Binding Sites of Resveratrol, Genistein, and Curcumin with Milk α- and β-Caseins

The binding sites of antioxidant polyphenols resveratrol, genistein, and curcumin are located with milk alpha- and beta-caseins in aqueous solution. FTIR, CD, and fluorescence spectroscopic methods and molecular modeling were used to analyze polyphenol binding sites, the binding constant, and the effects of complexation on casein stability and conformation. Structural analysis showed that polyphenols bind casein via hydrophilic and hydrophobic interactions with the number of bound polyphenol molecules (n) 1.20 for resveratrol, 1.42 for genistein, and 1.43 for curcumin with alpha-casein and 1.14 for resveratrol, 1.27 for genistein, and 1.27 for curcumin with beta-casein. The overall binding constants of the complexes formed are Kres-alpha-casein = 1.9 (+/- 0.6) x 10(4) M-1, Kgen-alpha-casein = 1.8 (+/- 0.4) x 10(4) M-1, and Kcur-alpha-casein = 2.8 (+/- 0.8) x 10(4) M-1 with alpha-casein and Kres-beta-casein = 2.3 (+/- 0.3) x 10(4) M-1, Kgen-beta-casein = 3.0 (+/- 0.5) x 10(4) M-1, and Kcur-beta-casein = 3.1 (+/- 0.5) x 10(4) M-1 for beta-casein. Molecular modeling showed the participation of several amino acids in polyphenol-protein complexes, which were stabilized by the hydrogen bonding network with the free binding energy of -11.56 (resveratrol-alpha-casein), -12.35 (resveratrol-beta-casein), -9.68 (genistein-alpha-casein), -9.97 (genistein-beta-casein), -8.89 (curcumin-alpha-casein), and -10.70 kcal/mol (curcumin-beta-casein). The binding sites of polyphenols are different with alpha- and beta-caseins. Polyphenol binding altered casein conformation with reduction of a-helix, indicating a partial protein destabilization. Caseins might act as carriers to transport polyphenol in vitro.