Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity.
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.
Güllçin
Ilhami
I
Atatürk University, Faculty of Science and Arts, Department of Chemistry, 25240, Erzurum, Turkey.
Küfrevioğlu
O Irfan
OI
Oktay
Münir
M
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*
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