Purification and characterization of polyphenol oxidase from nettle (Urtica dioicaL.) 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.

Carbonic anhydrase inhibitory properties of novel sulfonamide derivatives of aminoindanes and aminotetralins

“…The yield of protein during the purification steps was determined spectrophotometrically at 595 nm according to the Bradford method 31 , explained previously using bovine serum albumin as the standard protein 32 .…”

Synthesis and carbonic anhydrase isoenzymes I and II inhibitory effects of novel benzylamine derivatives

“…The yield of protein during the purification steps was determined spectrophotometrically at 595 nm according to the Bradford method 39 , using bovine serum albumin as the standard 40 .…”

The effects of some bromophenols on human carbonic anhydrase isoenzymes

“…After this process, a single band was observed for each isoenzyme 39 . This protein imaging method was previously described [40][41][42] . In this application, the imaging method was performed out in 10% and 3% acrylamide for the running and the stacking gel, respectively, with 0.1% SDS 43,44 .…”