Research Journal of Chemical Sciences ______ ______________________________ ______ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 68 - 70 , March (201 2 ) Res.J.Chem.Sci. International Science Congress Association 68 Short Communication Thermal investigation of the interaction between Phenyl Dithiocarbamate and mushroom tyrosinase Mirzaie M. 1 , Rezaei Behbehani G. 1 , Barzegar L. 1 , Mehreshtiagh M. 1 , Saboury A . A. 2 and Rezaei Behbehani Z. 1 1 Department of Chemistry, faculty of science, Islamic azad university, Takestan branch, Takestan, IRAN 2 Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IRAN Available online at: www.isca.in (Received 12 th January 2012 , revised 16 th January 2012 , accepted 31 st January 2012 ) Abstract A comprehensive, simple and rapid thermodynamic study on the interaction of Mushroom Tyrosinase (MT) with Phenyl Dithiocarbamate by using isothermal titration calorimetry at 27 and 37C in phosphate buffer (10 mM) at pH 6.8, was carried out to see whether Phenyl Dithiocarbamate induced conformational change of Mushroom Tyrosinase and how changes by ligand binding wer e occurred. The extended solvation theory can be used to elucidate the stability of enzyme by Phenyl Dithiocarbamate. The negative change of the Gibbs free energy at two temperatures of 27 and 37C shows that the binding process in both temperatures are sp ontaneous . The obtained results indicate that there are two identical and non - cooperative binding sites for Phenyl dithiocarbamate. Keywords : ushroom tyrosinase; phenyl dithiocarbamate; isothermal titration calorimetry. Introduction Tyrosinase is a bifunctional, copper - containing monooxygenase catalysing the o - hydroxylation of monophenols to the corresponding catechols and the oxidation of catechols to the corresponding o - quinones 1 . o - Quinones follow some reactions, which result in formation of biopolymers like melanin 2 . In mushrooms as well as in fruits and vegetables, this enzyme is responsible for browning, a commercially undesirable phenomenon 3 . Therefore, Tyrosinase inhibitors have attract ed interest recently due to undesired browning in vegetables and fruits in post - harvest handling. Among inhibitors, a distinction can be made between chelators of two Cu 2+ in the active site. The well - known heavy metal chelators, derivatives of dithiocarba mate have been found to possess a wide range of biological activities of Mushroom Tyrosinase. In view of the increasing importance of controlling Tyrosinase activities, we applied isothermal titration calorimetry (ITC) to obtain thermodynamic parameters fo r the interaction between Mushroom Tyrosinase and Phenyl Dithiocarbamate at two temperatures. We attempted to apply the extended solvation model to allow one to interpret the enzyme stability after binding to a ligand. Material and Methods Experimental : Mushroom t yrosinase was obtained from s igma and p henyl d ithiocarbamate was synthesized. All other materials and reagents were of analytical grade, and solutions were made in 10 mM phosphate buffer using double - distilled water. The isothermal titration cal orimetric experiments were performed with the four channel commercial calorimetric system, t hermal a ctivity m onitor 2277, t hermometric, s weden. The microcalorimeter is composed of two identical cells, reference cell and a sample cell of 1.8 mL in volume wh ich made of a highly efficient thermal conducting material surrounded by an idiabetic jacket. The sample cell was loaded with m ushroom t yrosinase solution (8.3 μM) an phosphate buffer sollution (10 mM) and the reference cell contained buffer sollution. Th e solution in the cell was stirred at 307 rpm by the syringe filled with p henyl d ithiocarbamate solution (25 mM) to ensure rapid mixing. Injection of p henyl d ithiocarbamate solution in to the perfusion vessel was repeated 20 times, with 20 μL per injection . To correct the thermal effects due to ligand dilution, control experiments were done in which identical aliquots were injected into the buffer solution with the exception of enzyme. The measured enthalpy changes associated with processes occurring at two constant temperatures of 27 0 C and 37C in kJ mol - 1 are shown in figure - 1. Results and Discussion Our efforts aimed at elucidation of whether Phenyl Dithiocarbamate induced conformational change of Mushroom Tyrosinase and how thermodinamical changes by ligand binding were occured. Obtained heats from the extended solvation model (Eq. 1) for interactions between a protein and ligand in the aqueous solvent systems, as we have shown before, are in principle compatible with ITC enthalpies 4 - 12 : Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 68 - 70 , March (201 2 ) Res.J.Chem.Sci International Science Congress Association 69 Where x' B , x' A can be defined as follows: x B is equal to the ligand concentrations divided by the maximum concentration of the ligand upon saturation of all enzyme as follows: It is worth noting that, the smallest relative standard coefficient error and the highest value of r 2 support p =1, this means that ligand binds at each site independently and the binding is non - cooperative. L A and L B are the contributions of unbound and bound ligand to the heats of dilution with the exclusion of enzyme and can be calculated from the heats of dilution of p henyl d ithiocarbamate in buffer as follows: and parameters have been optimized to fit the data and recovered from the coefficients of the second and third terms of Eq. 1, while they are indexes of m ushroom t yrosinase structural changes due to the reaction with Phenyl Dithiocarbamate in the low and high concentrations, respectively. The superscript θ in all cases refer to the quantities in the infinite dilution of the solute. It is interesting to note that the positive values of and at 27 0 C and 37C, exhibit th at p henyl d ithiocarbamate is able to stabilize the enzyme. These results of � at 27C and at 3 7 C indicate that low p henyl d ithiocarbamate concentration at 27 C and high p henyl d ithiocarbamate concentration at 37 C cause more stabilization of MT. According to f igure - 1 the calculated heats show a good agreement with the experimental data. For a set of identical and independent binding sites, we show three diff erent methods of ITC data analysis for providing the dissociation binding constant ( K d ). In the first method, using e q. 2, a plot of vs . should be a linear plot by a slope of and the vertical - intercept of , which the number of binding sites ( g ) and K d can be obtained: Our results suggest a set of two binding sites with non cooperativity. M 0 and L 0 are total concentrations of enzyme and ligand, respectively. While q represents the heat value at a certain L 0 and q max represents the heat value upon saturation of all enzyme, . The linearity of the plot has been examined by dif ferent estimated values for q max to find the best value for the correlation coefficient. If q max is calculated per mole of enzyme then the standard molar enthalpy of binding for each binding site will be . The calculated K d , g an d ∆ H are reported in t able 1. The change of the stanar Gibbs free energy of bining (∆ G ), which is shown in t able 1, is determined using K a , the association binding constant (the inverse of the K d ), In the euation ∆ G = - RT Ln K a . Where R is the gas constant and T is the absolute temperature. To compare all thermodynamic parameters, the value of ∆G° can use in e q. 5 for calculating the change in stanar entropy (∆S°) of this bining. ∆ G °=∆ H - T ∆ S (5) The binding process in b oth two temperatures is spontaneous (∆G°<0), which are only entropy riven that means the predominant interaction in the active site of the enzyme is hydrophobic. Conclusion p =1 indicates that the binding is non - cooperative in two binding sites. The posit ive values of and show that phenyl dithiocarbamate stabilizes the MT structure. The binding process for MT inhibition is only entropy driven, indicating that hydrophobic interaction is more important in the inhibition sites of MT. Acknowledgements Financial support from Islamic azad university of Takest an is gratefully acknowledged. References 1. Rescigno A., Sollai F., Pisu B., Rinaldi A. and Sanjust E. , Tyrosinase Inhibition: General and Applied Aspects., J. Enzym Inhib. Med. Chem. , 17(4) , 207 - 218 (2002) 2. Rezaei Behbehani G., Saboury A.A. and Taleshi E. , Determination of partial unfolding enthalpy for lysozyme upon interaction with dodecyltrimethylammonium bromide using an extended solvation model , J. Mol. Recogn , 21 , 132 - 135 (2008) 3. Rezaei Behbehani G., Divsalar A., Saboury A.A. and Hekmat A. , A thermodynamic study on the binding of PEG - stearic acid copolymer with lysozyme , J. Solution Chem . , 38 , 219 - 229 (2009) 4. Rezaei Behbehani G., Saboury A.A. and Yahaghi E. , A thermodynamic study of Nickel ion interaction with bovine carbonic anhydrase II molecule , J. Therm. Anal. Cal . , 100 , 283 - 288 (2010) Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 68 - 70 , March (201 2 ) Res.J.Chem.Sci International Science Congress Association 70 5. Rezaei Behbehani G., Saboury A.A., Barzegar L., Zarean O., Abedini J. and Payehghdr M. , A thermodynamic study on the interaction of nickel ion with myelin basic protein by isothermal tit ration calorimetry , J. Therm. Anal. Cal . 101 , 379 - 384 (2010) 6. Rezaei Behbehani G., Divsalar A., Saboury A.A., Faridbod F. and Ganjali M.R. , A thermodynamic study on the binding of human serum albumin with lanthanum ion , Chin. J. Chem . 28 , 159 - 163 (2010) 7. Saboury A.A. , A review on the ligand binding studies by isothermal titration calorimetry , J. Iran. Chem. 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Rezaei Behbehani G., Divsalar A., Saboury A.A., Hajian R., Rezaei Z., Yahaghi E. and Barzegar L. , A thermodynamic study on the binding of Cobalt and iron ions with bovine carbonic anhydrase II molecule at different temperatures , J Solution Chem . , 39 , 1142 - 1152 (2010) Table 1 Thermodynamic parameters for the interaction between Phenyl Dithiocarbamate and Mushroom Tyrosinase in Phenyl Dithiocarbamate solution with water Parameters T=300 K T=310 K 3.910.01 4.730.02 3.000.02 5. 310.02 2929.1154 3471.0262 g 2 2 P 10.04 10.04 12.120.06 12.520.02 - 19.900.04 - 21.000.05 0.110.01 0.100.01 Figure - 1 Comparison between the experimental heats, q, for the interaction between Phenyl Dithiocarbamate and Mushroom Tyrosinase at 27 C (  ), 37 C (O) and calculated datas (lines) at both temperatures via Eq. 1.