Effect of Sodium Dodecyl Sulphate on Partial Purified Polyphenol Oxidase Activity in Red and Green Tomatoes (Solanum Lycopersicum)

In order to better understand how to prevent enzymatic greenning, it is important to understand kinetic properties of polyphenol oxidase. we investigated the effect of SDS on the rate of catechol oxidation by small cherry tomato partial purified PPO. PPO activity increased with increasing SDS concentration. The most effective concentration of SDS was 0.8, 1 and 1.25 mM in according to pH and type of substrate, where the measured activity was 0.074 and 0.247 units/mg.protein at pH 6.7 and 0.159 and 0.118 unit/mg.protein at pH 8 for catechol and pyrogallol, respectively. The activation of field small cherry PPO increased linearly with the SDS concentration up to 1 – 1.5 mM and decreased thereafter. The activity of small cherry PPO was also enhanced 1.7-fold by exposure to SDS at pH 8.0 in presence of catechol, 1.8fold by exposure to SDS at pH 8.0 in presence of pyrogallol, 2.3-fold by exposure to SDS at pH 6.7 in presence of catechol and 2.6-fold by exposure to SDS at pH 6.7 in presence of pyrogallol. Therefore sodium dodesyl sulphate is an activator of polyphenol oxidase that can probably change latent form of enzyme to active form, so increases the activity of polyphenol oxidase. Key word: Polyphenol Oxidase, Catechol, Pyrogallol, SDS, Solanum Lycopersicum INTRODUCTION Polyphenol oxidase (PPO; EC 1.14.18.1), a member of type III copper proteins, catalyses orthohydroxylation of monophenols (cresolase activity) and oxidation of ortho-diphenols to orthodi-quinones (catecholase activity) at the expense of molecular oxygen. The resulting highly reactive quinones auto polymerize to form bgreenn polyphenolic catechol melanins, a process thought to protect damage to plants from pathogens and insects (Kessler and Baldwin, 2002). The enzyme can be found not only in different fungal, mammalian, avian and insect species but also in a variety of plant species (Moore and Flurkey, 1990). In plants, PPO is located in the chloroplast thylakoid membranes and often exists in multiple forms. An unusual and intriguing characteristic of the enzyme is its ability to exist in either a latent and/or an active form (Mayer and Harel, 1979). PPO can be released from latency or activated by acid and base shock (Kenten, 1958), detergents (Jiang et al. 2003), urea (Swain et al. 1966) and proteases (Robinson and Dry, 1992). SDS as an activating agent is intriguing because very few enzymes are known to be activated by SDS in contrast with the many that are inactivated by it. Kenten (1958) has reported that the activation of crude broad bean leaf PPO by SDS occurred below 1 mM SDS. Extending these observations further, Saeidian & Rashid zadeh Int J Adv Biol Biom Res. 2013; 1(7):691-700 692 | Page Robb et al (Robb et al. 1964) observed that this activation process is reversible and that prolonged incubation in the presence of SDS leads to a loss of activity. Laveda et al (2001) demonstrated the total reversibility of the SDS activation of latent peach PPO by SDS entrapment with cyclodextrins. Some authors have suggested that PPO plays a role in plant resistance against diseases (Melo et al. 2006) and against insect herbivory (Felton et al. 1992). Li and Steffens (2002) obtained direct evidence of such a role for PPO in plants. The biochemical and kinetic properties of many PPOs from fruits and vegetables have been investigated because their associated bgreenning reactions affect food flavor and quality. MATERIALS AND METHODS Materials and Reagents The g r e e n a n d r e d tomatoes used in this study were obtained from Kurdistan of Iran (Baneh) and frozen at -25 °C until used. Catechol, polyvinylpyrolidone (PVPP), pyrogallol, tyrosine were purchased from Merck (Darmstadt, Germany). Acetone, ammonium sulphate, L-cysteine, kojic acid, L-glycine, polyethylene glycol (PEG), phenylmethylsulfonyl fluoride (PMSF), cellulose membrane (76x49mm) and DEAE-cellulose were purchased from Sigma-Aldrich (St. Louis, USA). All chemicals were of analytical grade. Enzyme Extraction 200 grams of tomatoes were homogenized in 150 mL of 0.1M phosphate buffer (pH 6.8) containing 10 mM ascorbic acid and 0.5% polyvinylpyrrolidone with the aid of a magnetic stirrer for 1h. The crude extract samples were centrifuged at 30000 g for 20 min at 4oC. Solid ammonium sulphate (NH4)2SO4 was added to the supernatant to obtain 30 and 80% (NH4)2SO4 saturation, respectively. After 1 h, the precipitated proteins for each stage were separated by centrifugation at 30000 g for 30 min. The precipitate was redissolved in a small volume of distilled water and dialyzed at 4oC against distilled water for 24 h with 4 changes of the water during dialysis. Ion Exchange Chromatography The dialysate was applied to a column (2.5 cm x 30 cm) filled with DEAE-cellulose, balanced with 10 mM phosphate buffer, pH 6.8. In order to remove non adsorbed fractions the column was washed with 200 mL of the same buffer at the flow rate of 0.5 mL/min. Then, a linear gradient of phosphate buffer concentration from 20 to 180 mM was applied. 5 mL fractions were collected in which the protein level and PPO activity towards catechol as substrate were monitored. The fractions which showed PPO activity were combined and were used as enzyme source in the following experiments. Protein Determination Protein contents of the enzyme extracts were determined according to lowry method using bovine serum albumin as a standard (Lowry et al. 1984). Enzymatic activity assays Partial purified Polyphenol oxidase activity was determined spectrophotometrically by following, at a specific wavelength, the increase in absorbance due to the oxidation of a selected substrate to its corresponding o-quinone. Namely, the increase in absorbance was followed at 420 and 400 nm in order to Saeidian & Rashid zadeh Int J Adv Biol Biom Res. 2013; 1(7):691-700 693 | Page monitor the oxidation of, respectively, pyrogallol and catechol. Assays were conducted at room temperature (~ 22–25 C), in a 3-ml reaction mixture prepared as follows: to 2.9–2.97 ml of 0.1 M phosphate buffer, pH 6.7, containing the appropriate amount of substrate prepared in the same buffer, an aliquot (75 μl) of Small cherry tomato extract was added. Enzymatic activity was determined by measuring the increase in absorbance at 420 nm for pyrogallol and 400 nm for catechol with a spectrophotometer (6305 JENWAY). In order to correct for substrate autoxidation, the reaction mixture, was placed in the sample cuvette while the reference cuvette contained buffer and the substrate. Polyphenol oxidase activity was determined by measuring the amount of quinone produced, using an extinction coefficient of 12 M cm for pyrogallol and 4350 12 Mcm for catechol. Enzyme activity was calculated from the linear portion of the curve. One unit of PPO was defined as the amount of enzyme producing a change in absorbance of 0.001 min. Results were average of three different experiments. Appropriate aliquots of 5 mM SDS prepared in 0.1 M phosphate buffer, pH 6.7 and pH 8, were added to the reaction mixture just before addition of the small cherry tomato extract. The final volume of the reaction mixture was always 3 ml. The pH activity curve was determined using a citrate phosphate-borate buffer system (range 2–10) at a concentration of 0.1 M. SDS Activation of PPO The SDS solutions were prepared in 0.1M sodium phosphate buffer (pH 6.7 and pH 8). The enzyme assay solution contained different concentrations of the buffered SDS solutions and substrates (catechol and pyrogallol) in sodium phosphate. The sample cuvette contained 3 ml of substrate (pyrogallol or catechol) in constant concentrations and in presence of different concentration of SDS (below the critical micelle concentration), prepared in the phosphate buffer.