Analytical Sciences

Abstract − Analytical Sciences, 25(1), 115 (2009).

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 (CnX, 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. CnCOOH bound to all three ANS binding sites including the Trp-HSA site, however, it did not quench the fluorescence of Trp-HSA. CnCHO bound only to the Trp-HSA site with quenching of the fluorescence of Trp-HSA. By comparing the binding affinities of HSA for CnOH and CnCHO, it was concluded that the CnOH binding site is different from the CnCHO binding site. CnNH2 did not bind to any of the three ANS binding sites in HSA.