Analytical Sciences

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

Kô TAKEHARA,* Keiko YUKI,* Masumi SHIRASAWA,* Shinya YAMASAKI,* and Shuto YAMADA**

*Department of Chemistry, Faculty of Sciences, Kyushu University, 4-2-1 Ropponmatsu, Chuo, Fukuoka 810-8560, Japan
**Research and Development Center for Higher Education, Kyushu University, 4-2-1 Ropponmatsu, Chuo, Fukuoka 810-8560, Japan

Two fluorescence modes were combined to analyze the binding properties of terminally substituted alkanes (C_nX, X = COOH, OH, CHO, NH₂) 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_nCOOH bound to all three ANS binding sites including the Trp-HSA site, however, it did not quench the fluorescence of Trp-HSA. C_nCHO bound only to the Trp-HSA site with quenching of the fluorescence of Trp-HSA. By comparing the binding affinities of HSA for C_nOH and C_nCHO, it was concluded that the C_nOH binding site is different from the C_nCHO binding site. C_nNH₂ did not bind to any of the three ANS binding sites in HSA.

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