Probing α-amylase and lutein interaction using fluorescence spectroscopy: A thermodynamic study

Intermolecular interactions between enzymes and bioactive molecules (BM) can influence enzyme activity. Thus, is important to understand how these molecules interact. This study used fluorescence spectroscopy to investigate the thermodynamic binding between the digestive enzyme α-amylase (α-AMY) and the carotenoid lutein (LUT), between 293.15 to 313.15 K, and the effect of the complex formation on the inhibition of α-AMY activity. Increasing concentrations of LUT quenched the fluorescence emitted by α-AMY and the fluorescence-quenching mechanism was mainly static. α-AMY/LUT complex showed 1:1 stoichiometry and the binding constant was of the order of 104 L·mol−1. The standard Gibbs free energy change was negative at all temperatures, ranging from -23.20 to -24.45 kJ mol-1, indicating that α-AMY/LUT binding was more stable than free α-AMY and LUT molecules. As the temperature increased, the standard enthalpy (ΔH°) and entropy (TΔS°) changes exhibited a noteworthy trend: ΔH° values ranged from 17.30 to -19.31 kJ·mol-1, while TΔS° values ranged from 40.50 to 5.14 kJ·mol-1. This trend provides clear evidence that up to 299 K, α-AMY/LUT binding was primarily driven by entropy. Beyond this critical temperature, enthalpy became the dominant factor governing the process. This behavior can be elucidated by considering the prevalence of H-bonds and hydrophobic interactions during complex formation. As the temperature increases, molecular flexibility is enhanced, enabling the required orientations for H-bond formation to occur at higher temperatures. Additionally, it is worth noting that the reduction in ΔH° was accompanied by a corresponding decrease in TΔS°, indicative of an enthalpy-entropy compensation phenomenon. Regarding the inhibition of α-AMY activity, the inhibition of the enzyme activity raised with LUT concentration; however, the inhibition was very low, only reaching 3%. This study showed that understanding the interaction between BM and digestive enzymes is essential for achieving a better utilization and targeted intake of different BM for various purposes.