PURIFICATION AND PROPERTIES OF FATTY-ACID HYDROPEROXIDE LYASE FROM GREEN BELL PEPPER FRUITS

Fatty acid hydroperoxide lyase (HPO lyase) was purified to apparently homogeneity state from immature fruits of green bell pepper (Capsicum annuum L.) by differential centrifugation, ion-exchange chromatography , hydroxylapatite chromatography and gel filtration. The enzymatic act ivity was separated into two fractions (HPO lyases I and II) during th e chromatography on hydroxylapatite. Both the isoforms were deduced to be trimers of 55-kDa subunits and have similar enzymatic properties. Peptide maps revealed only slight differences between them. Furthermor e, immunoblot analysis showed that an antibody raised against HPO lyas e I reacted with HPO lyase II as strongly as with the original antigen . These results indicate that there is only limited heterogeneity in t erms of amino acid sequence and/or post-translational modification. Th e activities of both HPO lyases were considerably inhibited by lipophi lic antioxidants, such as nordihydroguaiaretic acid and a-tocopherol. The activities with 13-hydroperoxy-(9Z, 11E, 15Z)-octadecatrienoic aci d as substrate were about 12 times higher than those with 13-hydropero xy(9Z, 11E)-octadecadienoic acid. By contrast, no reactivity was detec table against the geometrical isomer, 13-hydroperoxy-(9E, 11E)-octadec adienoic acid or against the positional isomer, 9-hydroperoxy-(10E, 12 Z)-octadecadienoic acid. Tissue-print immunoblot analyses using antise rum against HPO lyase indicated that HPO lyase was most abundant in th e outer parenchymal cells of the pericarp.