Amyloid β-protein aggregation produces highly reproducible kinetic data and occurs by a two-phase process - PubMed (original) (raw)

Amyloid β-protein aggregation produces highly reproducible kinetic data and occurs by a two-phase process

Erik Hellstrand et al. ACS Chem Neurosci. 2010.

Abstract

Protein aggregation can lead to major disturbances of cellular processes and is associated with several diseases. We report kinetic and equilibrium data by ThT fluorescence and enzyme-linked immunosorbent assay of sufficient quality and reproducibility to form a basis for mechanistic understanding of amyloid β-peptide (Aβ) fibril formation. Starting from monomeric peptide in a pure buffer system without cosolvents, we find that the kinetics of Aβ aggregation vary strongly with peptide concentration in a highly predictable manner. The free Aβ concentration in equilibrium with fibrils was found to vary with total peptide concentration in a manner expected for a two-phase system. The free versus total Aβ concentration was linear up to ca. 0.2 μM, after which free Aβ decreased with total Aβ toward an asymptotic value. Our results imply that Aβ fibril formation arises from a sequence of events in a highly predictable manner.

Keywords: Alzheimer; Amyloid; aggregation; fibril; kinetics; mechanism.

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Figures

Figure 1

Figure 1

(A) Isolation of monomeric Aβ(M1−42) by gel filtration on a Superdex 75 column in 20 mM sodium phosphate buffer, pH 8, with 200 μM EDTA and 0.02% NaN3. The monomer is collected between the dashed red lines and is free from higher Aβ assembly forms (shoulder before monomer peak) and buffer and salts (tall peak after the monomer peak). (B) Kinetic traces by ThT fluorescence for 32 replicates at concentrations 2.4 (black), 1.2 (red), and 0.6 μM (green) Aβ(M1−42) in 20 mM sodium phosphate, pH 8, 200 μM EDTA, 0.02% NaN3, 20 μM ThT. The first 7 h are shown.

Figure 2

Figure 2

Concentration dependence of Aβ aggregation kinetics in 20 mM sodium phosphate, pH 8, 200 μM EDTA, 0.02% NaN3, 20 μM ThT. (A) Kinetics of aggregation monitored using ThT fluorescence. Data for the 13 highest concentrations in a single experiment are shown with Aβ(Μ1−42) concentrations of 5.8 (black), 4.9 (brown), 3.9 (red), 2.9 (orange), 2.6 (yellow), 2.2 (green-yellow), 1.85 (yellow-green), 1.65 (green), 1.46 (cyan), 1.31 (light blue), 1.17 (blue), 1.07 (marine blue), and 0.97 (purple) μM. (B) Fitting of eq 1 to one of the fibrillation traces in panel A, with data points as filled circles and the fitted curve as a solid line. The values for _t_1/2 as obtained by the fit and _t_lag by eq 2 are indicated. (C) Lagtime obtained by fitting eq 1 to 672 kinetic traces in seven sets of data (in black, red, green, magenta, cyan, yellow, and blue) versus Aβ(Μ1−42) concentration. Each point is average of 3−32 replicates of the same solution. The solid line is a power function with exponent −1.48 fitted to all data points. Inset: same data with logarithmic axes.

Figure 3

Figure 3

Concentration dependence of Aβ aggregation equilibrium in 20 mM sodium phosphate, pH 8, 200 μM EDTA, 0.02% NaN3. Samples were allowed to aggregate for 84−96 h, and large aggregates were removed by centrifugation. The concentration of soluble Aβ determined by ELISA is plotted versus total concentration (from acid hydrolysis): (A) low concentration samples, linear axes; (B) all samples, logarithmic axes.

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