12-Lipoxygenase activity in the muscle tissue of Atlantic mackerel (Scomber scombrus) and its prevention by antioxidants
2001
Lipoxygenase was prepared from Atlantic mackerel muscle using differential centrifugation, ammonium sulphate precipitation and gel permeation (phenyl Sephadex G-50) column chromatography. The crude lipoxygenase enzyme preparation was characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), which showed two prominent bands with molecular weights of 119 and 125 kDa. Fractions collected from the chromatography column were tested for enzyme activity by reacting with arachidonic acid and determining the production of hydroxyeicosatetraenoic acid (12-HETE) using reverse phase HPLC and GC-MS. The 12-HETE peak was absent from the fresh arachidonic acid control sample and from arachidonic acid treated with heat-inactivated lipoxygenase. Esculetin, a known inhibitor of lipoxygenase, inhibited the production of 12-HETE from the reaction of lipoxygenase with arachidonic acid, thus confirming that the enzyme was lipoxygenase. The HETE peak was partially reduced in the presence of antioxidants, namely synthetic butylated hydroxytoluene (BHT) and natural antioxidants vitamins C and E. The presence of lipoxygenase in Atlantic mackerel muscle indicates the possibility that the lipid oxidation mechanism is initiated enzymatically in chilled and frozen stored fillets of mackerel and that this oxidative deterioration could be inhibited by antioxidants (BHT, vitamins C and E) which are used widely in the food industry.
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