Determination of kinetic properties of polyphenol oxidase from Thymus (Thymus longicaulis subsp chaubardii var. chaubardii)

Abstract

A partial characterization of polyphenol oxidase (PPO) activity of Thymus longicaulis subsp. chaubardii var. chaubardii is described. Polyphenol oxidase of Thymus was isolated by (NH4)(2)SO4 precipitation and dialysis. The effects of substrate specificity, pH, temperature, heat-inactivation and glutathione inhibitor on polyphenol oxidase activity obtained from T longicaulis subsp. chaubardii var. chaubardii were investigated. Polyphenol oxidase showed activity toward catechol, 4-methylcatechol and pyrogallol. Pyrogallol was the most suitable substrate, due to the lowest Km (5.5 mM) and the biggest V-max/K-M (1260/min) values. It was found that the optimum pH values did not change with temperature, and were 6.5 for catechol and pyrogallol and 5.5 for 4-methycatechol at all temperatures. Optimum temperatures were 25 degreesC for catechol and 4-methylcatechol, and 35 degreesC for pyrogallol. Again, it was found that optimum temperature did not change with pH. Activation energy values were calculated from the Arrhenius equation and found to be in the range -1.72 and -7.48 kcal/mol for catechol, -3.56 and -9.17 kcal/mol for 4-methylcatechol, and -1.60 and -3.98 kcal/mol for pyrogallol as substrates, respectively. From heat-inactivation studies, the required times for 50% inactivation, using catechol, 4-methylcatechol and pyrogallol substrates, were 68.9, 66.4 and 96.3 min at 45 degreesC, 19.9, 17.9 and 34.3 min at 65 degreesC, and 4.1, 2.1 and 11.9 min at 85 degreesC, respectively. I-50 and K-i values for glutathione inhibitor, using catechol, 4-methylcatechol and pyrogallol substrates, were calculated, and it was found that the type of inhibition was competitive.