ABSTRACT
A polyphenol oxidase was purified and characterised from leaves of the common spiderflower. Purification sequentially with ammonium sulphate, dialysis, DEAE-Sepharose ion-exchange chromatography and Sephadex G-75 gel filtration chromatography resulted in 37.8-fold enrichment in the specific activity and 44.3% recovery of the total activity. Purified PPO is a monomeric protein of 52.6kDa revealed by Coomassie and active staining and Western blot. It was optimally active at pH 8.0 and 60°C, and stable from pH 3.0 to 9.0 and below 60°C. It displayed enzymatic activity towards monophenols, diphenols and triphenols, especially towards diphenols, and substrate specificity towards methylated and methoxylated substrates. Its activity was slightly increased by 0.1% SDS, heavily inhibited by Hg(2+) and Pb(2+), and completely inhibited by 1.0mM of ascorbic acid, l-cysteine, ß-mercaptoethanol, sodium diethyldithiocarbamate and thiourea, and by 10mM of dithioerythritol, sodium metabisulphite and sodium sulphite.
