12-lipoxygenase activity in the muscle tissue of Atlantic mackerel (Scomber scombrus) and its prevention by antioxidants

Lipoxygenase was prepared from Atlantic mackerel muscle using differential centrifugation, ammonium sulphate precipitation and gel permeation (phenyl Sephadex G-50) column chromatography. The crude lipoxgenase enzyme preparat ion was characterised by sodium dodecyl sulphate polyacrylamide gel electro phoresis (SDS-PAGE), which showed two prominent bands with molecular weight s of 119 and 125kDa. Fractions collected from the chromatography column wer e tested for enzyme activity; by reacting with arachidonic acid and determi ning the production of hydroxyeico-satetraenoic acid (12-HETE) using revers e phase HPLC and GC-MS. The 12-HETE peak was absent from the fresh arachido nic acid control sample and from arachidonic acid treated with heat-inactiv ated lipoxygenase. Esculetin, a known inhibitor of lipoxygenase, inhibited the production of 12-HETE from the reaction of lipoxygenase with arachidoni c acid, thus confirming that the enzyme was lipoxygenase. The HETE peak was partially reduced in the presence of antioxidants, namely synthetic butyla ted hydroxytoluene (BHT) and natural antioxidants vitamins C and E. The pre sence of lipoxygenase in Atlantic mackerel muscle indicates the possibility that the lipid oxidation mechanism is initiated enzymatically in chilled a nd frozen stored fillets of mackerel and that this oxidative deterioration could be inhibited by antioxidants (BHT, vitamins C and E) which are used w idely in the food industry. 2001 Society of Chemical Industry.