Purification and some properties of wheat germ lipoxygenase

Lipoxygenase from the germ of bread wheat was purified to near homogeneity by a classical scheme. After extraction at pH 4.5 from defatted germ, lipoxygenase activity was precipitated by 40% saturation (NH 4 ) 2 SO 4 from a 25% saturation supernatant. After dissolution in a phosphate buffer at pH 7 and extensive dialysis against this buffer, the extract was submitted to gel filtration on Ultrogel AcA 34. The final step of DEAE Sephadex A50 chromatography gave three peaks (L 1 , L 2 and L 3 ) with lipoxygenase activity. The total yield of the purification procedure was close to 30% and the degree of purification varied from 200 to 300 depending on the fraction considered. The three isoenzymes were also detected by disc electrophoresis using a specific staining method and were isolated by isoelectric focusing in a liquid medium. The molecular weight for each isoenzyme was determined to be 90 000–95 000 by gel filtration and 110 000 by electrophoresis in a gradient of acrylamide concentration. The stability, pH optimum and calcium effect have been studied. L 2 and L 3 were less stable than L 1 . Optimum pH ranged between 6–6.5 and neither isoenzyme activity was affected by calcium ions. L 1 was twice as active towards β‐carotene as L 2 and L 3 for the same level of oxygen uptake, but in comparison to lipoxygenase from horsebean the co‐oxidising power of wheat lipoxygenase was very poor.