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Journal of Fluorescence

, Volume 6, Issue 1, pp 33–40 | Cite as

Two distinguishable fluorescent modes of 1-anilino-8-naphthalenesulfonate bound to human albumin

  • Luis A. Bagatolli
  • Silvia C. Kivatinitz
  • Felipe Aguilar
  • Marco A. Soto
  • Patricio Sotomayor
  • Gerardo D. Fidelio
Article

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 (τ1) of 16 ns and a λ1max of 478 nm, with a quantum yield (фf1) of 0.67, and component 2, with a lifetime (τ2) of 2–4 ns and a λ2max of 483 nm, with an average quantum yield (фf2) of about 0.11. Considering these findings, the binding analysis is fitted with a model of two independent sites. Site 1 has an association constantKas1=0.87×106M−1 and a capacity of 1.04 mol of ANS/mol of HSA, and site 2 aKas2=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 theKas values for diazepam and aspirin are 0.113× 106 and 0.021×106M−1 respectively.

Key words

Human serum albumin 1-anilino-8-naphthalenesulfonate binding diazepam time-resolved fluorescence fluorescence lifetime 

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Copyright information

© Plenum Publishing Corporation 1996

Authors and Affiliations

  • Luis A. Bagatolli
    • 1
  • Silvia C. Kivatinitz
    • 1
  • Felipe Aguilar
    • 2
  • Marco A. Soto
    • 2
  • Patricio Sotomayor
    • 2
  • Gerardo D. Fidelio
    • 1
  1. 1.Departamento de Química Biológica-CIQUIBICUniversidad Nacional de CórdobaCórdobaArgentina
  2. 2.Institute de Quimica, Facultad de Ciencias Básicas y MatémáticasUniversidad Católica de ValparaísoValparaisoChile

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