Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 2(6), 88-90, June (2012) Res.J.Chem.Sci. International Science Congress Association 88 Short Communication Thermodynamic study on the interaction of Co2+ with Jack Bean Urease Rezaei Behbehani G.., Barzegar L., Mohebian M., Mirzaie M. and Taherkhani, A.Chemistry Department, Islamic Azad University, Takestan branch, Takestan, IRANAvailable online at: www.isca.in (Received 6th March 2012, revised 12th April 2012, accepted 16th April 2012)Abstract The interaction of Jack Bean Urease(IBU) with cobalt (II) ion was studied by isothermal titration calorimetry (ITC) at 300 K and 310 K in 30 mM Tris buffer, pH=7. The stability of the enzyme increases due to its binding with cobalt ions . The extended solvation model was used to reproduce the heats of Co2++JBU interaction. It was found that there is a set of 12 equivalent and non-interacting binding sites for Co2+ ions. The association equilibrium constant and the molar enthalpy of binding are 4260.76 -1 -16.5 kJmol-1 at 300 K and 3438 -1, -16 kJmol-1 at 310 K , respectively. Keywords: Isothermal titration calorimetry, jack bean urease cobalt ion Introduction Urease is found in bacteria, fungi and plants, and catalyzes the hydrolysis of urea yielding ammonia and carbamate as shown in equation 1 NHCONH + HO NHCOOH + NH3 (1) NHCOOH + HO NH3 + HCO3 The carbamate product is unstable and spontaneously degrades to ammonia and carbonic acid1, 2. There are some reports on the binding properties and structural changes of JBU due to its interaction with metal ions. Jack bean urease has many SH groups at its surface and this enzyme can be immobilized directly to the metal surface by adsorption3, 4. The interaction of JBU with some of divalent metal ions (Cu2+ and Cd2+) in aqueous solution was studied using different techniques. Cd2+ addition did not affect jack bean urease growth in plant5-7. The heavy metal ions were found to inhibit urease in the following decreasing order: Hg2+ � Cu2+ � Zn2+ � Cd2+ � Ni2+ � Pb2+ � Co2+ � Fe3+ � As3+ 8. In this paper, the interaction between Co2+ and JBU has been investigated in neutral tris buffer to clarify thermodynamics of metal binding properties. The binding parameters recovered from the extended solvation model were correlated to the effect of metals on the stability of protein5-9 Material and Methods Jack bean urease (JBU; MW=545.34 kDa) and Cobalt nitrate were obtained from Merck. The buffer solution used in the experiments was 30 mM Tris, pH=7.0, which was obtained from Merck. Experiments were carried out in 300 K and 310K. The isothermal titration microcalorimetric experiments were performed with the four channel commercial microcalorimetric system, Thermal Activity Monitor 2277, Thermometric, Sweden . The titration vessel was made from stainless steel. Cobalt solution (10 mM ) was injected by use of a Hamilton syringe into the calorimetric titration vessel, which contained 1.8 mL JBU (4 µM). Thin (0.15 mm inner diameter) stainless steel hypodermic needles, permanently fixed to the syringe, reached directly into the calorimetric vessel. Injection of cobalt solution into the perfusion vessel was repeated 30 times, with 20 µL per injection. The calorimetric signal was measured by a digital voltmeter that was part of a computerized recording system. The heat of each injection was calculated by the ‘‘Thermometric Digitam 3’’ software program. The heat of dilution of the Co2+ solution was measured as described above except JBU was excluded. Results and Discussion We have shown previously 4-10 that the enthalpies of the ligand+ JBU interactions in the aqueous solvent systems, can be calculated via the following equation: )(max (2) is the heat of Co2++ JBU interactions and max represents the heat value upon saturation of all JBU. The parameters and are the indexes of JBU stability in the low and high Co2+concentrations respectively. If the ligand binds at each site independently, the binding is non-cooperative. 1 or &#x-3.3;女1 indicate negative or positive cooperativity of macromolecule for binding with ligand respectively; = 1 indicates that the binding is non-cooperative. ˘ can be expressed as follows: pxpx (3) Bx ˘ is the fraction of bound Co2+ to the binding sites on JBU, and ˘ -= ˘ is the fraction of unbound Co2+. The model is a simple mass action treatment, with Co2+ molecules replacing Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(6), 88-90, June (2012) Res.J.Chem.SciInternational Science Congress Association 89 water molecules, at the binding sites. We can express fractions, as the total Co2+ concentrations divided by the maximum concentration of the Co2+ upon saturation of all JBU as follows: CoCoo][max (4) [Co2+] is the concentration of metal ions and [Co2+max is the maximum concentration of the Co2+ upon saturation of all JBU. In general, there will be "g" sites for binding of Co2+ per JBU molecule. L and L are the relative contributions due to the fractions of unbounded and bounded metal ions in the heats of dilution in the absence of JBU and can be calculated from the heats of dilution of Co2+ in buffer, qdilut, as follows: dilutdilut , dilutdilut (5) The heats of Co2++JBU interactions, , were fitted to equation 2 across the whole Co2+concenterations. In the fitting procedure the only adjustable parameter () was changed until the best agreement between the experimental and calculated data was approached. The optimized and values are recovered from the coefficients of the second and third terms of equation 2. The agreement between the calculated and experimental results (figure 1) is striking, and gives considerable support to the use of Eq. 2. value for Co2++JBU interactions is negetive, indicating that in the low concentration of the metal ions the JBU structure is destabilized. Destabilization by a ligand indicates that theligand binds preferentially to the unfolded (denatured) enzyme or to a partiallyunfolded intermediate form of the enzyme. Such effects are characteristicof nonspecific interactions, in that the nonspecific ligand bindsweakly to partiallyunfolded species of JBU. The negative values indicate that the nonspecific interactions are dominant in the low Co2+ ion concentration domain. The positive values for show that the JBU structure is stabilized by the addition of Co2+, indicate that JBU involvesspecific interactions with Co2+ions in the high Co2+ ion concentration region . values are one (table-1), indicating that there are a set of 12 identical and non-interacting binding sites for JBU + Co2+ interaction. According to the recently data analysis method, using equation 6, a plot of max D versus 0)(Lqq D should be a linear plot by a slope of 1/g and the vertical-intercept of , which g and can be obtained. max (6) Where is the number of binding sites, is the dissociation equilibrium constant, and are total concentrations of biomacromolecule and ligand, respectively, - = D max, represents the heat value at a certain and maxrepresents the heat value upon saturation of all biomacromolecule. If q and max are calculated per mole of biomacromolecule then the molar enthalpy of binding for each binding site (H) will be H= qmax/g. The linearly of the plot has been examined by different estimated values for maxto find the best value for the correlation coefficient (near to one). The best linear plot with the correlation coefficient value of 0.999 was obtained using amount of -1425.6µJ (equal to -198 kJmol-1 ) for max at 300 K and -1382.4µJ (equal to -192 kJmol-1) for qmax at 310 K. The amounts of g and obtained from the slope and vertical-intercept plot, are 12 and 234.78 µM, 290.84 µM at 300 and 310 K, respectively. Dividing the maxamounts of -198 kJmol-1 , -192 kJmol-1 by g=12, therefore, gives H= -16.5 kJmol-1 at 300 K and H= -16 kJmol-1 at 310 K. Binding parameters have been listed in table-1. Conclusion The agreement between the calculated and experimental results (figure-1) is striking, and gives considerable support to the use of equation 2. value for Co2++JBU interactions is negetive, indicating that in the low concentration of the metal ions the JBU structure is destabilized. The positive values for show that the JBU structure is stabilized by the addition of Co2+. AcknowledgementsFinancial support of Islamic Azad University of Takestan is gratefully acknowledged References1.Rescigno A., Sollai F., Pisu B., Rinaldi A. and Sanjust E. Tyrosinase inhibition: general and applied aspects. J.Enzym Inhib. Med. Chem.,17, 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, 277-283 (2010)3.Rezaei Behbehani G., Saboury A A., Taherkhani A., Barzegar L. and Mollaagazade A., A thermodynamic study on the binding of mercury and silver ions to urease, J. Therm. Anal. Cal., 105, 1081–1086 (2011) 4.Rezaei Behbahani G., Saboury A. A., Divsalar A., Faridbod F. and Ganjali M.R., A Thermodynamic Study on the Binding of Human Serum Albumin with Lanthanum Ion, Chinese Journal of Chemistry, 28, 159-163 (2009) Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(6), 88-90, June (2012) Res.J.Chem.SciInternational Science Congress Association 90 5.Rezaei Behbahani G., Saboury A. A., Barzegar L., and Yousefi O., A Thermodynamic investigation of Aspirin interaction with Human Serum Albumin at 298 and 310 K, Journal of Thermodynamics and Catalysis, , 1-4 (2011) 6.Rezaei Behbehani G., Saboury A. A., Taherkhani A., Barzegar L. and Mollaagazade A., A Thermodynamic Study on the binding of Mercury and silver Ions to urease,J. Therm. Anal. Cal., 105, 1081-1086 (2011) 7.Rezaei Behbahani G., Taherkhani A., Barzegar L., Saboury A. A., and Divsalar A., Refolding of lysozyme upon interaction with -cyclodextrin, Journal of Sciences, Islamic Republic of Iran, 22, 117-120 (2011)8.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 titration calorimerty. J. Therm. Anal. Cal., 101, 379-384 (2010)9.Rezaei Behbehani G., and Barzegar L., Thermal study of lysozyme binding with -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) Table-1 Binding parameters for JBU+ Co+2 interaction in 10 mM [Co (No solution. =1 suggests that Co2+ ion binds to JBU non-cooperatively Parameters JBU +Co 2+ (T=300 K) JBU +Co 2+ (T=310 K) qdA -0.051±0.010 -0.108±0.017 qdB 1.674±0.014 1.431±0.012 1/-MKa 4259.30±50 3438.32±42 g 12 12 P 1±0.04 1±0.04 kJmol -16.5 -16 kJmol-20.08 -20.98 kJmol0.02 0.016 Figure-1The heats of Co2+ ions binding with JBU at 300K() and 310K() for 30 automatic cumulative injections, each of 20 L, 10 mM of the cations solutions, into sample cell containing 1.8 ml of 4M JBU solution vs. total concentration of Co2+ ions [ Co2+] / mM 0123 q / µJ -1400-1200-1000-800-600-400-200