Partial purification and characterization of polyphenol oxidase from artichoke (Cynara scolymus L.) heads
Abstract
Partial characterization of polyphenol oxidase activity in artichoke heads is described. Stable and highly active PPO extracts were obtained using 1.0% (w/v) polyethylene glycol (PEG), 1.5% (w/v) Triton X-100 and 0.1% NaCl in 0.2 M potassium phosphate buffer, pH 6.0. Three isoenzymes of the artichoke PPO were detected by polyacrylamide gel electrophoresis. The pH optimum for artichoke PPO was found to be a very broad (5.0-7.0) and the enzyme activity was stable in the range 6.0-7.0 at 25°C for 60 min. The optimum temperature was 25°C. The enzyme was heat-stable between 20 and 30°C and completely inactivated at 80°C after 5 min. The activation energy (Ea) with catechol was 15.8 kJ/mol at pH 6.0. PPO showed activity to catechol, pyrogallol and 4-methylcatechol, DL-dopa, L-dopa and gallic acid. (Km and V max values were 10.2 mM and 19,662 U/ml min for catechol, 12.4 mM and 12,500 U/ml min for 4-methylcatechol, 14.3 mM and 8065 U/ml min for pyrogallol, 37.7 mM and 5865 U/ml min for L-dopa 36.3 mM and 6060 U/ml min for DL-dopa, 43.6 mM and 4620 U/ml min for gallic acid, respectively). L-tyrosine was also tested but was not oxidized by artichoke PPO. The I50 values of inhibitors studied on PPO were determined by means of activity percentage (I50) diagrams. The values were 6.17×10-5 M, 6.32×10-5 M, 9.11×10-5 M, 1.76×10 -5 M, 1.47×10-5 M, 8.33×10-5 M, 4.12×10-5 M, 1.94×10-4 M and 1.83×10-5 M for glutathione, thiourea, sodium azide, sodium metabisulfite, dithiothreitol, β-mercaptoethanol, sodium diethyl dithiocarbamate, oxalic acid and ascorbic acid, respectively. Therefore, the most effective inhibitor was dithiothreitol, followed in decreasing order by sodium metabisulphide and ascorbic acid. Metal ions (Zn++, Ba ++, Cu++) were poor inhibitors of the enzyme at 10 mM. © 2003 Elsevier Ltd. All rights reserved.
Description
Keywords
Cynara , Cynara scolymus , Scolymus , ascorbic acid , buffer , catechol , catechol oxidase , diethyldithiocarbamic acid , dithiothreitol , DOPA , dopamine , gallic acid , glutathione , macrogol , mercaptoethanol , methyl group , oxalic acid , potassium dihydrogen phosphate , pyrogallol , sodium azide , sodium metabisulfite , thiourea , tyrosine , acidity , alkalinity , artichoke , article , concentration response , controlled study , enzyme activity , enzyme assay , enzyme inactivation , enzyme purification , enzyme stability , food biotechnology , heating , nonhuman , oxidation , phytochemistry , polyacrylamide gel electrophoresis , temperature dependence