Research Journal of Chemical Sciences ______ ______________________________ ______ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 71 - 73 , March (201 2 ) Res.J.Chem.Sci. International Science Congress Association 71 Short Communication Research on Thermodynamic aspect of the Binding of p - Phenylene - bis dithiocarbamate to Mushroom Tyrosinase Rezaei Behbehani G . 1 , Bazegar L. 2 , Mehreshtiagh M . 1 and Mohebian M. 1 1 Chemistry Department, Imam Khomeini International University, Qazvin, IRAN 2 Department of Chemistry, Faculty of Science, Islamic Azad University, Takestan Branch, Takestan, IRAN Available online at: www.isca.in (Received 12 th January 2012 , revised 30 th January 2012 , accepted 31 st January 2012 ) Abstract The binding properties and structural changes of mushroom tyrosinase enzyme, MT, due to its interaction with p - phenylene - bis dithiocarbamate (I) was investigated at 27 and 37°C in phosphate buffer (10 mmol.L - 1 ) at pH 6.8 by isothermal titration calorimetric (ITC). The extended solvation model was used to calculate the solvation parameters, which were attributed to the stability of enzyme. Ther modynamic analysis indicated that the binding of I to MT essentially depends on electrostatic interactions. It was concluded that MT has two distinct sites for p - phenylene - bis and phenyl dithiocarbamate. Keywords: Mushroom tyrosinase; p - phenylene - bis dith iocarbamate; isothermal titration calorimetry. Introduction Tyrosinase is a copper monooxygenase, which catalyze the oxidation of mono - and o - diphenols to o - diquinones 1 . Tyrosinase plays an essential role in melanin production and responsible for the formation of pigment in the skin, hair and eye 2, 3 . The accumulation of an abnormal melanin amount in different specific parts of the skin, resulting in more pigmented patches, is an es thetic problem 4 . Tyrosinase inhibitors have attracted concern recently due to undesired browning in vegetables and fruits in post - harvest handling 3 . The di - copper centre of this enzyme has been the target of many inhibitors. Dithiocarbamate compounds act a s inhibitors of mushroom tyrosinase due to their ability to chelate copper ion 5 . Certain dithiocarbamate derivatives have been found to possess a wide range of biological activities, i.e, anti - bacterial, tuberculostatic, anti - diuretic and anti - hypertensive . n - Butyl dithiocarbamate, n - hexyl dithiocarbamate and n - octyl dithiocarbamate, as sodium salts, show inhibitory effect on mushroom tyrosinase 6 . Among various dithiocarbamates, we tried to elucidate the effect of p - phenylene - bis dithiocarbamate on mushroom tyrosinase stability at 27 and 37 °C applying the extended solvation model for the data analysis. Material and Methods Experimental : Mushroom tyrosinase was obtained from Sigma, p - phenylene - bis dithiocarbamate was synthesized. All other materials and re agents were of analytical grade, and solutions were made in 10 mmol.L - 1 phosphate buffer using double - distilled water. The isothermal titration calorimetric experiments were performed with the four channel commercial microcalorimetric system, Thermal Activ ity Monitor 2277, Thermometric, Sweden. The microcalorimeter is composed of two identical cell made of a highly efficient thermal conducting material surrounded by an adiabetic jacket. The sample cell containeļ¤ 1.8 mL MT (8.3 Ī¼mol.L - 1 ) and phosphate buffer solution (10 mmol.L - 1 ; pH 6.8) and the reference cell filled with phosphate buffer. The titration of MT with p - phenylene - bis dithiocarbamate involved 20 consecutive injections of the liganļ¤ anļ¤ each injection incluļ¤eļ¤ 20 Ī¼L p - phenylene - bis dithiocarbamate (2.5 mmol.L - 1 ). The heat of injection was calculateļ¤ by the ā€˜Thermometric ļ„igitam 3ā€™ software program. The measurements were performed at constant temperatures of 27 and 37°C and the temperature was controlled using a poly - science water bath. The microcal orimeter was frequently calibrated electrically during the course of the study. Results and Discussion The extended solvation model , the applicable, in principle, to a variety of protein + ligand systems, was used to analyize the heats of interaction of MT+II and MT+I mixtures as follows 7 - 10 x ' B can be expressed as follows: x ' B is a fraction of bound ligand with the protein molecule and x ' A =1 - x ' B is the fraction of unbound ligand. Where x B can be defined as follows: [ L ] is the concentration of p - phenylene - bis dithiocarbamate after every injection and [ L ] max is the maximum concentration of p - phenylene - bis dithiocarbamate upon saturation of all MT. In the fitting procedure, p was changed until the best agreement between the experimental and calculated data was approached Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 71 - 73 , March (201 2 ) Res.J.Chem.Sci International Science Congress Association 72 (figure - 1). If the binding of ligand at one site increases the affinity for ligand at another site, the macromolecule exhibits positive cooperativ ity ( p 1). Conversely, if the binding of ligand at one site lowers the affinity for ligand at another site, the enzyme exhibits negative cooperativity ( p ). If the ligand binds at each site independently, the binding is non - cooperative ( p =1). L A and L B are the relative unbound and bound I contributions to the heats of dilution in the absence of MT. Stability of the MT is discussed on the basis of the obtained results for and from the coefficients of the second and third terms of e q. 1. and values reflect to the MT structural changes due to its interaction with p - phenylene - bis dithiocarbamate in the low and high concentrations, respectively. The negative values of and indicate that MT is destabilized by p - phenylene - bis dithiocarbamate. Eq. 5 was used for isothermal titration calorimetric data anal ysis to obtain the number of binding sites ( g ) and the dissociation binding constant ( K d ) from the slope and the vertical - intercept of of the linear plot of vs. . Where āˆ† q = q max - q and q represents the heat value at a certain ligand and biomolecule concentration. M 0 and L 0 are total concentrations of enzyme and ligand, respectively. q max represents the heat value upon saturation of all MT and the molar enthalpy of binļ¤ing for each binļ¤ing site (āˆ† H °) will be . The linearity of the plot has been examined by different estimated values for q max to find the best value for the correlation coefficient (near to one). The standard Gibbs free energy, āˆ† G °, can be calculated from association constant as follows: āˆ† G = - R T ln K a (6) The negative values of āˆ† G ° suggest that the binding process of MT to I proceeds spontaneously in the forward direction. āˆ† S ° is ļ¤irectly calculateļ¤ from āˆ† G Ā° anļ¤ āˆ† H ° according to e q.7: All thermodynamic parameters of I binding to MT are summarized in t able - 1. The decreasing K a values (Table - 1) with increasing temperature ( t able - 1) indicates that the binding of I is an enthalpically driven process and consequently the electrostatic forces are dominant. Conclusion p =1 indicates that the binding is non - cooperative in two binding s ites. The negative values of and show that p - phenylene - bis dithiocarbamate destabilizes the MT structure. The binding process for MT inhibition is only enthalpy driven, indicating that electrostatic interactio n is more important in the inhibition sites of MT. Acknowledgement Financial support from Islamic azad university of Takestan 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. Alijanianzadeh M., Saboury A.A., Mansouri - Torshizi H., Haghbeen K., and Moosavi - Movahedi A.A. The inhibitory effect of some new synthesized xanthates on mushroom t yrosinase, J. Enzym Inhib. Med. Chem. , 22(2) , 239 - 246 (2007) 3. Saboury A.A., Enzyme Inhibition and Activation: A general theory, J. Iran. Chem. Soc. , 6(2) , 219 - 229 (2009) 4. Amin E., Saboury A.A., Mansouri - Torshizi H., Zolghadri S., and Bordbar A - Kh . Evaluation of p - phenylene - bis and phenyl dithiocarbamate sodium salts as inhibitors of mushroom tyrosinase. Acta Biochimica Polonica , 57(3) , 277 - 283 (2010) 5. Amin E., Saboury A.A., Mansouri - Torshizi H. and Moosavi - Movahedi. Potent inhibitory effects of be nzyl and p - xylidine - bis dithiocarbamate sodium salts on activities of mushroom tyrosinase, J. Enzym Inhib. Med. Chem. , 25(2) , 272 - 281 (2010) 6. Gheibi N., Saboury A.A. , Mansuri - Torshizi H ., Haghbeen K ., and Moosavi - Movahedi A.A ., The inhibition effect of some n - alkyl d ithiocarbamates on mushroom tyrosinase, J. Enzyme Inhib Med Chem. , 20(4), 393 - 399 (2005) 7. Rezaei Behbehani G., Saboury A.A., Mohebbian M., Tahmasebi S., and Poorheravi M., A Structural and Calorimetric Study on the Interaction Between Jack Bean Urease and Cyanide Ion., J. Solution Chem . , 38(12) , 1612 ā€“ 1621 (2009) 8. Rezaei Behbehani G., Saboury A.A., Mohebbian M., Abed ini J. and Tahmasebi Sarvestani S. Thermodynamic study on the interaction of cyanide ion and jack bean urease at different temperatures, J. Solution Chem. , 100(3) , 1079 ā€“ 1083 (2010) 9. Rezaei Behbehani G., and Barzegar L. Thermal study of lysozyme binļ¤ing with Ī² - cyclodextrin. Applied Mechanics and Materials, 110 , 1966 - 1969 (2012) 10. Mirzaie M., and Rezaei Behbehani G. Thermal Study of the nickel ion Interaction with Myelin Basic Protein. Applied Mechanics and Materials , 110 , 1963 - 19665 (2012) Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 71 - 73 , March (201 2 ) Res.J.Chem.Sci International Science Congress Association 73 Table - 1 Binding parameters for p - phenylene - bis dithiocarbamate +MT interaction recovered from Eqs. 1, 5, 6 and 7. p =1 indicates that the binding is non - cooperative in two binding sites. The negative values of and show that p - phenylene - bis dithiocarbamate destabilizes the MT structure at 27 and 37 °C. The binding process for MT inhibition is only enthalpy driven, indicating that electrostatic interaction is more important in the inhibition sit e. parameters T=27 °C T=37 °C p 1 1 g 2±0.01 2±0.01 K a / L.mol - 1 ±56 ±34 Ī” H / kJ mol - 1 - 39.2 - 39.8 Ī” G / kJ mol - 1 - 31.7 - 31.5 Ī” S / kJ mol - 1 K - 1 - 0.025 - 0.027 - 7.4 - 10.6 - 16.7 - 17.6 Figure - 1 Comparison between the experimental heats, q, for the interaction between p - phenylene - bis dithiocarbamate and Mushroom Tyrosinase at 27 °C (  ), 37 °C (O) and calculated datas (lines) at both temperatures via e q. 1