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Characterization of protofibrillar aggregates of bovine serum albumin by tryptophans fluorescence lifetime

  • Autori: Vetri, V; D'Amico, M; Leone, M; Cannas, M; Militello, V
  • Anno di pubblicazione: 2010
  • Tipologia: eedings
  • Parole Chiave: Fibril, Time resolve fluorescence, Aggregation
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We report an experimental study on the thermally induced aggregation of Bovine Serum Albumin at basic pH. In these conditions, we observe the growth of simple protofibrillar structures via the formation of intermolecular beta-sheets promoted by the increased electrostatic repulsion. Here we present a study on the time resolved fluorescence of Tryptophans (Trp) along the aggregation kinetics in the above reported conditions. We use the lifetimes distribution approach as a useful tool for the interpretation of the fluorescence decay in terms of protein conformational substates and interconversion dynamics. Trp fluorescence lifetime depends from protein conformations, also in relation with solvent interaction, and it reveals microenviroment properties around these chromophores. The observed changes in the fluorescence intensity and lifetime signify formation of complexes that are characterised by clustering and conformational rearrangement of the protein monomers. Particular interest is focused on the time evolution of lifetimes distribution width, which is related to the heterogeneity of Trp environment and gives information on the extent of motion of the fluorophore and/or to the number of microenvironments experienced by the chromophores. Our data indicate that, during the aggregation process, the Trp lifetimes distribution shows a significant decrease in the mean value and a simultaneous reduction of its width. This suggests that aggregation is accompanied by a reduction of accessible substates for the chromophores (reduction of the number of environments in the aggregated state) indicating the formation of more rigid, and possibly ordered, structures. Interestingly, these changes correlate with data relative to Circular Dichroism and Thioflavin T fluorescence emission, indicating the progressive growth of proto-fibrillar structures.