L-Ascorbic acid, α-tocopherol, procyanidin B3, β-carotene and astaxanthin are five classic dietary antioxidants. In this study, the interaction between the five antioxidants and ovalbumin was investigated by fluorescence spectroscopy, in combination with UV-vis absorption spectroscopy and circular dichroism (CD) spectroscopy. The quenching mechanism of ovalbumin by α-tocopherol is static quenching and the interaction between α-tocopherol and ovalbumin is synergistically driven by enthalpy and entropy. Electrostatic interactions and hydrophobic interactions play a major role in stabilizing the complex. For the other four antioxidants, the quenching mechanisms are all static quenching mechanisms at lower concentrations of antioxidants, but at higher concentrations of antioxidants, predominantly by the "sphere of action" quenching mechanisms. The binding processes of the other four antioxidants to ovalbumin are all entropy process and the major part of the action force is hydrophobic interactions. The binding constants of ovalbumin with the five antioxidants are in the following order as: astaxanthin > β-carotene > L-ascorbic acid > procyanidin B3 > α-tocopherol at 298 K. Synchronous fluorescence spectroscopy shows the interaction between L-ascorbic acid/β-carotene/astaxanthin and ovalbumin decreases the hydrophobicity of the microenvironment of tryptophan (Trp) and tyrosine (Tyr) residues. The hydrophobicity of Trp is increased while the hydrophility of Tyr is increased in the presence of α-tocopherol. However, the microenvironment of Trp and Tyr is not affected by procyanidin B3. The UV-vis absorption and CD spectra suggest that the interaction between the five antioxidants and ovalbumin leads to the loosening and unfolding of ovalbumin skeleton and exerts some influence on the natural secondary structure of ovalbumin. The study provides an accurate and full basic data for clarifying the binding mechanisms of L-ascorbic acid, α-tocopherol, procyanidin B3, β-carotene and astaxanthin interacting with ovalbumin and is helpful for understanding rational use of antioxidants as dietary supplements.
Keywords: Antioxidants; Circular dichroism spectroscopy; Fluorescence spectroscopy; Interaction; Ovalbumin; UV-vis absorption spectroscopy.