In vitro interactions of esculin and esculetin with bovine hemoglobin alter its structure and inhibit aggregation: insights from spectroscopic and computational studies

Hemoglobin serves a multitude of biological functions and its activity and stability are influenced by structural alterations. Fibrillation of this protein is one such complication that can hinder its physiological levels resulting in numerous health disorders. This study focuses on providing a comprehensive elucidation on the binding interaction of bioactive esculin and esculetin with bovine hemoglobin (BHb) and ultimately determining the anti-fibrillation properties of these compounds. UV/vis spectroscopy and fluorescence measurements verified the presence of a static form of quenching mechanism for the BHb-esculin complex and a combined quenching mechanism for the BHb-esculetin complex. The binding affinity (K-b) was determined to be higher for the BHb-esculetin complex than for the BHb-esculin complex. This could be due to the lower binding distance observed between a & beta;(2)-Trp37 residue and esculetin, as observed through Forster's theory, molecular docking, and molecular dynamic (MD) simulation. The calculated thermodynamic parameters indicated the presence of hydrophobic forces and hydrogen bonding stabilizing the complexes. Synchronous, 3D fluorescence, REES, and anisotropy measurements revealed microenvironmental alterations around the Trp residue induced by esculin/esculetin binding. Secondary structural changes of BHb in the presence of esculin/esculetin were observed through UV/vis absorption, CD, and FTIR studies. BHb was subjected to fibrillation in the presence of fructose, and ThT fluorescence, fluorescence microscopy, FESEM, Soret absorption, ANS measurements, and Congo red assay provided evidence of the anti-fibrillation effects of esculin and esculetin.