Investigation of some kinetic properties of polyphenol oxidase from parsley (Petroselinum crispum, Apiaceae)

Polyphenol oxidase was extracted and purified from parsley by a procedure that included (NH4)(2)SO4 precipitation followed by dialysis and gel filtration chromatography. These procedures led to 26.92-fold purification with 26.46% recovery. Optimum pH, temperature, substrate concentration, and ionic strength were determined with six substrates. Some kinetic properties of the enzyme such as V-max, K-M, and k(cat) were calculated for the substrates. The k(cat)/K-M values of the enzyme for catechol, 4-methyl catechol, progallol, L-dopa, dopamine, and catechin were 24,937, 2,680, 48.65, 10,000, 3.04, and 206.3 mM/min, respectively. The best substrate of the enzyme was found to be catechol. The native molecular weight of the enzyme was estimated to be 237 kDa based on its mobility in gel filtration column. The inhibitory effects of sodium azide, ascorbic acid, L-cysteine, and glutathione on the enzyme activity were tested, and IC50 values were estimated to be 23 mM, 51 mM, 62 mM. and 240 mM, and K-i constants were also calculated as 6.06 +/- 3.06 mM, 7.75 +/- 1.65 mM, 15.75 +/- 6.23 mM, and 23.60 +/- 8.25 mM, respectively by means of Lineweaver-Burk graphs. All inhibitors inhibited the enzyme noncompetitively and the most effective of them was sodium azide. (C) 2012 Elsevier Ltd. All rights reserved.