553 J. Indian Chem. Soc., Vol. 93, May 2016, pp. 553-561 Thermodynamics of excess binding of inorganic salts and organic solutes to crab hemocyanin A. Gani a,d , R. Bhadra b , D. K. Chattoraj* a , D. C. Mukherjee c and Atanu Mitra a,e a Department of Food Technology and Biochemical Engineering, Jadavpur University, Kolkata-700 032, India b Indian Institute of Chemical Biology, Kolkata-700 032, India c Department of Chemistry, University of Calcutta, 92, Acharya Prafulla Chandra Road, Kolkata-700 009, India Manuscript received 06 January 2016, accepted 23 March 2016 Abstract : Using isopiestic vapor pressure technique, extents of water bound to complex metaloprotein hemocyanin ob- tained from crab have been determined in the absence and presence of inorganic salts, sucrose and urea at a fixed temperature. The water vapor absorption curve for hemocyanin in the range of water activity varying between zero to unity is type III BET isotherm. Moles of water absorbed per kg of hemocyanin at unit water activity a 1 have been evaluated by extrapolation method and the results support several models of bound water for different ranges of a l . The standard free energies of adsorption G 0 for water-protein interaction at different temperatures have been calculated using Bull equation in integrated form. Based on Clausius-Clapeyron equation in integrated form, the integral enthalpy for water-hemocyanin interaction has also been evaluated. Using the isopiestic technique, values of excess binding of solute  2 1 and  1 2 excess binding of solvent per kg of hemocyanin in the presence of different bulk mole-fractions X 2 of solutes (LiCl, NaCl, KCl, NaBr, NaI, KSCN, urea, and sucrose) have been calculated in each case from the evaluated values of the Gibbs surface excess. In certain ranges of solute concentration, the plot of  1 2 .X 2 vs X 1 becomes linear so that moles of water and solute bound per kg of hemocyanin respectively can be calculated. X 1 and X 2 stand for the mole-fraction of the solvent and solute in the bulk phase of the sample. Also, using integrated form of the Gibbs adsorption equation, standard free energy change ( G 0 ) for the solute-hemocyanin and the solvent-hemocyanin interactions for different systems have been computed and the values have been compared critically. Keywords : Isopiestic vapor pressure techniques, hydration of crab hemocyanin, Gibbs surface excess of solutes, solvent bound to hemocyanin. Introduction Hemocyanin is a copper-containing respiratory pro- tein of numorous mollusks and arthopods. It resembles hemerythrin in the lack of porphyrins or any other pros- thetic group. It does not occur in blood cells but is present in a soluble form in the hemolymph of the animals. It combines with oxygen to form a blue oxygenated com- pound which contains one molecule of oxygen for two copper atoms 1 . Hemocyanin of many arthropods contain 0.17–0.18% copper 2 . The valence state of copper is not yet clear whether it is in the cupric or cuprous state. It has been suggested that copper atoms are bound to sul- Present address : d Department of Chemistry, A. P. C. Roy Govt. College, Siliguri-734 010, West Bengal, India. e Department of Chemistry, Sree Chaitanya College, Habra-743 268, West Bengal, India. phur atoms of cysteine residues. The determination of molecular weight of hemocyanin, by several methods like sedimentation technique with ultracentrifuge, osmometry and the light-scattering gives values varying from 5.0 × 10 6 to 1.0 × 10 7 . Hemocyanin from the crab, limulas polyphemus, is found to consist of almost spherical par- ticle with an average diameter 200 A, in which six sub- units are joined together 3 . Structure of hemocyanin and its subunits have been discussed extensively in a recent review by Decker et al. 4 . There are several domains in this protein having helical, bundle and copper unit 4 . Recently binding of water and various types of solutes