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

Abstract

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.

Interaction between Alizarin and Human Serum Albumin by Fluorescence Spectroscopy

“…28,29 Some papers indicated that there were three fluorescence-active ANS binding sites in HSA. 30,31 The ANS binds most preferentially to the Trp-HSA site compared to the other two sites in HSA, and Trp-HSA is located in the subdomain II A. 31 In a competitive binding assay of alizarin with ANS-HSA, it decreased about 40% of the initial intensity of the ANS-HSA fluorescence.…”

“…30,31 The ANS binds most preferentially to the Trp-HSA site compared to the other two sites in HSA, and Trp-HSA is located in the subdomain II A. 31 In a competitive binding assay of alizarin with ANS-HSA, it decreased about 40% of the initial intensity of the ANS-HSA fluorescence. This value corresponds to the release of one point two ANS among the total three binding sites in HSA.…”

Thermodynamics of Nucleic Acid Structural Modifications for Biotechnology Applications

“…The point of this comparison is that hydrophobic or amphipathic binding is possible, but it is difficult to obtain specificity as reported in a study of the binding affinity of these aptamers [81]. Studies of the binding of 1-anilino-8-naphthalene sulfonate (ANS) , a small amphipathic molecule that is widely used as a probe of protein structure, show that it has a predominantly electrostatic mode of binding [82,83]. Thus, one might interpret this as a general observation that electrostatic interactions supercede hydrophobic interactions.…”

Interaction of ANS with human serum albumin under confinement: Important insights and relevance

“…In this study we have probed the effect of the confinement provided by the water pools of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) reverse micelles on the multidomain protein human serum albumin (HSA), well-known for its ligand binding properties, using the fluorescent probe ANS. Here, ANS has been used as a dual probe, that is (i) as a reporter of hydrophobic patches of proteins [7][8] and (ii) as a representative drug molecule that shows appreciable affinity for HSA [9][10].…”