Bovine hemoglobin thermal stability in the presence of naringenin: Calorimetric, spectroscopic and molecular modeling studies

Abstract

Calorimetric, spectroscopic and dynamic light scattering experiments, together with complementary computational investigations afforded a detailed analysis of the thermal stability and conformation of the bovine hemoglobin (BHb)–naringenin (NAR) complex. Differential scanning microcalorimetry (μDSC) revealed complex, multi-step aggregation processes above 330 K. There is a definite enhancement and alteration of the thermal signature of protein aggregation with increasing NAR concentration. Circular dichroism (CD) spectroscopy indicated minor conformational changes in the protein secondary structure upon ligand binding at 298 K. Temperature-dependent CD measurements evidenced steep variations of the α-helix (decrease) and unordered structures (increase), with only a slight increase of β-structure as a result of unfolding and aggregation. UV–Vis absorbance and DLS measurements pointed to large size aggregates formation in the presence of ligand in high concentration.Molecular docking data identified the binding site of NAR, located in the internal cavity of BHb. Molecular dynamics simulations indicated hydrogen bonds, electrostatic and hydrophobic interactions as main contributions in the binding process; the bound ligand produced no sizable structural alterations of the protein molecule. Therefore, the clearly evidenced (μDSC, CD, DLS) influence of the flavonoid on hemoglobin thermal aggregation is exerted as a free (not bound) component of the studied systems.