Establishment of a kinetic model of dialysis-related amyloid fibril extension in vitro

beta(2)-microglobulin (beta(2)M) is a major structural component of dialysis-related amyloid fibrils (fA beta(2)M). In order to make clear the mechanism of fA beta(2)M 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 fA beta(2)M formation in vitro. We first determined the optimum conditions for quantitative fluorometry of fA beta(2)M with thioflavine T (ThT). Optimum fluorescence measurements of fA beta(2)M were obtained at the excitation and emission wavelengths of 455 nm and 485 nm, respectively, with the reaction mixture containing 3 mu M ThT and 50 mM of glycine-NaOH buffer pH 8.5. We then focused our study on the extension phase of fA beta(2)M formation in vitro. When fA beta(2)M were incubated with monomeric beta(2)M, the extension of fA beta(2)M was observed with electron microscopy. quantitative fluorometry revealed that: (a) extension of fA beta(2)M 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 fA beta(2)M number concentration and the beta(2)M concentration; (d) the net rate of extension was the sum of the rates of polymerization and depolymerization. These results show that fA beta(2)M formation can be explained by a first-order kinetic model: that is, the extension of fA beta(2)M proceeds via the consecutive association of beta(2)M onto the ends of existing fibrils. We propose that this model could be generally applied for the extension of all types of amyloid fibrils in vitro.