Investigation of some kinetic properties of polyphenol oxidase from parsley (Petroselinum crispum, Apiaceae) Yusuf Ziya Doğru a, b , Mustafa Erat c, ⁎ a Atatürk University, Department of Physiology, Faculty of Medicine, 25240 Erzurum, Turkey b Atatürk University, Narman Vocational College, Food Processing Technologies, 25200 Narman, Turkey c Atatürk University, Erzurum Vocational College, Chemistry and Chemical Processing Technologies, 25240 Erzurum, Turkey abstract article info Article history: Received 26 April 2012 Accepted 17 July 2012 Keywords: Polyphenol oxidase Parsley Kinetic Inhibition Polyphenol oxidase was extracted and purified from parsley by a procedure that included (NH 4 ) 2 SO 4 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 IC 50 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. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Enzymatic browning of damaged tissues of fresh fruits and vegeta- bles mainly occurs from the oxidation of phenolic compounds by polyphenol oxidase (PPO) and contributes significantly to quality loss (Ding, Chachin, Ueda, & Imahori, 1998). PPO is a major copper enzyme which catalyzes the hydroxylation of monophenols to o-diphenols (cresolase activity; E.C. 1.14.18.1.) and the oxidation of o-diphenols to their corresponding o-quinones (catecholase activity; E.C. 1.10.3.2.) in the presence of oxygen (Fuerst, Anderson, & Morris, 2006). The quinones are highly reactive electrophilic molecules that can rapidly react with themselves and with amino acids or proteins, polymerizing to red, brown or black pigments (Spagna, Barbagallo, Chisari, & Branca, 2005). This browning phenomenon is generally undesirable in food technology because of the unpleasant appearance and the concomitant development of off flavor. Owing to its techno- logical importance, therefore, numerous studies have been devoted to the inhibition of the enzyme from different sources by different chemical compounds (Vamos-Vigyazo, 1995). Enzymatic browning caused by PPO can be controlled not only via inhibition of the enzyme with a wide range of chemicals but also by removal of one of its necessary components such as, oxygen, enzyme, copper and substrate (Dogan, Turan, & Dogan, 2006; Vamos-Vigyazo, 1995). Parsley (Petroselinum crispum, Apiaceae) is a well-known herb used to give fragrance to different food products. It has also been used as a medicinal plant for ailments and complaints of the gastrointestinal tract, as well as the kidney and lower urinary tracts, and for stimulating digestion (Popovic, Kaurinovic, Jakovljevic, Mimica-Dukic, & Bursac, 2007). Parsley is an important source of phytochemicals such as carotenoids, flavonoids, and vitamin C. β-carotene is a fat soluble plant component that is easily converted by the body into vitamin A whenever required (Brigitte, Paul, & Jeffrey, 2005). Diets with β-carotene-rich foods are also associated with a reduced risk for the development and progression of conditions like atherosclerosis, diabe- tes, and colon cancer (Daly, Jiwan, O'Brien, & Aherne, 2010). β-carotene may also be helpful in reducing the severity of asthma, osteoarthritis, and rheumatoid arthritis. The compound is an important nutrient to form a strong immune system. The flavonoids in parsley have been shown to function as antioxidants that combine with highly reactive oxygen species and help in the prevention of oxygen-based damage to cells (Brigitte et al., 2005). Vitamin C is the body's primary water-soluble antioxidant, rendering harmless dangerous free radicals in all water-soluble areas of the body. High levels of free radicals contribute to the development and progression of a wide variety of diseases, including atherosclerosis, colon cancer, diabetes, and asthma. Food Research International 49 (2012) 411–415 ⁎ Corresponding author. Tel.: +90 442 2311966; fax: +90 442 2360948. E-mail address: eratm@atauni.edu.tr (M. Erat). 0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2012.07.028 Contents lists available at SciVerse ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres