consisting of emulsified linoleic acid are described. Oxygen consumption was measured by a polarographic technique using hemin to accelerate the lipid oxidation. The formation of conjugated diene compounds during oxidation at 37° C was determined by spectrophotometric measurement of absorption at 234 nm. A sensitive gas chromatographic procedure was used for direct recording of the development of different volatile compounds by analyzing the headspace gas over the reaction medium. In the latter case denatured horseradish peroxidase was used as the catalyst. The advantages and limitations of the different methods are discussed.

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Lipid oxidation catalysis with peroxidase heat treated in vitro was strongly influenced by the pH at which the treatment was carried out, particularly in the range 5.5-6.5. Below pH 5 no increase in lipid oxidation activity occurred due to masking of the heine groups. Electron microscopy studies showed differences in size and shape of the thermal aggregates produced at pH 7.2 and 4.9. Increased lipid oxidation activity of peroxidase on heat treatment at pH 6.5 was almost exclusively associated with the aggregates, which were separated by, gel chromatography from nonaggregated material containing the residual enzyme activity. Heine migration during heat treatment led to relatively higher heme content of the aggregates, thus increasing the number of catalytic sites. Thermal destruction of the heme group decreased its lipid oxidation activity.

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