Thermodynamic Properties of Antifluorescyl Antibodies
The thermodynamic properties of three monoclonal antifluorescyl antibodies (4-4-20,20-19-1,20-20-3) were determined over a temperature range of 2°-70°C., using fluorescence methodology. Curvilinear van’t Hoff plots (In Ka vs. T-1 were observed for all three antibodies indicating that their standard enthalpy changes (ΔH°) were temperature dependent. This phenomenon was further investigated by plotting the changes in unitary free energy (ΔGu), standard enthalpy (ΔH°), and unitary entropy (ΔSu) versus temperature. At low temperatures (4°C), entropy played a major role in the binding of fluorescein by all three antibodies, while enthalpy dominated at higher temperatures. Negative heat capacity values (ΔCp°) were observed for all three antibodies, which indicated that the hydrophobic effect was instrumental in the binding of fluorescein. However, ΔSu values were lower than expected for hydrophobic binding alone, suggesting that other forces were acting to mitigate the hydrophobic effect. One possibility was that the binding of fluorescein acted to restrain vibrational fluctuations in the active site region, producing negative changes in both heat capacity and entropy. This hypothesis was confirmed by determining the hydrophobic and vibrational contributions to binding, using the method of Sturtevant (1977). The phenomenon was most pronounced with the 20-20-3 protein, and the least with the 4-4-20 protein. Considering that 4-4-20 exhibited the highest affinity of the three proteins, it is possible that one mechanism of generating high affinity active sites may involve structural changes which reduce the size of the unfavorable vibrational condition.