Purification, substrate specificity, and products of a Ca2+-stimulating lipoxygenase from sea algae (Ulva lactuca)

A calcium-stimulated lipoxygenase (LOX) was isolated and purified 103-fold from sea algae (Ulva lactuca) using 40-55% saturation of ammonium sulfate fractionation, MacroPrep-Q ion exchange, and gel filtration on Sephacryl S-300. When this partially purified algal LOX was run on 4-20% polyacrylamide gel electrophoresis (PAGE), two bands with LOX activity were found using activity staining. The apparent molecular masses were estimated as 41 and 116 kDa by PAGE. The LOX activities from the sea algae were stimulated by Ca2+ at 0.2 mM to a maximal activity of about 8.8-fold. The optimal pH of the algal LOX was 7.5, and optimal temperature was 33 degrees C. At pH ranging from 5.5 to 9.0 and temperature below 40 degrees C, the algal LOX was stable. The algal LOX showed the highest reactivity toward 18:2 fatty acid followed by 20:4, 20:5, 22.6, and 18:3. Linoleic acid and its esters were peroxidized by algal LOX in the order linoleic acid >> monolinolein > dilinolein, methyl linoleate, but no activity was observed on trilinolein. On the basis of retention time in normal phase HPLC, the products of 18:2 reacting with algal LOX were 9- and 13-hydroxyoctadecadienoic acid at a ratio of 86:14; the product from 18:3 was 9-hydroxyoctadecatrienoic acid. Products derived from 20:4 were 12- and 15-hydroxyeicosatetraenoic acid at a ratio of 90:10 using reversed phase HPLC. Fatty acids of 20:5 and 22:6 yielded 12-hydroxyeicosapentaenoic acid and 14-hydroxydocosahexaenoic acid, respectively. It seems that n-9 LOX is the dominant LOX and n-6 LOX is the minor LOX in sea algae.