Comparative study of the hydrophobic interaction effect of pH and ionic strength on aggregation/emulsification of Congo red and amyloid fibrillation of insulin.

Amyloid fibrillation is provoked by the conformational rearrangement of its source. In our previous study, we claimed that the conformational rearrangement of hen egg white lysozyme requires intermolecular aggregation/packing induced. Our proposed causality of the aggregation and amyloid formation was demonstrated by the quantitative dependence of amyloid fibrillation on pH difference from its isoelectric point (pI) and on the square root of ionic strength in order to reduce the intermolecular repulsion due to the shielding effect of electrolytes (DLVO effect). When Congo red has dianionic form at the pH higher than its pKa, it forms ribbon-like micelle colloids under lower ionic strength, while it loses electrostatic repulsion and aggregates to be emulsified in the octanolic phase under the higher ionic strength. These behaviors of Congo red were resembling to molecular assembly of surfactants. In contrast, the amyloid formation of insulin was proportional to the square root of ionic strength at the pH lower than its isoelectric point. Therefore, the trigger for conformational rearrangement of amyloid fibrillation is predominantly gripped by hydrophobic hydration and an electrostatic shielding effect. We concluded that the both behaviors of Congo red and insulin were derived from a driving force related to the hydrophobic hydration.

Singular value decomposition analysis of the secondary structure features contributing to the circular dichroism spectra of model proteins.

Amyloid fibril formation occurs in restricted environment, such as the interface between intercellular fluids and bio-membranes. Conformational interconversion from α-helix to ß-structure does not progress in fluids; however, it can occur after sedimentary aggregation during amyloid fibril formation induced by heat treatment of hen egg white lysozyme (HEWL). Secondary structures of various proteins and denatured proteins titrated with 2,2,2-trifluoroethanol (TFE) were examined using their CD spectra. Gaussian peak/trough and singular value decompositions (SVD) showed that the spectral pattern of the α-helix comprised a sharp trough at wavelength 207 nm and a broad trough at 220 nm. Conversely, we distinguished two patterns for ß-sheet-a spread barrel type, corresponding to ConA, and a tightly weaved type, corresponding to the soybean trypsin inhibitor. Herein, we confirmed that the spectral/conformational interconversion of the heat-treated HEWL was not observed in the dissolved fluid.

Effects of ionic and reductive atmosphere on the conformational rearrangement in hen egg white lysozyme prior to amyloid formation.

In this study, the combined effects of pH and salt concentration on the aggregation and amyloid formation of a charge-bearing protein (hen egg white lysozyme, HEWL) were investigated, as well as the inhibition of amyloid formation by using dithiothreitol (DTT) as a denaturing agent. Amyloid formation was found to depend on the ion strength and pH of the sample solution. Rather than the total charge, the partial charge of the amyloid related residues contributes to amyloid formation at pH < isoelectric point (pI). On the other hand, at pH> pI HEWL only undergoes alkaline denaturation regardless of the ionic strength. The effect of adding different amounts of DTT at different times on amyloid formation was also investigated. These results suggested that the positions of charges on a protein and the protein secondary structure are critical for protein aggregation and amyloid formation.