THERMODYNAMIC ANALYSIS OF ANTIGEN-ANTIBODY BINDING USING BIOSENSOR MEASUREMENTS AT DIFFERENT TEMPERATURES

The thermodynamic parameters of the interaction between hen egg white lysozyme and Fab D1,3 were determined by measuring the temperature dep endence of the ratio of its kinetic association and dissociation rate constants. Biosensor technology (BIAcore 2000) was used to measure the rate constants at temperatures ranging from 5 to 40 degrees C. The va lue of Delta G degrees at 25 degrees C (-49 kJ M(-1)) calculated by th is method was very close to that obtained previously from fluorescence quenching measurements (-48.5 kJ M(-1)). However, the value of Delta H degrees measured at 25 degrees C by biosensor technology (-35 kJ M(- 1)) was smaller than that determined previously by microcalorimetry (- 90 kJ M(-1)). Another difference was the limited variation of In K and Delta G with temperature observed with BIAcore compared to the steady decrease of In K with temperature found by calorimetry. Our data show ed that the binding reaction was driven only by enthalpy below 23 degr ees C, by enthalpy and entropy between 23 and 35 degrees C, and only b y entropy above 35 degrees C. This suggests, inter alia, that the cont ribution from the enthalpy of hydration due to the water molecules pre sent at the interface in the lysozyme-antibody complex is progressivel y eliminated as the temperature increases. Whereas calorimetric data p ertain to all the components present in the sample, including solvent molecules, BIAcore measurements monitor only the physical association and dissociation of the two macromolecular species. The difference bet ween the two sets of data may also reflect the complexity of the bindi ng mechanism between lysozyme and Fab D1.3. (C) 1997 Academic Press.