Thermodynamic properties of ligand binding by monoclonal anti-fluorescyl antibodies.
The effects of temperature on the binding of fluorescein by three monoclonal anti-fluorescyl antibodies (4-4-20, 20-19-1, and 20-20-3) were assessed by measurements of affinity constants (Ka) over a temperature range of 2-70 degrees C. Values for Ka were determined from the degree of ligand association by using fluorescence methodology. Curvilinear van't Hoff plots (ln Ka vs. T-1) were observed for all three antibodies, indicating that their standard enthalpy changes (delta Ho) were temperature dependent. This phenomenon was further investigated by plotting the changes in unitary free energy (delta Gu), standard enthalpy (delta Ho), and unitary entropy (delta Su) vs. temperature. Strong temperature dependencies were observed for enthalpy and entropy values, while free energy plots were only weakly dependent on temperature. At low temperatures (4 degrees C), entropy played a major role in the binding of fluorescein by all three antibodies, while enthalpy dominated at higher temperatures. This was a consequence of the negative heat capacity changes (delta Cpo approximately equal to -320 cal K-1 mol-1) observed for these antibodies, which produced a negative trend in both enthalpy and entropy values with increasing temperature. The negative heat capacity values also indicated that the hydrophobic effect was instrumental in the binding of fluorescein. Entropy changes 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000911:Antibodies, Monoclonal; D019793:Fluorescein; D005452:Fluoresceins; D006825:Hybridomas / Q000276:immunology; D008024:Ligands; D008433:Mathematics; D051379:Mice; D011485:Protein Binding; D011487:Protein Conformation; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D008024:Ligands; D019793:Fluorescein