Mutant human lysozymes (Ile56Thr & Asp67His)
have been reported to form amyloid deposits in the viscera.
From the standpoint of understanding the mechanism of amyloid
formation, we searched for conditions of amyloid formation
in vitro using hen egg lysozyme, which has been extensively
studied from a physicochemical standpoint. It was found
that the circular dichroism spectra in the far-ultraviolet
region of the hen egg lysozyme changed to those characteristic
of a β-structure from the native α-helix rich spectrum
in 90% ethanol solution. When the concentration of protein
was increased to 10 mg/mL, the protein solution formed
a gel in the presence of 90% ethanol, and precipitated
on further addition of 10 mM NaCl. The precipitates were
examined by electron microscopy, their ability to bind
Congo red, and X-ray diffraction to determine whether amyloid
fibrils were formed in the precipitates. Electron micrographs
displayed unbranched protofilament with a diameter of ∼70
Å. The peak point of the difference spectrum for
the Congo red binding assay was 541 nm, which is characteristic
of amyloid fibrils. The X-ray diffraction pattern showed
a sharp and intense diffraction ring at 4.7 Å, a
reflection that arises from the interstrand spacing in
β-sheets. These results indicate that the precipitates
of hen egg lysozyme are amyloid protofilament, and that
the amyloid protofilament formation of hen egg lysozyme
closely follows upon the destruction of the helical and
tertiary structures.