A soluble pyropheophorbide a cleavage enzyme in broccoli (Brassica oleracea L.) flower buds

Acetone powder extracts (APE) prepared from broccoli newer buds, contained a soluble pyropheophorbide a cleavage enzyme. The enzyme catalyses pyropheo phorbide 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 th e cleavage and protein concentration range from 0 similar to 450 mug protei n per 3.0 ml of the reaction mixture. The Km for the pyropheophorbide a, PC A and H2O2 was ca. 10.6 muM, ca. 382 muM, and ca. 352 muM, respectively. The enzymatic cleavage of pyropheophorbide a was inhibited by ascorbate, n- propyl gallate, tiron, potassium cyanide (KCN) and hydroquinone. We confirm ed that the enzymatic cleavage of pyropheophorbide a is involved with free radicals. The cleavage by PCA-H2O2-APE was accompanied by the opening of th e 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 rea ction systems of pyropheophorbide a participate in the pyropheophorbide a d egradation process in broccoli flower buds. Moreover, ethylene-induced chlo rophyll catabolism in broccoli flower buds is discussed.