Purification and properties of violaxanthin de-epoxidase from spinach

Abstract

The enzyme violaxanthin de-epoxidase (VDE) catalyzes the conversion of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z). It has been purified 194-fold with a yield of 1.4% from a sonicate of thylakoids of Spinacea oleracea to a specific activity of 19 μmol Z + A/min per mg protein. Purification steps included chromatography on DEAE-Sephadex, hydrophobic interaction chromatography on Butyl Sepharose, isoelectric-focusing, and gel filtration on Sephadex G-100. A single peptide band of 43.3 kDa was detected on SDS-PAGE, the apparent molecular mass of native enzyme was 45.8 kDa by gel filtration, and the pI was 4.95 on isoelectric focusing. The enzyme required ascorbate for activity, had a pH optimum of 5.2 and was stimulated three-fold by the addition of monogalactosyldi-acylglycerol (MGDG). The K ms for V and A were 5 and 5.3 μM, respectively. VDE was inhibited 50% by dithiothreitol (DTT), mercaptoethanol, cysteine and o-phenanthroline at 0.055, 0.68, 2.7 and 0.025 mM, respectively. With both DTT and o-phenanthroline, the rate of conversion of V to A was relatively unchanged whereas the conversion of A to Z was 50–80% inhibited. The enzyme also exhibited product inhibition by Z.