Enthalpy of antibody--cytochrome c binding.
High-sensitivity titration calorimetry is used to measure changes in enthalpy, 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 proton uptake, while there is no change in protonation for MAb 5F8 binding to the opposite side of the molecule. Both antibodies have association rate constants with the activation enthalpy and viscosity dependence expected of diffusion-limited reactions [Raman et al. (1992) Biochemistry 31, 10370-10379], and bind with high affinity (delta Gzerob = -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 entropy contributions to the free energy of binding are negative for both antibodies (delta Hzerob = -21.0 kcal mol-1, delta Szerob = -28.2 cal mol-1 K-1 for MAb 2B5; and delta Hzerob = -21.7 kcal mol-1, delta Szerob = 26.3 cal mol-1 K-1 for MAb 5F8). The enthalpy of MAb 2B5-cytochrome c association exhibits a marked temperature dependence (delta Cp = -580 cal mol-1 K-1), but the enthalpy for MAb 5F8 binding is much less dependent on temperature (delta Cp = -172 cal mol-1 K-1). The large differences in delta Cp for binding of the two antibodies suggest corresponding differences in the mode of binding, or in the molecular surfaces buried in the binding reactions. In particular, factors other than hydrophobic effects may be significant contributors to the thermodynamics of antibody-cytochrome c binding, especially when delta Cp is small (MAb 5F8).
Raman
C S
CS
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Allen
M J
MJ
Nall
B T
BT
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000941:Antigens / Q000378:metabolism; D001666:Binding Sites, Antibody; D002151:Calorimetry; D003574:Cytochrome c Group / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D008958:Models, Molecular; D011522:Protons; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D000941:Antigens; D003574:Cytochrome c Group; D011522:Protons; D006418:Heme