The isolation and specificity of alfalfa lipoxygenase

Partial purification of the lipoxygenase from alfalfa seed was accomplished by fractionation of the protein with (NH4)2SO4, phosphate, heavy metal salts and ultracentrifugation. About 24% of the original activity was recovered. The partially‐purified alfalfa lipoxygenase enzyme was free of hydroperoxide‐decomposing activity and was used to determine the positional specificity of linoleic acid oxidation by alfalfa lipoxygenase. Combined gas liquid chromatography‐mass spectrometry was used to analyze known mixtures of 10‐ and 12‐hydroxystearic acid derivatives and was satisfactory for the quantitative determination of the ratio of each component. This combination was used to analyze mixtures containing position isomers of hydroxy fatty acids without separation of each individual compound by other methods. Hydroperoxides produced from linoleic acid oxidation catalyzed by alfalfa lipoxygenase were converted by sodium borohydride reduction, catalytic hydrogenation and bis(trimethylsilyl)acetamide silylation to their corresponding trimethylsilyl either esters and the positional distribution was studied. The 9‐ and 13‐linoleate hydroperoxides produced by alfalfa lipoxygenase were in equal concentrations (50∶50) whereas the distribution for soybean lipoxygenase was 70% 13‐ and 30% 9‐hydroperoxides.