Thermodynamic Properties of Ligand Binding by Monoclonal Anti-fluorescyl Antibodies

James N. Herron, David M Kranz, David M. Jameson, Edward W. Voss

Research output: Contribution to journalArticle

Abstract

The effects of temperature on the binding of fluorescein by three monoclonal anti-fluorescvl 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 °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 (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) 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 °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 (ΔCp° ≈ -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. Sturtevant (1977) developed an analytic method for evaluating both the hydrophobic and vibrational contributions to binding. When Sturtevant's procedure was applied to anti-fluorescyl antibodies, unfavorable vibrational contributions were observed for all three antibodies. The effect 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 in the active site which reduce the size of the unfavorable vibrational contribution. The effects of pressure on the binding of fluorescein by anti-fluorescyl antibodies were assessed by measurements of affinity constants (Ka) over a pressure range of 10-3-3 kbar. Standard volume changes (ΔF°) were determined at 25 °C from ΔGu vs. pressure plots. Positive ΔK° values were observed for 4-4-20 and 20-20-3, while 20-19-1 exhibited a negative volume change. It was not clear from the pressure plots why the standard volume change of the 20-19-1 protein was opposite in sign to the other two antibodies. In keeping with our hydrophobic and vibrational analysis of thermodynamic parameters, we analyzed standard volume changes in terms of hydrophobic and vibrational components. All three antibodies exhibited positive (~90 mL mol-1) contributions from the hydrophobic effect and negative contributions (~-80 mL mol-1) from vibrational effects. This result was analogous to the relative hydrophobic and vibrational contributions observed for thermodynamic parameters and indicative of hapten-induced conformational changes in the antigen binding region.

Original languageEnglish (US)
Pages (from-to)4602-4609
Number of pages8
JournalBiochemistry
Volume25
Issue number16
DOIs
StatePublished - Aug 1986
Externally publishedYes

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Thermodynamics
Anti-Idiotypic Antibodies
Thermodynamic properties
Monoclonal Antibodies
Entropy
Ligands
Temperature
Fluorescein
Enthalpy
Antibodies
Specific heat
Pressure
Catalytic Domain
Hot Temperature
Free energy
Proteins
Haptens
Fluorescence
Antigens

ASJC Scopus subject areas

  • Biochemistry

Cite this

Thermodynamic Properties of Ligand Binding by Monoclonal Anti-fluorescyl Antibodies. / Herron, James N.; Kranz, David M; Jameson, David M.; Voss, Edward W.

In: Biochemistry, Vol. 25, No. 16, 08.1986, p. 4602-4609.

Research output: Contribution to journalArticle

Herron, James N. ; Kranz, David M ; Jameson, David M. ; Voss, Edward W. / Thermodynamic Properties of Ligand Binding by Monoclonal Anti-fluorescyl Antibodies. In: Biochemistry. 1986 ; Vol. 25, No. 16. pp. 4602-4609.
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N2 - The effects of temperature on the binding of fluorescein by three monoclonal anti-fluorescvl 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 °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 (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) 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 °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 (ΔCp° ≈ -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. Sturtevant (1977) developed an analytic method for evaluating both the hydrophobic and vibrational contributions to binding. When Sturtevant's procedure was applied to anti-fluorescyl antibodies, unfavorable vibrational contributions were observed for all three antibodies. The effect 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 in the active site which reduce the size of the unfavorable vibrational contribution. The effects of pressure on the binding of fluorescein by anti-fluorescyl antibodies were assessed by measurements of affinity constants (Ka) over a pressure range of 10-3-3 kbar. Standard volume changes (ΔF°) were determined at 25 °C from ΔGu vs. pressure plots. Positive ΔK° values were observed for 4-4-20 and 20-20-3, while 20-19-1 exhibited a negative volume change. It was not clear from the pressure plots why the standard volume change of the 20-19-1 protein was opposite in sign to the other two antibodies. In keeping with our hydrophobic and vibrational analysis of thermodynamic parameters, we analyzed standard volume changes in terms of hydrophobic and vibrational components. All three antibodies exhibited positive (~90 mL mol-1) contributions from the hydrophobic effect and negative contributions (~-80 mL mol-1) from vibrational effects. This result was analogous to the relative hydrophobic and vibrational contributions observed for thermodynamic parameters and indicative of hapten-induced conformational changes in the antigen binding region.

AB - The effects of temperature on the binding of fluorescein by three monoclonal anti-fluorescvl 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 °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 (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) 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 °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 (ΔCp° ≈ -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. Sturtevant (1977) developed an analytic method for evaluating both the hydrophobic and vibrational contributions to binding. When Sturtevant's procedure was applied to anti-fluorescyl antibodies, unfavorable vibrational contributions were observed for all three antibodies. The effect 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 in the active site which reduce the size of the unfavorable vibrational contribution. The effects of pressure on the binding of fluorescein by anti-fluorescyl antibodies were assessed by measurements of affinity constants (Ka) over a pressure range of 10-3-3 kbar. Standard volume changes (ΔF°) were determined at 25 °C from ΔGu vs. pressure plots. Positive ΔK° values were observed for 4-4-20 and 20-20-3, while 20-19-1 exhibited a negative volume change. It was not clear from the pressure plots why the standard volume change of the 20-19-1 protein was opposite in sign to the other two antibodies. In keeping with our hydrophobic and vibrational analysis of thermodynamic parameters, we analyzed standard volume changes in terms of hydrophobic and vibrational components. All three antibodies exhibited positive (~90 mL mol-1) contributions from the hydrophobic effect and negative contributions (~-80 mL mol-1) from vibrational effects. This result was analogous to the relative hydrophobic and vibrational contributions observed for thermodynamic parameters and indicative of hapten-induced conformational changes in the antigen binding region.

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