Biophysical Characterization of Drug–Protein Interactions through UV, Fluorescence, and Circular Dichroism Spectroscopy

Abstract Understanding drug–protein interactions is critical for predicting pharmacokinetics and therapeutic performance. This study investigates the interaction between Favipiravir (FAV) and human hemoglobin (HHb) using UV–Visible absorption, fluorescence quenching, and circular dichroism (CD) spectroscopy. UV–Vis spectra revealed hyperchromicity with a slight red shift in the Soret band, indicating ground-state complex formation. Fluorescence quenching analysis confirmed a static mechanism with moderate binding affinity (Kb ≈ 3.2 × 10⁴ M⁻¹ at 298 K) and 1:1 stoichiometry. Thermodynamic parameters (ΔH° < 0, ΔS° < 0) suggest hydrogen bonding and van der Waals forces dominate the interaction. CD analysis showed a small decrease in α-helical content (72.0% → 68.1%), confirming minor conformational adjustments without denaturation. The integrated spectroscopic approach provides a reliable framework for elucidating drug–protein binding mechanisms relevant to drug design and pharmacokinetic prediction.