FIGURE 2.

T560M and Q294L exchanges inactivate human ALOX15. Wild-type and mutant enzymes were expressed and purified as described under “Materials and Methods.” LOX concentrations of each preparation were quantified by immunoblotting, and equal amounts of enzyme were used in the activity assays. Representative example chromatograms are shown. The y axis was scaled for the product amount formed by the wild-type enzyme. In A, the mobile phase of the HPLC system consisted of 80% methanol, 20% water, and 0.05% acetic acid, resulting in longer retention times. For B, the solvent composition was 85% methanol, 15% water, and 0.05% acetic acid as described under “Materials and Methods.”