The oligomers formed during the early steps of amyloid aggregation are thought to be responsible for the neurotoxic damage associated with Alzheimer's disease. It is therefore of great interest to characterize this early aggregation process and the aggregates formed, especially for the most significant peptide in amyloid fibrils, Amyloid-β(1-42) (Aβ42). For this purpose, we directly monitored the changes in size and concentration of initially monomeric Aβ42 samples, using Fluorescence Correlation Spectroscopy. We found that Aβ42 undergoes aggregation only when the amount of amyloid monomers exceeds the critical aggregation concentration (cac) of about 90 nM. This spontaneous, cooperative process resembles surfactants self-assembly and yields stable micellelike oligomers whose size (≈50 monomers, R h ≈ 7-11 nm) and elongated shape are independent of incubation time and peptide concentration. These findings reveal essential features of in vitro amyloid aggregation, which may illuminate the complex in vivo process.Alzheimer's disease (AD) is a neurodegenerative disease characterized by the presence of Amyloid-β plaques in the brain. Although the causal relationship between these protein fibrillar aggregates and the neurodegenerative disease has not been established yet, the 'amyloid hypothesis' , that accumulation and aggregation of amyloid-β peptide initiates a cascade of neurodegenerative events, has been widely accepted [1][2][3] . Impairment of Amyloid-β clearance in AD patients seems to be the main cause for accumulation of the peptide 4,5 . It is thought that the neurotoxic species that trigger the amyloid cascade leading to neurodegeneration are early non-fibrillar aggregates, which may also be the precursors of the amyloid fibrils 2,6-8 . The dominant peptides in amyloid fibrils are Amyloid-β(1-42) (Aβ42) and Amyloid-β(1-40) (Aβ40), with Aβ42 being the more fibrillogenic of the two, with a much stronger tendency to aggregate [9][10][11] .There is ample literature on the mechanism underlying amyloid fibril formation 12,13 . Most kinetic studies agree on a complex nucleation-growth mechanism, where the differences in the microscopic rates and in the relevance of secondary nucleation processes determine the degree of aggregation and can account for the differences between Aβ40 and Aβ42 11,14 . For such nucleation-dependent processes, a critical aggregation concentration (cac) is predicted, above which aggregation takes place 10 . For Aβ40 the formation of micelle-like intermediates was reported, with a critical concentration in the micromolar range [15][16][17] , whereas recent studies have found nanomolar cac values for both Aβ40 and Aβ42 [18][19][20] . The latter values fit better with the reported physiological concentrations of Aβ in the picomolar to nanomolar range which may be locally higher due to accumulation or impairment of clearance 4,5,18,21,22 . A possible reason for the discrepancy in the cac values may be the strong adsorption of Amyloids Aβ40 and Aβ42 to interfaces 23 , which can lead to great ...
Thioflavin T is a highly sensitive fluorescent marker of amyloid fibrils that has been widely used for in vitro biomedical assays. However, neither its complex photophysical behavior nor its binding mode to amyloid fibrils are still well understood. We present a detailed analysis of the photophysical properties of Thioflavin T in various media, including solvents and solvent mixtures of different viscosities as well as fibrillar and globular proteins. We propose a model that explains the strong wavelength dependency of the Thioflavin T fluorescence and the large fluorescence enhancement in certain environments. We determine the binding affinities and the fluorescence properties of Thioflavin T bound to amyloid-β (1-42) fibrils and to bovine serum albumin and discuss the sensitivity and the specificity of this probe to amyloid aggregates. These results allow us to assess the suitability of Thioflavin T for quantitative determinations in biomedical studies.
The nature and strength of the interactions between a cationic fluorophore, Rhodamine 123 (R123), and surfactants of different head charge are investigated. Series of absorption and fluorescence emission spectra and of fluorescence decays are measured. R123 does not interact with the monomers of both nonionic and cationic surfactants but it presents affinity to their micelles. A partition equilibrium model was proposed and the corresponding equilibrium constants were obtained, as well as the photophysical properties of the dye bound to the micelles. In the case of the cationic surfactants, changes of the fluorescence properties were already observed below the critical micellar concentration (CMC) due to dynamic quenching caused by the free counterions. In the presence of anionic surfactants R123 shows a very different behaviour with dramatic spectral changes below the CMC. The observed variations are attributed to a first, strong interaction between R123 and the surfactant monomers, which yields ionic pairs of singular photophysical properties and dominates at low surfactant concentrations, followed by the association of R123 with the surfactant premicellar and micellar aggregates at higher surfactant concentrations near the CMC. This behaviour results from the competition between the strong electrostatic interactions of the cationic dye with the anionic surfactant head groups and the hydrophobic forces stabilizing the dye inside the micelles. The results of this work illustrate the complex physicochemical and photophysical behaviour of a charged dye in micellar systems, which resembles the expected situation in similar systems such as biological membranes.
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