title: Binding of organic dyes with human serum albumin: a single-molecule study.
abstract: Kinetics of binding of dyes at different sites of human serum albumin (HSA) has been studied by single-molecule spectroscopy. The protein was immobilized on a glass surface. To probe different binding sites (hydrophobic and hydrophilic) two dyes, coumarin 153 (C153, neutral) and rhodamine 6G (R6G, cationic) were chosen. For both the dyes, a major (ca. 96-98%) and minor (ca. 3%) binding site were detected. Rate constants of association and dissociation were simultaneously determined from directly measuring fluctuations in fluorescence intensity (τ(off) and τ(on)) and from this the equilibrium (binding) constants were calculated. Fluorescence lifetimes at individual sites were obtained from burst-integrated lifetime analysis. Distributions of lifetime histograms for both the probes (C153 and R6G) exhibit two maxima, which indicates the presence of two binding domains in the protein. Unfolding of the protein has been studied by adding guanidinium hydrochloride (GdnHCl) to the solution. It is observed that addition of GdnHCl affects the dissociation and association kinetics and hence, binding equilibrium of the association of C153. However, the effect of binding of R6G is not affected much. It is proposed that GdnHCl affects the hydrophobic binding sites more than the hydrophilic site.
authors.affiliation: Physical Chemistry Department, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India.
authors.forename: Dibyendu Kumar
authors.initials: DK
authors.lastname: Das
authors.forename: Tridib
authors.initials: T
authors.lastname: Mondal
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Mandal
authors.forename: Kankan
authors.initials: K
authors.lastname: Bhattacharyya
mesh_terms: D001665:Binding Sites; D002412:Cations; D004396:Coloring Agents; D003374:Coumarins / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D031541:Fluorescence Resonance Energy Transfer; D019791:Guanidine / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008853:Microscopy; D008958:Models, Molecular; D015335:Molecular Probes / Q000378:metabolism*; D011485:Protein Binding; D012235:Rhodamines / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics
chemical_list: D002412:Cations; D004396:Coloring Agents; D003374:Coumarins; D015335:Molecular Probes; D012235:Rhodamines; D012709:Serum Albumin; C488877:coumarin 153; C026188:rhodamine 6G; D019791:Guanidine