Thermodynamic Analysis of Lactoperoxidase activity in camel milk H. Tayefi-Nasrabadi Faculty of Veterinary Medicine University of Tabriz, Tabriz, Iran tayefi@tabrizu.ac.ir M. A. Hosseinpour-Feizi, M. Mohasseli Faculty of Natural Sciences University of Tabriz, Tabriz, Iran pourfizi@eastp.ir Abstract—Lactoperoxidase (LP) is a glycoprotein that occurs naturally in colostrum, milk, and many other human and animal secretions. The present study was undertaken to explore thermodynamic parameters of LP in camel milk in a temperature range of 67-73°С. The H 2 O 2 -mediated oxidation of pyrogallol at 430 nm was used to assess the peroxidase activity. Thermal denaturation of LP, measured by loss in activity, showed good agreement with a first-order reaction. The low values obtained for activation energy (349.05 kJ/mol) and change in enthalpy of activation (~346 kJ/mol) indicate that low amount of energy is required to initiate denaturation, probably due to the molecular conformation of camel LP. Keywords- Camel milk; Lactoperoxidase; Thermal stability I. INTRODUCTION Lactoperoxidase (LP) is an oxidoreductase enzyme secreted into milk, saliva, and tears [1]. LP catalyzes the peroxidation of thiocyanate, generating hypothiocyanite and other products that impair the function of bacterial metabolic enzymes [2, 3]. The biological significance of LP is its involvement in the natural host defense system against invading microorganisms [4]. Also degradation of various carcinogens and protection of animal cells against peroxidative effects for LP system reported by Tenovuo [5]. LP system can be activated in milk after heat treatment, thus contributing to extend the shelf-life of pasteurized milk in locations with inefficient cold storage conditions [6]. The purpose of this study was to determine thermodynamic parameters of LP in camel milk in a temperature range of 67 to 73 ◦C. II. MATERIALS AND METHODS A. Milk Sampling Fresh raw camel (Camelus bactrianus) milk were supplied from Khokhor and Tabriz (East-Azerbaijan province, Iran) and divided into small portions (50 ml) and stored at -20 °C until analysis. B. Enzymatic activity assay Milk LP activity was measured by following the H2O2- dependent oxidation of pyrogallol at 430 nm, using an extinction coefficient of 2470 M -1 cm -1 . 3 mL of TS buffer (0.1 M Citrate-phosphate-borate buffer, pH 6.5), 0.15 mL pyrogallol (200 mM) and milk sample (0.05 mL) were added together in cuvette. The reaction was initiated by the addition of 0.03 mL hydrogen peroxide solution (61 mM) and immediately the measurement of absorbance started at 430 nm as a function of time for 2 min at 15 sec intervals using an UNICO UV-2100 PC (USA) spectrophotometer. Measurements were carried out against the reagent blank containing pyrogallol and enzyme solution only. Reaction velocity was computed from linear slopes of absorbance- time curve. One unit of activity is defined as the amount of enzyme that catalyzes the oxidation of 1 μmol of pyrogallol per min at room temperature (~22-25°C). C. Heat incubation study Thermal stability of milk LP was studied by incubating aliquots of milk at various temperatures (67, 69, 71 and 73 °C) up to 60 min in a thermostatic water bath and measuring their activity at room temperature after brief cooling in ice. The incubation was carried out in sealed vials to prevent change of volume of the sample and, hence, the enzyme concentration due to evaporation. Assays at the different temperatures were done at least in 3 separate experiments and the mean values of data were used to obtain the different kinetic and thermodynamic parameters. D. Kinetic data analysis Inactivation kinetics of milk LP toward thermal processes was subjected to reaction kinetic analysis. This process behaves in an analogous way to a general rate reaction of order n according to this equation: -dA/dt = k.A n (1) where -dA/dt represents the loss of LP activity rate, k the inactivation rate constant (min -1 ), A the LP activity at each time of treatment, and n the order of reaction. The experimental points are plotted according to the equation lnA/A 0 = k.t derived from Eq. 1, where A 0 is the initial response value (e.g initial enzyme activity at isothermal condition at time t 0 ), A is the response value after heating treatment and t is the exposure time (min). Linear regressions were performed using the SigmaPlot for windows version 10.0 (Systat software, Germany). The rate constant in a denaturation process and the temperature of treatment are related according to the Arrhenius equation: ln k = lnA −Ea/ RT (2) where k is the rate constant, A is the Arrhenius constant, Ea the apparent activation energy, R the universal gas constant, and T the absolute temperature. The slope of the line obtained permits to calculate the activation energy. The values of the activation energy (Ea) allow the determination of different thermodynamic parameters such 4 2011 International Conference on Life Science and Technology IPCBEE vol.3 (2011) © (2011) IACSIT Press, Singapore