[7] Arachidonic acid-12-lipoxygenase from bovine platelets

This chapter describes the assay, partial purification, and properties of arachidonic acid-12-lipoxygenase that is available in large quantities from blood platelets of slaughterhouse animals. In partial purification of arachidonate lipoxygenase described in the chapter, the enzyme was found for the greater part in the 100,000g supernatant after freezing and thawing the platelets three times. Further fractionation was possible with (NH4)2SO4. The lipoxygenase in intact platelets was rather stable and can be kept for at least 1 week at 0°. Lipoxygenase activity can be determined in intact platelets or crude, turbid enzyme preparations by incubations with radioactive arachidonic acid. The products were separated by thin-layer chromatography, and their radioactivity was measured. A simpler and quicker method can be used in more purified, clear enzyme solutions: the increase in UV absorption at 234 nm due to the formation of a conjugated system of double bonds was followed continuously.