Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 2(9), 90-92, Sept. (2012) Res.J.Chem.Sci. International Science Congress Association 90 Short CommunicationCalorimetric Studies on the Interaction between two n-alkyl Xanthates and Mushroom TyrosinaseRezaei Behbehani G., Barzegar L., Mehreshtiagh M.1 and Saboury A.A.Department of Chemistry, Imam Khomeini International University, Qazvin, IRAN Department of Chemistry, Faculty of science, Islamic Azad University, Takestan branch, Takestan, IRAN Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IRAN Available online at: www.isca.in Received 6th March 2012, revised 31st March 2012, accepted 16th April 2012Abstract Thermodynamics of the interaction between two iso-alkyl dithiocarbonates (xanthates), COCSNa (I), C11OCSNa (II) with mushroom tyrosinase was investigated at 27°C, pH 6.8 and in phosphate buffer (10 mM) by isothermal titration calorimetry to clarify thermodynamics of this binding as well as structural changes of the enzyme due to its interaction with xanthates. These compounds are potent inhibitors of MT with K values of 9.07 x 10, 1.68 x 10 M-1 for I and II, respectively. The MT inhibition is related to the chelating of the copper ions at the active site by a negative head group (S-) of the anion xanthate. Different K values for MT inhibition are related to different interactions of the aliphatic chains of I and II with hydrophobic pockets in the active site of the enzyme. The obtained results indicate that there are two identical and non-cooperative binding sites for both xanthates. The extended solvation theory was used to elucidate the effect of these xanthates on the stability of enzyme. These compounds are potent inhibitors of MT with association equilibrium constant ( ) values of 9.07  10 and 1.68 × 105 L.mol-1 for I and II, respectively. Different K values for MT inhibition are related to different interactions of the aliphatic chains of I and II with hydrophobic pockets in the active site of the enzyme. It is possible to ascribe the values of A and for I and II to the type of inhibition. The obtained results indicate that there are two identical and non-cooperative binding sites for both xanthates. Keywords: Mushroom tyrosinase, iso-propyl xanthate, iso-pentyl xanthate, the extended solvation theory. Introduction Tyrosinase is the common name for enzyme that is formally termed monophenol mono oxygenase and is listed as enzyme (1.14.18.1) in the standard enzyme nomenclature. Tyrosinase is bifunctional metalloenzyme and deals with catalyzing ortho hydroxylation of monophenols to diphenols and the oxidation of o-diphenols to o-quinones, also it is a necessary enzyme for synthesizing melanin. Melanin is a pigment, which is responsible for hyper pigmentation in skin and the undesirable browning of fruits and vegetables after harvest-handling. Tyrosinase has two Cu2+, that are each coordinated by histidine residues in the active sites and these two Cu2+ are essential for the enzyme to participate in catalysis, so chelating the Cu2+ in this enzyme by different inhibitors has been embattled to reduce tyrosinase activity. Incidentally, many investigators have been paying special attention to tyrosinase inhibition because of its potential use in medicinal and cosmetic applications as well as its usefulness for agricultural purposes. Previous studies on the inhibitory effect of xanthates reported that iso-propyl xanthate and iso-pentyl xanthate show mixed and competitive inhibition, respectively3,5. Among many inhibitors of mushroom tyrosinase, we applied isothermal titration calorimetry (ITC) to obtain thermodynamic parameters of the interaction between two new n-alkyl xanthates (iso-propyl xanthate and iso-pentyl xanthate) and mushroom tyrosinase. To understand the relationship between the structure and stability of biological macromolecules, thermodynamic investigating of bindings is very useful. Material and MethodsMushroom tyrosinase was obtained from Sigma, iso-propyl xanthate and i so-pentyl xanthate sodium salts were synthesized. All other materials and reagents were of analytical grade, and solutions were made in 10 mM buffer phosphate using double-distilled water. The isothermal titration calorimetric experiments were performed with the four channel commercial calorimetric system, Thermal Activity Monitor 2277, Thermometric, Sweden. A solution of ligand (20 µL) was injected by use of a Hamilton syringe into the calorimetric titration vessel, which contained 1.8 mL tyrosinase (8.3 µmol.L-1). The heat of each injection was calculated by the “Thermometric Digitam 3” software. Results and DiscussionWe have shown previously that the heats of interactions between a protein and ligand in the aqueous solvent systems can be analyzed by the following equation6-10: Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(9), 90-92, Sept. (2012) Res.J.Chem.SciInternational Science Congress Association 91  =-(+)-(-)(+)(1) maxBBBBBBB AAAAAA qqxxLxLxLxLx¢¢¢¢¢¢Where and A can be defined as follows: '' =1-(2) Bxxxpx is equal to the ligand concentrations divided by the maximum concentration of the ligand upon saturation of all enzyme as follows: [L] =(3) [L]maxIt is worth noting that, the smallest relative standard coefficient error and the highest value of r support the extended solvation model (figure-1). p=1, this means that ligand binds at each site independently and the binding is non-cooperative. and are the relative contributions of unbound and bound ligand in the heats of dilution with the exclusion of enzyme and can be calculated from the heats of dilution of ligands in buffer as follows: dilutdilut =+()=-()(4) BBAdilutdilut BB qqLqxLqxxx¶¶¶¶Parameters have been optimized to fit the data and recovered from the coefficients of the second and third terms of equation 1 (table-1), while they are indexes of MT structural changes due to the reaction with xanthates in the low and high concentrations, respectively. The negative values of A and exhibit that iso-propyl and iso-pentyl xanthate destabilizes MT structure. From another point of view, the approximately identical values of A and for iso-propyl xanthate (=-4.99, =-4.23), can be related to the mixed inhibition, whereas the large difference between the obtainedA and values for iso-pentyl xanthate (=-4.23, =-8.66), can be related to the competitive mode of inhibition. In the competitive inhibition type, similarities between substrate and inhibitor exclude simultaneous binding of inhibitor and substrate. Double reciprocal Lineweaver-Burk plots confirm mixed and competitive inhibitions for iso-propyl and iso-pentyl xanthate, respectively3,5. For a set of identical and independent binding sites, using equation 5, a plot of  M 0 maxvs.  ()L 0 should be a linear plot by a slope of 1 g and the vertical-intercept of - d () K g , so we can obtain the number of binding sites () and 10:  M=()L-(5) 00maxqqqqggOur results suggest a set of two binding sites with non cooperativity. M and L are total concentrations of enzyme and ligand, respectively. represents the heat value at a certain Land max represents the heat value upon saturation of all enzyme, =- max qqq . The linearity of the plot has been examined by different estimated values for max to find the best value for the correlation coefficient. If max is calculated per mole of enzyme then the standard molar enthalpy of binding for each binding site will be  max =qH g . The change of the standard Gibbs free energy of binding (G°), which is shown in table-1, is determined by using , the association binding constant (the inverse of the ), in the equation 6: °= - Ln a (6) The change in standard entropy (°) of this binding can be calculated as equation 7: °- °=(7) HGAll calculated thermodynamic parameters are reported in table-1. Conclusion The extended solvation theory was used to obtain the effect of iso-propyl and iso-pentyl xanthate on the stability of MT. The agreement between the experimental heats and the calculated results via equation 1 is strong and support the extended solvation model. The results of this study also show non-cooperative binding between two identical binding sites of MT. The binding processes of both ligands are spontaneous (G°0) and both enthalpy and entropy driven. Negative and B values forthe interaction, essential for many non-specific ligand-proteininteractions (in the nonspecific interaction, ligand bindsweakly to many different groups at the protein/water interface), indicating that both xanthates destabilize mushroom tyrosinase structure. The approximately identical values of A and for iso-propyl xanthate can be related to the mixed inhibition, whereas the large difference between the obtainedA and values for iso-pentyl xanthate can be related to the competitive mode of inhibition. AcknowledgementFinancial support from the Universities of Tehran and Imam Khomeini (Qazvin) are gratefully acknowledged. References1.Sánchez-Ferrer A., Rodríguez-Lopez J.N., Garcia-Canovas F. and Garcia-Carmona F., Tyrosinase: a comprehensive review of its mechanism, J. Biochim Biophys Acta., 1247(1), 1-11 (1995) 2.Korner A.M., and Pawelek J.M., Mammalian tyrosinase catalyzes three reactions in the biosynthesis of melanin, J. Science, 217, 1163–1165 (1982) Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(9), 90-92, Sept. (2012) Res.J.Chem.SciInternational Science Congress Association 92 3.Saboury A.A., Enzyme Inhibition and Activation: A general theory, J. Iran. Chem. Soc., 6(2), 219-229 (2009)4.Seo S., Sharma V.K. and Sharma N., Mushroom Tyrosinase: Recent Prospects, J. Agric. Food Chem., 51, 2837-2853 (2007)5.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. Chem., 22(2), 239-246 (2007) 6.Rezaei Behbehani G., Divsalar A., Saboury A.A. and Gheibi N., A new approach for Thermodynamic Study on binding some metal ions with human growth hormone, J. Solution Chem., 37(12), 1645-1655 (2008)7.Rezaei Behbehani G., Divsalar A., Saboury A.A. and Bagheri M.J., A Thermodynamic Study on the Binding of human Serum Albumin with New synthesized Anti cancer Pd (II) complex, J. Solution Chem., 37(12), 1785-1794 (2008)8.Rezaei Behbehani G., Saboury A.A., Barzegar L., Zarean O., Abedini J. and Payehghdr M., A thermodynamic study on the interaction of nickel with myelin basic protein by isothermal titration calorimetry, J. Therm. Anal. Cal., 101(1), 379-384 (2010)9.Rezaei Behbehani G. and Mirzaie M., A high performance method for Thermodynamic Study on the Binding of Copper Ion and Glycine with Alzheimer's amyliod peptide, J. Therm. Anal. Cal., 96(2), 631-635 (2009)10.Saboury A.A., Atri M.S., Sanati M.H. and Sadeghi M., Application of a simple calorimetric data analysis on the binding study of calcium ions by human growth hormone, J. Therm. Anal. Cal., 83(1)175-179 (2006)Table-1 Binding parameters for xanthates+MT interactions recovered from Eqs. 1, 5, 6 and 7. =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 parameters I II p 1±0.01 1±0.01 g 2±0.02 2±0.02 K a / M - 1 9.07 × 10 4 ±241.68 × 10 5 ±12 °/ kJ.mol - 1 -18.70±0.06 -1.16±0.03 °/ kJ.mol - 1 -28.47±0.12 -30.02±0.13 °/ kJ.mol - 1 .K - 1 0.03±0.01 0.10±0.02 -4.99±0.02 -4.23±0.06 -4.23±0.02 -8.66±0.08 Figure-1 Comparison between the experimental heats, q, for the interaction between mushroom tyrosinase and iso-propyl xanthate ¡¡), iso-pentyl xanthate () at 27°C and calculated data (lines) via equation [L]/ mol.L-1 050100150200250300350400 / kJ.mol-1 -45-40-35-30-25-20-15