Inhibition kinetic of Ocimum basilicum L. polyphenol oxidase

Abstract

The paper reports the inhibition model of the purified polyphenol oxidase (PPO) activity from basil (Ocimum basilicum L.) with L-cysteine, ethylenediaminetetraacetic acid (EDTA), ascorbic acid, gallic acid, D,L-dithiothreitol, tropolone, glutathione, sodium azide, benzoic acid, salicylic acid and 4-aminobenzoic acid inhibitors using 4-methylcatechol, catechol and pyrogallol as substrates. The inhibitors such as salicylic acid, benzoic acid and EDTA did not inhibit Ocimum basilicum L. PPO for all substrates used in this study. Purification was carried out by precipitation of contaminating proteins with (NH4)(2)O-4 dialysis of the supernatant and a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity chromatography. The enzyme-catalysed browning reaction was significantly inhibited in the presence of L-cysteine, ascorbic acid, gallic acid, D,L-dithiothreitol, tropolone, glutathione, sodium azide and 4-aminobenzoic acid inhibitors. It was found that the inhibition types were (i) competitive inhibition for L-cysteine, ascorbic acid, D,L-dithiothreitol, tropolone and sodium azide inhibitors using 4-methylcatechol as a substrate; for L-cysteine, ascorbic acid, gallic acid, tropolone and glutathione inhibitors using catechol as a substrate; and for ascorbic acid inhibitor using pyrogallol as a substrate, (ii) uncompetitive inhibition for gallic acid inhibitor using 4-methylcatechol as a substrate; for 4-aminobenzoic acid inhibitor using catechol as a substrate; for tropolone and 4-aminobenzoic acid inhibitors using pyrogallol as a substrate, (iii) noncompetitive inhibition for 4-aminobenzoic acid inhibitor using 4-methylcatechol as a substrate; for D,L-dithiothreitol and sodium azide inhibitors using catechol as a substrate; and for L-cysteine, glutathione and sodium azide inhibitors using pyrogallol as a substrate. Furthermore, tropolone was the most effective inhibitior for Ocimum basilicum L. PPO because of its low K-I value. Results showed that the type of inhibition depended on the origin of the PPO studied and also on the substrate used.