[20] Kinetic analysis of amyloid fibril formation

A nucleation-dependent polymerization model has been proposed to explain the mechanisms of amyloid fibril formation in vitro. This model consists of two phases—the nucleation and extension phases. Nucleus formation requires a series of association steps of monomers that are thermodynamically unfavorable, representing the rate-limiting step in amyloid fibril formation. Once the nucleus (n-mer) has been formed, the further addition of monomers becomes thermodynamically favorable, resulting in a rapid extension of amyloid fibrils. This chapter developes a first-order kinetic model of amyloid fibril extension in vitro and confirmed that the extension of amyloid fibrils proceeds via the consecutive association of monomeric precursor proteins onto the ends of existing fibrils. A characteristic sigmoidal time-course curve of amyloid fibril formation from monomeric precursor proteins at a physiological pH is widely believed to represent the essence of a nucleation-dependent polymerization model—that is, an initial lag phase represents the thermodynamically unfavorable nucleus formation. However, no convincing kinetic models to explain the sigmoidality of the curve have been reported. This chapter describes the paradigm to analyze the kinetic aspect of amyloid fibril formation in vitro. During the investigation of murine senile amyloidosis observed in the senescence-accelerated mouse (SAM), it develops a novel fluorometric method to determine amyloid fibrils in vitro based on the unique characteristics of thioflavin T (ThT). In recent years, this method has been applied to the analysis of several human amyloidoses, including gelsolin-derived amyloidosis, dialysis-related amyloidosis, and Alzheimer's β-amyloidosis. With this fluorometric method, this chapter successfully measures the polymerization velocity of three types of amyloid fibrils—that is, murine senile amyloid fibrils (fAApoAII), Alzheimer's β-amyloid fibrils (fAβ), and dialysis-related amyloid fibrils (fAβ2-m), and performs kinetic analysis of amyloid fibril polymerization in vitro.