  The importance of understanding the mechanism of protein aggregation into
insoluble amyloid fibrils relies not only on its medical consequences, but also
on its more basic properties of self--organization. The discovery that a large
number of uncorrelated proteins can form, under proper conditions, structurally
similar fibrils has suggested that the underlying mechanism is a general
feature of polypeptide chains. In the present work, we address the early events
preceeding amyloid fibril formation in solutions of zinc--free human insulin
incubated at low pH and high temperature. Aside from being a easy--to--handle
model for protein fibrillation, subcutaneous aggregation of insulin after
injection is a nuisance which affects patients with diabetes. Here, we show by
time--lapse atomic force microscopy (AFM) that a steady-state distribution of
protein oligomers with an exponential tail is reached within few minutes after
heating. This metastable phase lasts for few hours until aggregation into
fibrils suddenly occurs. A theoretical explanation of the oligomer
pre--fibrillar distribution is given in terms of a simple
coagulation--evaporation kinetic model, in which concentration plays the role
of a critical parameter. Due to high resolution and sensitivity of AFM
technique, the observation of a long-lasting latency time should be considered
an actual feature of the aggregation process, and not simply ascribed to
instrumental inefficency. These experimental facts, along with the kinetic
model used, claim for a critical role of thermal concentration fluctuations in
the process of fibril nucleation.
