ITC measurements involved use of a MicroCal iTC200 microcalorimeter (GE Healthcare) at 25uC. In individual titration, 1-2 ml carbohydrate solution was added at 180-s intervals by use of a computer-controlled 40 ml syringe to a cell containing 280 ml IPO protein solution under constant stirring at 1,000 rpm. The concentration of IPO protein was 1-3 mM and that of Me-Man, Me-Glc, Me-Gal, Man, Glc and Gal 25-75 mM. The titration of carbohydrate solution in this range of concentration to the dialysis buffer was used as a control. Measurements of the heat change determined from the binding constant (K A ), reaction stoichiometry (n), and enthalpy (DH). The 18 experimental data were fitted for a 1:1 binding model (one-site of fitting) with Microcal Origin 7.0 software. Free energy (DG) and binding entropy (DS) were calculated by the equations DG = -RTlnK A and DG = DH -TDS. R is the gas constant and T the absolute temperature. The optimal c-value in ITC calculation varied between 1 and 10. However, for titrations with Man, Glc and Gal, the c-values were ,1.