Concentration dependence of NaCl salting of lysozyme was investigated in the range of 0.5-9 mM lysozyme concentration in 0.1 M sodium acetate buffer, pH = 4.25and the concentration of NaCl up to 0.1 M. Calorimetric experiments were performed with the use of a titration ITC Omega MicroCal calorimeter. It was found that the estimated number of bonding sites depended on the lysozyme concentration. For infinitely diluted lysozyme solution, the number of binding sites could be roughly estimated to similar to50. In the range of 2-9 mM protein concentration, the number of weakly binding (K = 2.7 +/- 0.8 M-1) sites on the protein surface was estimated to 35 +/- 7. McMillan and Mayer's approach reduced to the third order virial coefficients demonstrates that besides the dominating effect of the protein-salt interaction (a(11)) the coefficient describing the lysozyme aggregation upon salt addition (a(12)) is statistically significant. (C) 2003 Elsevier B.V. All rights reserved.