CONVERSION OF LINOLEIC ACID HYDROPEROXIDE BY FRENCH BEAN HYDROPEROXIDE ISOMERASE

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Abstract
Hydroperoxide isomerase (HPI) was extracted from the cotyledon portion of sprouted French bean seeds after five days of incubation and partially purfied by ammonium sulphate fractionation and ultracentrifugation. Linoleic acid hydroperoxides (LAHPO) were produced using commercial soybean lipoxygenase and purified using a silicic acid column and identified by HPLC. The characterized LAHPOs were used as substrates for the French bean HPI. The purified French bean extract showed a three fold increase in activity over the crude preparation and a pH optimum of 7.2. The primary end-products of the enzyme reaction were separated into ketol and carbonyl compounds and then further purified by preparative TLC. Subsequent GC-MS analysis of the ketol fraction indicated that HPI converted the 13-hydroperoxyoctadec-9, 11-dienoic acid primarily into 10,13-dihydroxyoctadec-11-enoic acid (.gamma.-ketol) and to a lesser extent into 12,13-dihydroxyoctadec-9-enoic acid (.alpha.-ketol). Trace amounts of 9,12-dihydroxyoctadec-10-enoic acid resulting from the isomerization of 9-hydroperoxyoctadec-10,12-dienoic acid were also found. Hexanal was revealed to be the principal carbonyl compound resulting from the decomposition of 13-hydroperoxyoctadec-9,11-dienoic acid, suggesting that a lyase was also present in the French bean extract.