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.

Diffusion of organic dyes in bovine serum albumin solution studied by fluorescence correlation spectroscopy

“…Recently, Bhattacharyya and co-workers have shown a 1.2-fold decrease in the binding constant between R6G and HSA upon addition of 5 M guanidinium hydrochloride. 44 Hence, it is not surprising to find a situation where the two factors counter-balance each other resulting in very little or negligible change in the D t value of R6G in BSA.…”

“…FCS has been previously used to study the diffusion of fluorescent probes in many organized assemblies, which include vesicles and membranes, [22][23][24] colloidal particles, 25 ionic liquids, [26][27][28][29] agarose gel, 30,31 polymer matrices, 32,33 micelles and [34][35][36][37] microemulsions, 38 and to study protein aggregation 39,40 and conformational changes of the protein. [41][42][43][44][45][46] As BSA is a carrier protein, the mobility of the molecules in BSA solution is of fundamental importance in drug delivery and for the design of drug molecules. Considering the fact that a protein solution consists of both hydrophilic and hydrophobic domains, the diffusion of a probe may be location dependent with fast diffusion near the surface and slow diffusion in the core region.…”

“…Bhattacharyya and co-workers recently studied the interaction of C153 and R6G with immobilized human serum albumin (HSA) and determined the association sites of the probes in HSA by studying the effect of guanidinium hydrochloride on the binding kinetics of the probes with HSA. 44 Salts, such as Na 2 SO 4 , NaCl, NaI, NaSCN, at high concentrations have significant effects on the conformational stability of biomacromolecules. 49,50 Salt-induced association or dissociation of many biological macromolecules depends upon their ''salting out'' and ''salting in'' properties.…”

Dispersion and Interaction of Charged Fluorescent Dyes in Protein‐Polymer Surfactant‐based Non‐Aqueous Liquid

“…Also, selective interaction of fluorescent probes with certain region of a protein has enormous utility in site‐specific binding related photo‐physical studies. Rhodamine 6G (Rh6G) and fluorescein (FL) are well known positively and negatively charged hydrophilic dyes, respectively, with their structures shown in Figure b and c …”

Unusual Binding of a Potential Biomarker with Human Serum Albumin

“…Conventional ligand‐binding studies that consider the optical properties of HSA are generally performed by monitoring circular dichroism (CD) and fluorescence 6. 15, 16 Biophysical techniques, such as isothermal titration calorimetry (ITC) and NMR‐based screening methods, can also provide useful information regarding the specific binding of drugs to proteins 17. 18 In spite of the complex structure of HSA, fluorescence measurements are possible, owing to the presence of a lone tryptophan (Trp) group in subdomain IIA at position 214.…”