Amyloid protofilament formation of hen egg lysozyme in highly concentratedethanol solution

Mutant human lysozymes (IleS6Thr & Asp67His) have been reported to form amy loid deposits in the viscera. From the standpoint of understanding the mech anism 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 spect ra in the far-ultraviolet region of the hen egg lysozyme changed to those c haracteristic of a beta-structure from the native ct-helix rich spectrum in 90% ethanol solution. When the concentration of protein was increased to 1 0 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 e xamined by electron microscopy, their ability to bind Congo red, and X-ray diffraction to determine whether amyloid fibrils were formed in the precipi tates. Electron micrographs displayed unbranched protofilament with a diame ter of similar to 70 Angstrom. The peak point of the difference spectrum fo r the Congo red binding assay was 541 nm, which is characteristic of amyloi d fibrils. The X-ray diffraction pattern showed a sharp and intense diffrac tion ring at 4.7 Angstrom, a reflection that arises from the interstrand sp acing in beta-sheets. These results indicate that the precipitates of hen e gg lysozyme are amyloid protofilament, and that the amyloid protofilament f ormation of hen egg lysozyme closely follows upon the destruction of the he lical and tertiary structures.