Protein binding study of isoflavones by high-performance frontal analysis

High-performance frontal analysis was used for protein binding study of isoflavones (daidzein, genistin, and genistein) to human serum albumin. The analysis was performed on a Develosil 100-Diol-5 column (10 cm x 4.6 mm). Sodium phosphate solution (pH 7.4, ionic strength 0.17) was used as the mobile phase at a flow rate of 1 mL min^-1. UV wavelength was set at 260 nm. To ensure the drug to be eluted as a trapezoidal peak with a plateau, injection volumes of 700 μL for daidzein and genistin, 900 μL for genistein were chosen, respectively. Experimental data were fitted by Scatchard equation. The binding constants (K) and binding affinities (nK) of isoflavones to HSA were: K= 1.581 × 10⁵ (L mol^-1), nK = 0.77 × 10 ⁴ (L mol^-1) for daidzein, K = 1.082 × 10⁵ (L mol^-1), nK = 0.32 × 10⁴ (L mol^-1) for genistein, and K = 3.533 × 10⁵ (L mol^-1), nK = 0.70 × 10⁴ (L mol^-1) for genistin, respectively.