Purification and Characterization of Two Isozymes of Chlorophyllase from Mature Leaves of Chenopodium album

Chlorophyllase (Chlase) was purified from mature leaves of Chenopodium album, and its enzymatic properties were investigated. Chlase was extracted from acetone powder of C. album and purified by the following chroma-tographic procedures: hydrophobic chromatography, Con A Sepharose, Heparin affinity chromatography, Mono Q ion-exchange chromatography, and gel-filtration. Con A Sepharose affinity chromatography and gel-filtration were the most effective steps on the purification. On Mono Q chromatography, the Chlase preparation separated into two major and one minor fractions that exhibited Chlase activity. The two major Chlases were purified to homogeneity. Their molecular masses were estimated as 41.3 kDa and 40.2 kDa by SDS-PAGE. The optimum pH and Km values of these two Chlases were similar. Their N-terminal amino acid sequences were almost identical except for a deletion in the tenth amino acid residue in one of the Chlase; there was no homologous protein detected by database search.