Abstract: Alpinetin (Alp), a main active component of Alpinia katsumadai, was used to study the mechanism underlying its interaction with hemoglobin (BHG) by fluorescence polarization, synchronous fluorescence, 3D fluorescence and absorption spectroscopy combined with molecular modeling technique under physiological conditions. The data from the overlapping spectra confirmed that the interaction between Alp and BHG could be matched with the non-radioactive energy transfer theory. Various binding constants (5.880 × 104, 4.337 × 104 and 4.935 × 104 L/mol for quenching mechanism; 3.239 × 103, 5.225 × 103 and 7.692 × 103 L/mol for sensitization effect) and thermodynamic parameters (ΔS: 182.073 J/(mol·K), ΔH: 34.320 kJ/mol, ΔG: −20.119 (299 K), −21.940 (309 K) and −23.760 kJ/mol (319 K)) for Alp-BHG systems were hereby obtained under different temperatures (299, 309 and 319 K) based on different action mechanisms. Molecular docking was performed to reveal the binding of Alp moiety to the hydrophobic cavity of BHG and the acting force was mainly a hydrophobic interaction. The low anisotropy values suggested that there was shorter relaxation time and Alp molecules were observed in a motionally unrestricted environment introduced by BHG. The synchronous fluorescence and absorption spectra showed that the addition of Alp impacted the microenvironment around BHG in aqueous solution. The 2D and 3D fluorescence spectroscopy of Alp-BHG system indicated that Alp strongly quenched the intrinsic fluorescence of BHG causing a conformational change of the protein. The results indicated that the interaction between Alp and BHG was strong, implying that Alp may be stored and transferred by BHG in some degree. In addition, the effects of common ions on the constants of alpinetin-BHG complex were also discussed, indicating an increasing trend for binding constants in the present of different metal ions.

Key words: alpinetin, bovine hemoglobin, energy transfer, interaction