title: Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity.
abstract: Polyphenol oxidase (PPO) of nettle (Urtica dioica L.) was extracted and purified through (NH4)2SO4 precipitation, dialysis, and CM-Sephadex ion-exchange chromatography and was used for its characterization. The PPO showed activity to catechol, 4-methylcatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), L-tyrosine, p-cresol, pyrogallol, catechin and trans-cinnamic acid. For each of these eight substrates, optimum conditions such as pH and temperature were determined and L-tyrosine was found to be one of the most suitable substrates. Optimum pH and temperature were found at pH 4.5 and 30 degrees C respectively and Km and Vmax values were 7.90 x 10(-4) M, and 11290 EU/mL for with L-tyrosine as substrate. The inhibitory effect of several inhibitors, L-cysteine chloride, sodium azide, sodium cyanide, benzoic acid, salicylic acid, L-ascorbic acid, glutathione, thiourea, sodium diethyl dithiocarbamate, beta-mercaptoethanol and sodium metabisulfite were tested. The most effective was found to be sodium diethyl dithiocarbamate which acted as a competitive inhibitor with a Ki value of 1.79 x 10(-9)M. In addition one isoenzyme of PPO was detected by native polacrylamide slab gel electrophoresis.
authors.affiliation: Atatürk University, Faculty of Science and Arts, Department of Chemistry, 25240, Erzurum, Turkey.
authors.forename: Ilhami
authors.initials: I
authors.lastname: Güllçin
authors.forename: O Irfan
authors.initials: OI
authors.lastname: Küfrevioğlu
authors.forename: Münir
authors.initials: M
authors.lastname: Oktay
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology; D004156:Catechol Oxidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D004226:Dithioerythritol / Q000494:pharmacology; D004050:Ditiocarb / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004795:Enzyme Stability; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011190:Potassium Cyanide / Q000494:pharmacology; D019810:Sodium Azide / Q000494:pharmacology; D013379:Substrate Specificity; D013447:Sulfites / Q000494:pharmacology; D013696:Temperature; D013890:Thiourea / Q000494:pharmacology; D026982:Urtica dioica / Q000737:chemistry / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; D013447:Sulfites; C005200:sodium metabisulfite; D004226:Dithioerythritol; D019810:Sodium Azide; D004050:Ditiocarb; D004156:Catechol Oxidase; D005978:Glutathione; D013890:Thiourea; D011190:Potassium Cyanide; D001205:Ascorbic Acid