Inhibition of amyloid formation of bovine hemoglobin by bioactive phenolic acids: an elaborate investigation into their binding properties with the protein using multi-spectroscopic and computational techniques

Abstract

Studying the interactions of bioactive phenolic acids [ferulic acid (FA), caffeic acid (CA), and chlorogenic acid (CGA)] with the carrier protein bovine hemoglobin (BHb) helps to understand their modes of binding and transport, which affect their bioavailability. The protein–ligand complexes exhibited binding constants (K_b) in the range of 10⁴ M⁻¹, indicating a reversible mode of association. BHb–CA complexation attenuated the inherent fluorescence of BHb (β₂-Trp37 dominant fluorophore) through a static quenching mechanism. However, a combined (static and dynamic) quenching mechanism was observed for the BHb–FA/CGA complexes. In this study, the thermodynamic parameter, ΔG°, determined from fluorescence spectroscopy, was negative, suggesting that the complexation of BHb with phenolic acids was spontaneous. The negative change in enthalpy [ΔH° = –(8.539 ± 1.331) kJ mol⁻¹, –(10.620 ± 1.028) kJ mol⁻¹, and –(8.637 ± 0.232) kJ mol⁻¹ for BHb–FA, BHb–CA, BHb–CGA complexes, respectively] and positive entropy change [ΔS° = + (63.862 ± 1.218) J mol⁻¹ K⁻¹, +(67.108 ± 1.681) J mol⁻¹ K⁻¹, and +(62.540 ± 1.058) J mol⁻¹ K⁻¹ for BHb–FA, BHb–CA, BHb–CGA complexes, respectively] indicated that hydrogen bonding and hydrophobic forces stabilized the complexes. Computational studies also illustrated similar interactive forces in BHb–ligand complexes. Changes around the microenvironment of the Trp residues in the binding process were verified using various spectral measurements. Circular dichroism and Fourier infrared spectroscopies illustrated a decrease in the α-helical content of BHb during complexation. Fibril inhibition properties of the ligands were verified using ThT assay, ANS binding studies, Congo Red assay, turbidity assay, Soret absorption measurements, and CD spectral measurements. The structure–activity relationship, binding affinity, and interactive forces between the protein and phenolic acids could explain the order of anti-fibrillation properties.