Two distinguishable fluorescent modes of 1-anilino-8-naphthalenesulfonate bound to human albumin |
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Authors: | Luis A Bagatolli Silvia C Kivatinitz Felipe Aguilar Marco A Soto Patricio Sotomayor Gerardo D Fidelio |
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Institution: | (1) Departamento de Química Biológica-CIQUIBIC, Universidad Nacional de Córdoba, Córdoba, Argentina;(2) Institute de Quimica, Facultad de Ciencias Básicas y Matémáticas, Universidad Católica de Valparaíso, Valparaiso, Chile;(3) Departamento de Química Biológica-CIQUIBIC, Facultad de Ciencias Químicas, Ciudad Universitaria, Casilla de Correo 61, 5016 Córdoba, Argentina |
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Abstract: | We study the interaction of 1-anilino-8-naphthalenesulfonate (ANS) with human (HSA) and bovine serum albumin (BSA) by phase and modulation fluorescence spectroscopy. We determined that both HSA and BSA show one or two distinguishable fluorescent sites, depending of the ANS/serum albumin ratio. At above a 1 1 ANS/HSA molar ratio, the steady-state emission spectra for ANS can be resolved in two components: component 1, emitting with a lifetime ( 1) of 16 ns and a 1max of 478 nm, with a quantum yield ( f1) of 0.67, and component 2, with a lifetime ( 2) of 2–4 ns and a 2max of 483 nm, with an average quantum yield ( f2) of about 0.11. Considering these findings, the binding analysis is fitted with a model of two independent sites. Site 1 has an association constantK
as1=0.87×106M–1 and a capacity of 1.04 mol of ANS/mol of HSA, and site 2 aK
as2=0.079×106M–1 and a capacity of 2.34 mol of ANS/mol of HSA. Analysis of fluorescence lifetime distributions shows that the rigidity of the fluorophore environment at site 1 changes when site 2 is occupied. These findings suggest an interconnection between the two sites and that ligands can stabilize the protein's globular structure. To assess the identity of the ANS binding sites we used diazepam as a marker of the site located at the IIIA HSA subdomain and aspirin as a marker of sites located at the IIIA and IIA HSA subdomains. Both ligands displace ANS only from site 1, suggesting that it corresponds to the binding site located at the IIIA sub-domain of the protein. We determined that theK
as values for diazepam and aspirin are 0.113× 106 and 0.021×106
M
–1 respectively. |
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Keywords: | Human serum albumin 1-anilino-8-naphthalenesulfonate binding diazepam time-resolved fluorescence fluorescence lifetime |
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