Characterization of lipoxygenase from mackerel (Scomber scombrus) muscle

The polyunsaturated fatty acids (PUFA) in fish render them potentially susceptible to enzymatic oxidation. Lipoxygenase (LOX) activity from mackerel (Scomber scombrus) was characterized with respect to pH stability and activity, temperature stability, buffer and substrate preferences and inhibition patterns. The partially purified enzyme was stable over the pH range 4.0-11.0 and had a pH-activity optimum pH of 5.6. Mackerel muscle LOX (mmLOX) was stable at 0-50C but lost activity at higher temperatures. Enzymatic assays using different buffers revealed that MES-NaOH and Bistris-HCl were the suitable buffers for reaction. Substrate preference was for linoleic acid compared to linolenic, arachidonic, docosahexaenoic or oleic acid. Km values were 0.69 and 0.57 MM and V-max was 0.058 and 0.019 mumol min(-1) for linoleic and linolenic acid, respectively. mmLOX was inhibited by classical LOX inhibitors such as BHA, BHT, NDGA, esculetin and also by the heme protein inhibitor KCN. Values for the IC50 (concentration for half maximal inhibition) was between 0.02 muM and 41 muM. Normal phase high performance liquid chromatography (HPLC) analyses revealed that both 13- and 9-hydroperoxides were formed during mmLOX catalyzed oxidation with linoleic acid substrate although a higher proportion of the 13-isomer was formed. With enzymes from different mackerel tissues (skin, gills and muscle), mmLOX had the highest hydroperoxide forming ability.