A Soluble Pyropheophorbide a Cleavage Enzyme in Broccoli (Brassica oleracea L.) Flower Buds

Acetone powder extracts (APE) prepared from broccoli flower buds, contained a soluble pyropheophorbide a cleavage enzyme. The enzyme catalyses pyropheophorbide a cleavage in the presence of H2O2 and p-coumaric acid (PCA). The optimum pH was 5.0 with an acetate buffer. Linearity between the rate of the cleavage and protein concentration range from 0a450μg protein per 3.0 ml of the reaction mixture. The Km for the pyropheophorbide a, PCA and H2O2 was ca. 10.6μM, ca. 382μM, and ca. 352μM, respectively. The enzymatic cleavage of pyropheophorbide a was inhibited by ascorbate, n-propyl gallate, tiron, potassium cyanide (KCN) and hydroquinone. We confirmed that the enzymatic cleavage of pyropheophorbide a is involved with free radicals. The cleavage by PCA-H2O2-APE was accompanied by the opening of the chlorophyll-ring and a decrease in the red and Soret bands of the UV/VIS differential spectrum of the reaction mixture. These results indicate that the peroxidase-catalysed oxidative cleavage reaction systems of pyropheophorbide a participate in the pyropheophorbide a degradation process in broccoli flower buds. Moreover, ethylene-induced chlorophyll catabolism in broccoli flower buds is discussed.