Original article Inhibition kinetic and mechanism of polyphenol oxidase from various sources by diethyldithiocarbamic acid Serap Dog ˘an, 1 Pınar Turan, 2 Mehmet Dog ˘an, 2 * Mahir Alkan 2 & Oktay Arslan 2 1 Balikesir University Faculty of Science and Literature, Department of Biology, 10145 Balıkesir ⁄ Turkey 2 Balikesir University Faculty of Science and Literature, Department of Chemistry, 10145 Balıkesir ⁄ Turkey (Received 18 September 2006; Accepted in revised form 23 April 2007) Summary Inhibition kinetics and mechanism of polyphenol oxidases (PPO) partially purified from various sources such as Thymbra spicata L. var. spicata and Ocimum basilicum L., and of mushroom PPO bought from Sigma by diethyldithiocarbamic acid have been described using catechol, 4-methylcatechol and pyrogallol as substrates. The inhibition type was competitive for O. basilicum L. PPO using catechol and 4-methylcatechol as substrates, for mushroom PPO using catechol, 4-methylcatechol and pyrogallol as substrates, and for T. spicata L. var. spicata PPO using 4-methylcatechol as a substrate; uncompetitive inhibition for T. spicata L. var. spicata PPO using pyrogallol as a substrate; and non-competitive inhibition for O. basilicum L. and T. spicata L. var. spicata PPO using pyrogallol and catechol as substrates, respectively. The inhibition effect of diethyldithiocarbamic acid on enzymatic browning varied greatly from one phenol to another and from one enzyme to another. Hence, no general rule can easily be established with regard to the type of inhibition observed. Keywords Diethyldithiocarbamic acid, inhibition, inhibitors, polyphenol oxidase, substrates, type of inhibition. Introduction Polyphenol oxidase (monophenol, dihydroxyphenylala- nine: oxygen oxidoreductase, EC 1.14.18.1; PPO) is a bifunctional, copper-containing enzyme widely distri- buted in the phylogenetic scale which, in the presence of molecular oxygen, catalyses both the o-hydroxyla- tion of monophenols to give o-diphenols (cresolase activity) and the further oxidation of o-diphenols to o-quinones (catecholase activity). The o-quinones thus generated are very unstable and rapidly react with themselves and with amino acids or proteins, polymer- ising to the brown or black pigments (Mason & Peterson, 1965; Matheis & Whitaker, 1984; Garcia- Carmona et al., 1988) that are responsible for melanisation in animals and browning in plants. This browning phenomenon is generally undesirable in food technology because of the unpleasant appearance and the concomitant development of off flavour. Owing to its technological importance, therefore, numerous stud- ies have been devoted to the inhibition of the enzyme from different sources by different chemical compounds (Ferrar & Walker, 1996). The prevention or inhibition of enzymatic browning is a major concern of the food industry all over the world, and attempts have been made to eliminate from the reaction one or more of its essential components: oxygen, enzyme, copper, or (poly)phenols (Richardson & Hyslop, 1985). Oxygen can be excluded from the reaction by immersing the fruits and vegetables in water, syrup, and brine or by exposure to vacuum or modified atmospheric packing (Langdon, 1987). However, this treatment is not definitive, because when the package is opened, oxygen is reintroduced and browning will restart. The enzyme can be effectively heat-denatured by steam blanching before freezing or carrying food (McEvily et al., 1992) at the expense of adverse off flavours and texture changes in fresh material (Langdon, 1987). Thermal processing is generally considered as the most effective method to inactivate PPO and, conse- quently, to inhibit enzymatic browning (McEvily et al., 1992). Thermal treatments can, however, be responsible for considerable sensorial and nutritional quality losses in fruits and vegetables (Lund, 1977; Sapers, 1993). In this regard, molecular biology is starting to produce antisense PPO mRNA in transgenic plants, to inhibit enzymatic browning in fresh fruits. However, develop- ment of this technology will depend on a better knowledge of PPO genes in different plant sources *Correspondent: E-mail: sdogan@balikesir.edu.tr International Journal of Food Science and Technology 2008, 43, 1316–1322 1316 doi:10.1111/j.1365-2621.2007.01610.x Ó 2008 The Authors. Journal compilation Ó 2008 Institute of Food Science and Technology Trust Fund