The pure 15-LOX (0.5 mg/mL) was preincubated in 0.9 mL 50 mol/L Tris-HCl buffer, pH 7.4 for 3 minutes with the different additives listed in the table. The lipoxygenase reaction was started by the addition of a linoleic stock solution (final substrate concentration 0.26 mmol/L). Calcium was added as calcium chloride stock reaching a final concentration of 0.5 mmol/L. The concentration of SMP used as model membranes was adjusted to 53 μg/mL assay mixture. When the enzyme was incubated with SMP in the absence of linoleic acid (no linoleic acid, control 1) and when linoleic acid was incubated with SMP (no enzyme, control 2), no increase in absorbance at 235 nm was observed. One of two experiments performed with different LOX charges is shown. Both of them gave similar results, but are not strictly comparable.

Presence of 1 mmol/L EDTA.