Probing the interactions of hemoglobin with antioxidant flavonoids via fluorescence spectroscopy and molecular modeling studies.

Steady state and time resolved fluorescence spectroscopy, combined with molecular modeling computations, have been used to explore the interactions of two therapeutically important flavonoids, fisetin (3,7,3',4'-OH-flavone) and 3-hydroxyflavone (3-HF), with normal human hemoglobin (HbA). Distinctive 'two color' fluorescence signatures and fairly high fluorescence anisotropy (r=0.12-0.28) of fisetin and 3-HF reveal their specific interactions with HbA. Binding constants estimated from the fluorescence studies were ≈4.00 × 10(4)M(-1) and 9.83 × 10(3)M(-1) for fisetin and 3-HF respectively. Specific interactions with HbA were further confirmed from flavonoid-induced static quenching of the protein tryptophan fluorescence as indicated by: (a) bimolecular quenching constant K(q)≫diffusion controlled limit (b) closely matched values of Stern-Volmer quenching constant and binding constant (c) τ(o)/τ≈1 (where τ(o) and τ are the unquenched and quenched tryptophan fluorescence lifetimes respectively). Molecular docking and electrostatic surface potential calculations reveal contrasting binding modes of fisetin and 3-HF with HbA.