Binding Properties of Hydrophobic Molecules to Human Serum Albumin Studied by Fluorescence Titration

Two fluorescence modes were combined to analyze the binding properties of terminally substituted alkanes (C _n X, X = COOH, OH, CHO, NH ₂ ) to human serum albumin (HSA). A competitive binding assay using an 8-anilino-1-naphthalenesulfonate (ANS) fluorescence probe provides information on all the hydrophobic binding sites in HSA. A binding assay using the intrinsic fluorescence of the tryptophan residue in HSA (Trp-HSA) provides information on the specific binding site close to the tryptophan residue. There are three fluorescence-active ANS binding sites in HSA, which can be classified into two types by their affinity for ANS. C _n COOH bound to all three ANS binding sites including the Trp-HSA site, however, it did not quench the fluorescence of Trp-HSA. C _n CHO bound only to the Trp-HSA site with quenching of the fluorescence of Trp-HSA. By comparing the binding affinities of HSA for C _n OH and C _n CHO, it was concluded that the C _n OH binding site is different from the C _n CHO binding site. C _n NH ₂ did not bind to any of the three ANS binding sites in HSA.