Binding Sites of Fluorescent Probes on Human Serum Albumin

Abstract

Human serum albumin is known to have two major and selective drug binding sites, termed sites I and II. The fluorescent probes, dansylamide and dansylsarcosine selectively interact with sites I and II, respectively. However, the binding site of the fluorescent probe dansylglycine on human serum albumin is not clear from the literature. This study investigated whether dansylglycine interacts tightly with site I or II. Spectrofluorimetric titrations (quenching and complex) and circular dichroism measurements were performed to determine the binding characteristics of dansylglycine to human serum albumin. Modification in probe fluorescence was described by fluorescence titrations to be a result of competitive displacement by ligands. The pattern of displacement of this probe by several ligands whose primary binding sites are exactly known, enabled the identification of its specific binding site. The fluorescence of dansylglycine is only extensively changed when ligands of site II are added, suggesting that it strongly interacts with the benzodiazepine/indole binding site on human serum albumin.

Characterization of the binding sites of the anticancer ruthenium(III) complexes KP1019 and KP1339 on human serum albumin via competition studies

“…Based on the molar fluorescence spectra, which are only valid under the conditions and instrument settings applied, stability constants were calculated using PSEQUAD [44]. The conditional HSA binding constants (logK') for WF, BR and DG are comparable with data obtained by us and others [26][27][28][29][30][31][32]36,47,48] (Table S1) accompanied by a considerable decrease in the emission intensity. Therefore, the displacement of the site markers at various concentrations of the Ru(III) complexes could be followed via intensity changes of the emission spectra.…”

“…A typical site marker for site I is the anticoagulant drug warfarin (WF) [26][27][28][29], while dansylglycine (DG) shows specificity for site II (see Fig. S1) [30][31][32]. Initial rapid binding of KP1019 takes place in a non-covalent manner at the hydrophobic binding sites of HSA, however after a longer incubation time the type of the interaction is converted slowly to a protein-coordinated form [33].…”

Practical Aspects of the Ligand-Binding and Enzymatic Properties of Human Serum Albumin.

“…If the latter is the case, the probe-to-protein molar ratios should be kept well below unity to minimize secondary probe binding. Examples of site I probes, in addition to warfarin, are 5-dimethylaminonaphthalene-1-sulfonamide (DNSA), 6) dansylamide, 40,41) dansyl-L-glutamine, 40) dansyl-Lasparagine, 10) dansyl-L-lysine, 42) n-butyl p-aminobenzoate, 10) prodan, 43) and phenol red, 7) and among the site II probes are dansylsarcosine, 6) dansyl-L-proline, 40) dansylglycine, 41) and 7-alkylaminocoumarin-4-acetic acids. 44) Care should be exercised with respect to site I especially, because, as mentioned above, several examples of independent co-binding of two ligands to this site have been reported.…”

A Study on the Interaction of the DAS‐K with Bovine Serum Albumin by On‐line Ultrafiltration and Chemiluminescence

“…22 Spectrophotometry and fluorometry have been extensively used for direct study of drug-protein interaction based on monitoring the change of a physicochemical property of the drug-protein system. 23,24 Ion-selective electrode and quartz crystal resonant sensor techniques have also been developed for the direct study of drug-protein interaction. 25,26 Each of them often presents different advantages and disadvantages.…”