FTIR spectroscopy for the determination of interaction of albumin with antioxidant

FTIR spectroscopy has been performed to examine the eventually changes in secondary structure of Human Serum Albumin in the presence of antioxidant and to supply relevant information on the complex binding mechanism. Infrared spectroscopy is a powerful tool to analyze proteins secondary structures and their dynamics or modifications. In particular, the Amide I′ band between 1600 and 1700 cm-1 is mainly due to C=O stretch. The secondary structure of HSA, obtained by the deconvolution of this band, consists approximately of 67% α-helix, 10% turn, significant change or shift in band intensity at 1652 and 1680 cm-1 of the FTIR spectra suggesting a rapid change in the association and dissociation properties of the antioxidant agent (phloretin) that induces little changes on secondary structure of the protein. This is further supported by the observed increase of Amide II' and Amide II absorbance values. The vibration modes of these two bands are composed differently from the internal coordinate vibrations and will be affected differently by the conformation and the environment of the amide group. This increase is due to H–D exchange that involves hydrogen remained within the core and justifies the transient formation of intermediate protein conformational states during HSA-phloretin complex formation. Moreover the increase in Amide II band may be indicative of hydrogen bonding formation during the process. In fact hydrogen bonding influences predominantly the NH bending vibration, which contributes to the amide II but not to the amide II' vibration. The FTIR spectrum of HSA in the absence or in the presence of phloretin shows interesting peculiarity that make able us to evidence new interaction and prospective in the complex formation.