Establishment of a kinetic model of dialysis-related amyloid fibril extensionin vitro

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Abstract
β2-microglobulin (β2M) is a major structural component of dialysis-related amyloid fibrils (fAβ2M). In order to make clear the mechanism of fAβ2M deposition in vivo, as well as to assess the effects of several biological factors on it, it is essential to build up a kinetic experimental system to analyze fAβ2M formation in vitro. We first determined the optimum conditions for quantitative jluorometry of fAβ2M with thioflavine T (ThT). Optimum fluorescence measurements of fAβ2M were obtained at the excitation and emission wavelengths of 455 nm and 485 nm, respectively, with the reaction mixture containing 3 μM ThT and 50 mM of glycine-NaOH buffeer, pH 8.5. We then focused our study on the extension phase of fAβ2M formation in vitro. When fAβ2M were incubated with monomeric β2M, the extension of fAβ2M was observed with electron microscopy. Quantitative fiuorometry revealed that: (a) extension of fAβ2M proceeded by a pseudo-first order exponential increase as measured by the fluorescence of ThT; (b) the rate of extension was maximum around pH 2.5, and was dependent on the incubation temperature; (c) the rate of polymerization was found to be proportional to the product of fAβ2M number concentration and the β2M concentration; (d) the net rate of extension was the sum of the rates of polymerization and depolymerization. These results show that fAβ2M formation can be explained by a first-order kinetic model: that is, the extension of fAβ2M proceeds via the consecutive association of β2M onto the ends of existing jibrils. We propose that this model could be generally applied for the extension of all types of amyloid fibrils in vitro.