Abstract
To elucidate the mechanism of degreening in flower buds of broccoli (Brassica oleracea L.), high performance liquid chromatography (HPLC) was used to separate chlorophyll derivatives in acetone extract derived from broccoli flower buds. Low levels of chlorophyll a derivatives, i.e. chlorophyllide a, pheophorbide a, pyropheophorbide a, C132-hydroxy-chlorophyll a, and pheophytin a were detected in extracts with or without treatment with ethylene for 48hr. Chlorophyll degrading reaction, the conversion of chlorophyllide a to pheophorbide a, was catalyzed by soluble enzyme in the acetone powder extract from flower buds in the presence of chlorophyllin, Mg-chlorin prepared from chlorophyll a as a substrate. The release of Mg2+ is associated with a shift of absorption from 658 to 687 nm. Changes in absorption at 687 nm are taken as a measure of Mg-dechelatase activity. Heating the extract prevented the reaction from proceeding, indicating that the protein/enzyme in the extract was inactivated. Mg-dechelatase did not catalyze the releasing reaction of Cu2+ from Cu-chlorophyllin as substrate. Hence, Mg-dechelatase of the flower buds appears to recognize Mg-chlorin specifically. Moreover, a high negative correlation which existed between the total chlorophyll contents in flower buds and Mg-dechelatase activity is expressed as : Y=-0.73X+97.37, r2=0.94, where X and Y are Mg-dechelatase activity and the total chlorophyll contents, respectively. These findings indicate that chlorophyll degradation in the flower buds is caused by the enhancement of Mg-dechelatase activity.
