Interaction of 3’-O-caffeoyl D-quinic acid with multisubunit protein helianthinin

Abstract

Chlorogenic acid, 3’-O-caffeoyl D-quinic acid, is an inherent ligand present in Helianthus annuus L. The effect of pH on chlorogenic acid binding to helianthinin suggests that maximum binding occurs at pH 6·0. The protein-polyphenol complex precipitates as a function of time. The association constant of the binding of chlorogenic acid to helianthinin, determined by equilibrium dialysis, at 31°C has a value of 3·5 ± 0·1 × 104M–1 resulting in a ΔG value of – 6·32 ±0·12 kcal /mol. The association constant Ka is 1·0 ± 0·1 × 104M-1 as determined by ultraviolet difference spectral titration at 25°C with ΔGo of -5·46 ± 0·06 kcal/mol. From fluorescence spectral titration at 28°C, the Ka value is 1·38 ± 0·1 × 1 0 4M-1 resulting in a ΔG of – 5·70 ± 0·05 kcal/mol. The total number of binding sites on the protein are 420 ± 50 as calculated from equilibrium dialysis. Microcalorimetric data of the ligand-protein interaction at 23°C suggests mainly two classes of binding. The thermal denaturation temperature, Tm of the protein decreases from 76°C to 72°C at 1 × 10-3M chlorogenic acid concentration upon complexation. This suggests that the complexation destabilizes the protein. The effect of temperature on Ka of chlorogenic acid shows a nonlinear increase from 10·2°C to 45°C. Chemical modification of both lysyl and tryptophanyl residues of the protein decreases the strength of binding of chlorogenic acid. Lysine, tryptophan and tyrosine of protein are shown to be present at the binding site. Based on the above data, it is suggested that charge-transfer complexation and entropically driven hydrophobic interaction are the predominant forces that are responsible for binding of chlorogenic acid to the multisubunit protein, helianthinin.