Research Journal of Chemical Sciences ______ ______________________________ ______ ____ ISSN 2231 - 606X Vol. 2 ( 5 ), 81 - 84 , May (201 2 ) Res.J.Chem.Sci. International Science Congress Association 81 Short Communication Thermal Investigation of three n - alkyl Xanthates binding with Mushroom Tyrosinase Rezaei Behbehani G. 1 , Barzegar L. 2 , Mehreshtiagh M. 1 , Saboury A.A. 3 and Rezaei Behbehani Z. 2 1 Department of Chemistry, Imam Khomeini International University, Qazvin, IRAN 2 Department of Chemistry, Faculty of science, Islamic Azad University, Takestan branch, Takestan, IRAN 3 Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IRAN Available online at: www.isca.in (Received 6 th March 201 2 , revised 26 th March 201 2 , accepted 31 st March 201 2 ) Abstract The interaction between three iso - alkyldithiocarbonates (xanthates), as sodium salts, C 3 H 7 OCS 2 Na (I), C 4 H 9 OCS 2 Na (II), C 5 H 11 OCS 2 Na (III) and mushroom tyrosinase enzyme, MT, have been investigated by isothermal titration calorimetry to clarify thermodynamics of these bindings as well as structural changes of the enzyme due to i ts interaction with xanthates at 27 C in phosphate buffer (10 mmol.L - 1 ; pH=6.8). The extended solvation model was used to elucidate the effect of these xanthates on the stability of the enzye. The values of δ θ A an δ θ B were attributed to the type of inhi bition for I , II and III. The obtained results indicate that there are two identical and non - cooperative binding sites for three xanthates. Keywords : Mushroom tyrosinase , iso - propyl xanthate , i so - butyl xanthate , iso - pentyl xanthate . Introduction Tyrosinase is ubiquitously distributed among animals, plants and fungi and plays a pivotal role in catalysis the hydroxylation of monophenols and the oxidation of o - diphenols to o - diquinones 1 . Tyrosinase is a bifunctional, copp er - containing enzyme involved in pigment biosynthesis of various organisms such as melanin 2,3 . Besides, tyrosinases are important subjects of many ongoing researches mainly due to their key role in the enzymatic browning phenomenon which affects the qualit y of fruits, vegetables and crop products 4 . The inhibitors of this enzyme have been utilized in cosmetics, especially as depigmenting agents in the case of hyperpigmentation 5, 6 . The inhibitory effect of xanthates on mushroom tyrosinase was elucidated, whi ch is related to the chelating of the copper ions at the active site by a negative head group (S - ) of the anion xanthate. The inhibitory effects of three synthetic n - alkyl xanthates, sodium salts, with different aliphatic tails of C 3 , C 4 and C 5 were descri bed. Lineweaver - Burk plots showed different patterns of mixed for iso - propyl and competitive inhibition for iso - butyl and iso - pentyl xanthate 3, 4 . To see whether three new n - alkyl xanthates (iso - propyl, iso - butyl and iso - pentyl xanthate) induced structura l changes of tyrosinase and how thermodynamical changes by ligand binding are occurred, the analysis of isothermal titration calorimetric data by the extended solvation model is conducted. Material and Method s Mushroom tyrosinase was purchased from Sigma and iso - propyl xanthate, iso - butyl xanthate, iso - pentyl xanthate sodium salts were synthesized 4 . All other materials and reagents were of analytical grade, and solutions were made in 10 mmol.L - 1 buffer phosphate using double - disti lled water. The isothermal titration calorimetric experiments were performed with the four channel commercial calorimetric system, Thermal Activity Monitor 2277, Thermometric, Sweden. Injection of iso - propyl, iso - butyl or iso - pentyl xanthate solution was repeated 20 times by use of a Hamilton syringe into the calorimetric titration vessel, which contained 1.8 mL tyrosinase (8.3 mol.L - 1 ) and phosphate buffer solution (10 mmol.L - 1 ; Ph 6.8). E ach injection included 20 L xanthate solution. The measurements w ere performed at constant temperature of 27C. The heat of each injection was calculated by the “Theroetric Digita 3” software. Results and Discussion The interaction between a protein and ligand in the aqueous solvent system can be analyzed by the extended solvation equation 7 - 20 : x ' B is a fraction of bound ligand with the protein molecule and x ' A is the fraction of unbound ligand. Where x ' B and x ' A can be defined as follows: x B is equal to the concentration of ligand after every injection divided by the maximum concentration of ligand upon saturation of all enzyme as follows: Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 5 ), 81 - 84 , May (201 2 ) Res.J.Chem.Sci International Science Congress Association 82 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 relative contributions of unbound and bound ligand in the heats of d ilution with the exclusion of enzyme and can be calculated from the heats of dilution of ligands in buffer as follows: Recovered values of δ θ A and δ θ B from the coefficients of the second and third terms of equation 1, are indexes of MT structural changes due to the reaction with xanthates in the low and high concentrations, respectively. It is possible to attribute the approximately identical values of δ θ A and δ θ B for iso - propyl xanthate ( t able - 1) to the mixed inhibi tion, whereas the different δ θ A and δ θ B values for iso - butyl and iso - pentyl xanthate ( t able - 1) can be related to the cooperative manner of inhibition. These interpretations are in agreement with previous reports 3,4 . The negative values of δ θ A and δ θ B exhib it that iso - propyl, iso - butyl and iso - pentyl xanthates destabilize MT structure. A simple graphical method was applied for ITC data analysis in the protein - ligand interaction f or a set of identical and independent binding sites to provide the number of b inding sites (g), the dissociation binding constant (K d ) and the molar enthalpy of bining site (∆H°). using E. 5, a plot of vs. should be a linear plot by a slope of and the vertical - intercept of 11 - 13 : 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 indicates the heat value upon sat uration of all enzyme, . The linearity of the plot has been examined by different 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 change of the stanar Gibbs free energy of bining (∆ G ) is determined by using the association binding constant ( K a ), obtained from the inverse of K d value, in the Eq. 6, where R is the gas constant and T is the absolute temperature 14 : ∆ G = - R T Ln K a (6) The change in stanar entropy (∆ S ) of this binding can be calculated as Eq. 7 15 : All calculated thermodynamic parameters are reported in t able - 1. Conclusion The extended solvation theory was used to predict enzyme destabilization and binding non - cooperativity in two identical binding sites. The close agreement is found between the calculated and experimental results ( f igure - 1) and gives considerable support to the use of theory. The binding processes for three xanthates are spontaneous in the forward direction (∆ G ). Three xanthate binding processes are both enthalpy and entropy driven, with negative δ θ A and δ θ B values, indicating that three xanthates destabilize mushroom tyrosinase structure. It is possible to attribute the values of δ θ A and δ θ B to the type of inhibition. Acknowledgement Financial s upport from the Universities of Tehran and Imam Khomeini (Qazvin) are 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. 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, J. Acta Biochimica Polonica , 57(3) , 277 - 283 (2010) 3. S aboury A . A. , Enzyme Inhibition and Activation: A general theory, J. Iran. Chem. Soc. , 6(2) , 219 - 229 (2009) 4. 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 tyrosinase, J. Enzym Inhib. Med. 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Yao Dongliang, Wang Yutian and dong Yan , The Evaluation of Soil Cementation Generated from the Function of Microorganism , Res. J. Chem. Sci. , 1(1), 13 - 17 (2011) 19. Parmar Kokila, Prajapati Sarju, Patel Rinku, Patel Rekha , A Simple and Efficient Procedure for Synthesis of Biologically Active 1,2,4 - Triazolo - [3,4 - b] - 1,3,4 - thiadiazole - 2 - aryl - thiazolidine - 4 - one Derivatives , Res. J. Chem. Sci ., 1(1), 18 - 24 (2011) 20. Sonawane Vilas Y . , Mechanistic study of chromium (VI) catalyzed oxidation of benzyl alcohol by polymer supported chromic acid , Res. J. Chem. Sci., 1(1), 25 - 30 (2011) Table - 1 Binding parameters for xanthates+MT interactions 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 xanthates destabilize the MT structure. The binding process for MT inhibition is both enthalpy and entropy - driven but the electrostatic interact ions are more im portant than hydrophobic forces parameters I II III p 10.01 10.01 10.01 G 20.02 20.02 20.02 K a / M - 1 9.07 10 4 24 1.26 10 5 12 1.68 10 5 12 Δ H / kJ.mol - 1 - 18.700.06 - 19.300.07 - 1.160.03 Δ G / kJ.mol - 1 - 28.470.12 - 29.280.14 - 30.020.13 Δ S / kJ.mol - 1 .K - 1 0.030.01 0.030.01 0.100.02 - 4.990.02 - 4.470.06 - 4.230.06 - 4.230.02 - 6.580.08 - 8.660.08 Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 5 ), 81 - 84 , May (201 2 ) Res.J.Chem.Sci International Science Congress Association 84 Figure - 1 Comparison between the experimental heats, q, for the interaction between mushroom tyrosinase and iso - propyl xanthate (  ) , i so - butyl xanthate (  ) and iso - pentyl xanthate (  ) at 27 C and calculated data (lines) via equation 1