CHARACTERIZATION OF LIPOXYGENASE FROM MACKEREL (SCOMBER SCOMBRUS) MUSCLE

Abstract

The polyunsaturated fatty acid (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 prefeences and inhibition patterns. The partially puriped enzyme was stable over the pH range 4.0-1 I. 0 and had a pH-activity optimum pH of 5.6. Mackerel muscle LOX (&OX) was stable ot 0-50C but lost activity at higher temperatures. Enzymatic assays using diflereru buffers revealed that MES-NaOH and Bistris-HCl were the suitable b@ers for reaction. Substrate preference was for linoleic acid compared to linolenic, arachidonic, docosahexaenoic or oleic acid. K,,, values were 0.69 and 0.57 mM and V, was 0.058 and 0.019 pmol min for linoleic and linolmic acid, respectively. &OX was inhibited by classical LOX inhibitors such as BHA, BHT, NDGA, esculetin and also by the heme protein inhibitor KCN. Values for the IC,, (concentration for halfmaximclr inhibition) was between 0.02 pM and 41 pM. Normal phase high pegormance liquid chromatography (HPLC) analyses revealed that both 13- and 9-hydroperoxides were formed during mmLOX catalyzed oxidation with linoleic acid substme although a higher proportion of the 13-isomer was formed. With enzymes from different mackerel tissues (skin, gills and muscle), &OX had the highest hydroperoxide forming ability.