Substrates and carboxylic acid inhibitors of a partially purified polyphenol oxidase from gum Arabic

Polyphenol oxidase (PPO) was extracted from gum arabic, and two isoenzymes were partially purified by ammonium sulfate treatment and hydrophobic and ion-exchange chromatographies. Both fractions displayed an optimum at pH 5.3. PPOs showed activity toward o-diphenolic substrates but not on monophenols or p-diphenols. Activity was maximum with 4-methylcatechol (4MC) followed by the two catechins. Both enzymes showed apparent K-m of 0.8 mM for (+)-catechin and 2.4 mM for (-)-epicatechin and 4MC. Aromatic acids of the benzoic, cinnamic, and phenylalkanoic series and sorbic acid were mixed-type inhibitors. Benzoic acid was the most effective one (K-1 = = 0.44 mM and K-1 = 1.3 mM). Inhibition efficiency increased when pH was lowered indicating that both neutral (AH) and dissociated (A-) forms are responsible for inhibition. For all compounds tested, AH forms were more potent inhibitors than A- forms, and their affinity was higher for free enzyme than for the complex enzyme-substrate.