CONFIGURATIONAL EFFECTS IN ANTIBODY-ANTIGEN INTERACTIONS STUDIED BY MICROCALORIMETRY

In this paper we study the binding of two monoclonal antibodies, E3 an d E8, to cytochrome c using high-sensitivity isothermal titration calo rimetry. We combine the calorimetric results with empirical calculatio ns which relate changes in heat capacity to changes in entropy which a rise from the hydrophobic effect. The change in heat capacity for bind ing E3 is -350 +/- 60 cal K-1 mol-l while for E8 it is -165 +/- 40 cal K-1 mol(-1). This result indicates that the hydrophobic effect makes a much larger contribution for E3 than for E8. Since the total entropy change at 25 degrees C is very similar for both antibodies, it follow s that the configurational entropy cost for binding E3 is much larger than for binding E8 (-77 +/- 15 vs, -34 +/- 11 cal K-1 mol(-1)). These results illustrate a case of entropy compensation in which the cost o f restricting conformational degrees of freedom is to a large extent c ompensated by solvent release. We also show that the thermodynamic dat a can be used to make estimates of the surface area changes that occur upon binding. The results of the present study are consistent with pr evious hydrogen-deuterium exchange data, detected using 2D NMR, on the two antibody-antigen interactions. The NMR study indicated that prote ction from exchange is limited to the binding epitope for E8, but exte nds beyond the epitope for E3. These results were interpreted as sugge sting that a larger surface area was buried on cytochrome c upon bindi ng to E3 than to E8, and that larger changes in configurational entrop y occur upon binding of E3 than E8. These findings are confirmed by th e present study using isothermal titration calorimetry. (C) 1995 Wiley -Liss, Inc.