The interaction of human serum albumin (HAS) with divalent nickel ion was studied by isothermal titration calorimetry (ITC) in 30 mM Tris buffer, pH 7.0. There is a set of eight identical and independent binding sites for nickel ions on the protein at the temperature of 300 K. A new calorimetric data analysis allows the determination of the complete set of thermodynamic parameters. The binding isotherm for nickel-HSA interaction is easily obtained by carrying out two different ITC experiments. In the first experiment, the enthalpy of binding for one mole of nickel ion to one mole of binding site on HSA (DeltaH = -36.5 kJ) is obtained, and is used in a second experiment to determine the binding isotherm and to find the number of binding sites (g = 8) and the equilibrium constant (K = 0.57 muM(-1)). (C) 2003 Elsevier Ltd. All rights reserved.