Characterization of violaxanthin de-epoxidase purified in the presence of Tween 20: Effects of dithiothreitol and pepstatin A

Violaxanthin de-epoxidase (VDE) was purified from thylakoid membranes of sp inach by conventional column chromatography in the presence of Tween 20. Th e neutral detergent was necessary to prevent non-specific interaction of VD E with column resins. In anion-exchange chromatography on Mono Q, VDE appea red in two peaks. Both peaks exhibited a polypeptide of 41 kDa when fully r educed with 5 mM dithiothreitol. Re-chromatography of either peak gave rise to both peaks, suggesting that the two forms of VDE are interconvertible. VDE characteristically changed its electrophoretic mobility depending on th e concentration of dithiothreitol with which the protein was treated. When non-reduced, it showed two polypeptides of 43 and 42 kDa. These polypeptide s moved down to the position of 40 kDa, and then up to the position of 41 k Da, along with the increase in the dithiothreitol concentration from 0 to 2 mM. These findings suggest that VDE has more than one disulfide bond and t akes multiple forms depending on the extent of the reduction. Studies with various types of protein-modifying reagent revealed that VDE is sensitive t o pepstatin A, a specific inhibitor of aspartic protease. This finding sugg ests that the reaction center of VDE contains a reactive aspartic acid resi due(s).