ENTHALPY OF ANTIBODY - CYTOCHROME-C BINDING

High-sensitivity titration calorimetry is used to measure changes in e nthalpy, heat capacity, and protonation for binding of two monoclonal antibodies (MAbs) to topologically distinct surfaces of cytochrome c. MAb 2B5 binds near the exposed heme crevice in a reaction involving pr oton uptake, while there is no change in protonation for MAb 5F8 bindi ng to the opposite side of the molecule. Both antibodies have associat ion rate constants with the activation enthalpy and viscosity dependen ce expected of diffusion-limited reactions [Raman et al. (1992) Bioche mistry 31, 10370-10379], and bind with high affinity (Delta G(b)(o) = -12.6 kcal mol(-1) for MAb 2B5 and -13.9 kcal mol(-1) for MAb 5F8, at pH 7, 25 degrees C). At 25 degrees C, the equilibrium enthalpy and ent ropy contributions to the free energy of binding are negative for both antibodies (Delta H-b(o) = -21.0 kcal mol(-1), Delta S-b(o) = -28.2 c al mol(-1) K-1 for MAb 2B5; and Delta H-b(o) = -21.7 kcal mol(-1), Del ta S-b(o) = -26.3 cal mol(-1) K-1 for MAb 5F8). The enthalpy of MAb 2B 5-cytochrome c association exhibits a marked temperature dependence (D elta C-p = -580 cal mol(-1) K-1), but the enthalpy for MAb 5F8 binding is much less dependent on temperature (Delta C-p = -172 cal mol(-1) K -1). The large differences in Delta C-p for binding of the two antibod ies suggest corresponding differences in the mode of binding, or in th e molecular surfaces buried in the binding reactions. In particular, f actors other than hydrophobic effects may be significant contributors to the thermodynamics of antibody-cytochrome c binding, especially whe n Delta C-p is small (MAb 5F8).