Inverse Temperature Dependence in Static Quenching versus Calorimetric Exploration: Binding Interaction of Chloramphenicol to β‑Lactoglobulin

Abstract

The binding interaction between the whey protein bovine β-lactoglobulin (βLG) with the well-known antibiotic chloramphenicol (Clp) is explored by monitoring the intrinsic fluorescence of βLG. Steady-state and time-resolved fluorescence spectral data reveal that quenching of βLG fluorescence proceeds through ground state complex formation, i.e., static quenching mechanism. However, the drug–protein binding constant is found to vary proportionately with temperature. This anomalous result is explained on the basis of the Arrhenius theory which states that the rate constant varies proportionally with temperature. Thermodynamic parameters like Δ<i>H</i>, Δ<i>S</i>, Δ<i>G</i>, and the stoichiometry for the binding interaction have been estimated by isothermal titration calorimetric (ITC) study. Thermodynamic data show that the binding phenomenon is mainly an entropy driven process suggesting the major role of hydrophobic interaction forces in the Clp−βLG binding. Constant pressure heat capacity change (Δ<i>C</i>_p) has been calculated from enthalpy of binding at different temperatures which reveals that hydrophobic interaction is the major operating force. The inverse temperature dependence in static quenching is however resolved from ITC data which show that the binding constant regularly decreases with increase in temperature. The modification of native protein conformation due to binding of drug has been monitored by circular dichroism (CD) spectroscopy. The probable binding location of Clp inside βLG is explored from AutoDock based blind docking simulation