Deciphering the fluorescence quenching mechanism of a flavonoid drug following interaction with human hemoglobin

The present work demonstrates comprehensive spectrofluorometric characterization of interaction of a bioactive flavonoid quercetin (QCT) with the transport protein human hemoglobin (Hb) by protein‐induced quenching phenomenon. Interaction between such a pharmacologically essential drug and Hb has been enlightened by means of steady‐state and time‐resolved fluorescence spectroscopy. The protein‐induced Stern–Volmer quenching study of QCT depicts an upward curvature that cannot be an outcome of only the static quenching arising from ground‐state complexation. Therefore, an extensive time‐resolved fluorescence characterization of the quenching process has been performed to discern the actuating quenching mechanism. However, the estimated value of bimolecular quenching constant (kq) from the diffusion‐controlled quenching study hints for dynamic quenching phenomenon. Finally, the significant amplification of the average lifetime of QCT as a function of protein concentration aids to resolve this conflict affirming the quenching phenomenon to be an exquisite drug–protein binding interaction. Not only the quenching mechanism has been established, but also the binding parameters and location of binding within the microheterogeneous cavity of Hb has been explicated. AutoDock‐based “blind docking” simulation has been employed to conquer probable binding location of the drug within protein cavity. Nevertheless, circular dichroism (CD) spectroscopy depicts the modification of the native conformation of Hb, which is indicative of this binding phenomenon.