Amyloid gels: precocious appearance of elastic properties during the formation of an insulin fibrillar network.

The formation of insulin amyloid fibrils is important not only for the development of reliable drugs but also for modeling the basic properties of protein self-assembly. Fibrillation kinetics is typically characterized by an initial apparent lag phase related to the formation of oligomers, protofibrils, and aggregation nuclei. Afterwards, aggregation proceeds over a wide range of length scales via fibril elongation, thickening, and/or flocculation and eventual gelation. By light scattering and rheological techniques, we reveal the structural details hidden in the apparent lag phase and we show the unexpected appearance of noteworthy elastic properties concurrently with initial fibril nucleation and elongation preceding the formation of the larger structures and the gel network.

Ordering of agarose near the macroscopic gelation point.

Gel formation and spatial structure is an important area of study in polymer physics and in macromolecular and cellular biophysics. Agarose has a sufficiently complex gelation mechanism to make it an interesting prototype for many other gelling systems, including those involved in amyloid fibrillogenesis. Static (over a scattering vector range of 0.1-30 microm(-1)) and dynamic light scattering and rheology methods were used to follow the gelation kinetics of agarose at 0.5% in water or in the presence of 25 mM NaCl and quenched to temperatures of 20-43 degrees C. Light scattering results on gelling samples are fully described by a fractal aggregate model with four physically meaningful parameters. In all cases aggregates, with fractal dimensions at or near 3, form more rapidly and are smaller in characteristic size at lower quench temperatures. A region three to four times larger than the aggregate becomes depleted of agarose as the gelation proceeds. Below about 30 degrees C the aggregation process freezes spatial ordering rapidly, resulting in fragile macroscopic gels as determined by rheology. Salt effects are seen to be minimal and not important in the fundamental aggregation mechanism.

Inhibitory effects of arginine on the aggregation of bovine insulin.

Static and dynamic light scattering were used to investigate the effects of L-arginine, commonly used to inhibit protein aggregation, on the initial aggregation kinetics of solutions of bovine insulin in 20% acetic acid and 0.1 M NaCl as a model system for amyloidosis. Measurements were made as a function of insulin concentration (0.5-2.0 mM), quench temperature (60-85°C), and arginine concentration (10-500 mM). Aggregation kinetics under all conditions had a lag phase, whose duration decreased with increasing temperature and with increasing insulin concentration but which increased by up to a factor of 8 with increasing added arginine. Further, the initial growth rate after the lag phase also slowed by up to a factor of about 20 in the presence of increasing concentrations of arginine. From the temperature dependence of the lag phase duration, we find that the nucleation activation energy doubles from 17 ± 5 to 36 ± 3 kcal/mol in the presence of 500 mM arginine.