Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins.

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Polarization imaging method, PIM, can provide us with two-dimensionally spatial distributions of birefringence and extinction angle. In a previous study, we reported a new rheo-optical apparatus based on conbination of PIM and a conventional rheometer. The appratus was very effective to visualize the stress field of shear induced sturucture. However, there was a problem in the quantitative evaluation of birefringence, which can be attributed to non-ideality of the wave plate for the circuler polarizer. In this paper, we improved the wave plate and reports a new data for a wormlike micelle solution which is composed of 30 mol/m 3 cetyltrimethylammonium bromide and 60 mol/m 3 sodium salicylate. While the previous optical system provided us with 2-fold symmetry images, the new one provided us with almost uniform birefringence distributions. The spatially mean value of the birefringence distribution, [Δn], in low strain range in the new optical system meets the stress-optical rule, and the stress-optical coefficient is consistent to literature value in linear regime. In non-linear regime where the finite extensibility effect significantly contribute to the stress, the stress-optical coefficient can be described as a unique function of orientation factor of micelles.