The formation of amyloid-beta fibrils within the human brain has been reported to be a possible cause for Alzheimer’s disease. Amyloid fibrils can also be formed from various non-disease causing proteins. In this study, in vitro formation of amyloid fibrils from bovine gamma globulin and hen egg white lysozyme (HEWL) and the subsequent proteolytic degradation provided an exploration of amyloid fibril properties. The fibril formation from bovine gamma globulin at pH 2 was monitored using thioflavin T (ThT), which is known to bind amyloid fibril to form a complex, resulting in an increase of ThT fluorescence without a lag phase. The excitation wavelength used was 440 nm with an emission wavelength of 482 nm. The proteases, pepsin and trypsin were subsequently added to the pre-formed amyloid fibril to induce degradation. Pepsin’s properties allowed for the pH of the fibril networks to remain at 2, while for trypsin an adjusted pH of 7 ensured that the trypsin would be active. A decrease in fluorescence of the amyloid fibril-ThT complex confirmed that proteolytic degradation occurred to the amyloid fibril. The rate constant and the half-life for the degradation were calculated to compare influences from changes in pH to that of the proteases. Final half-life calculations for degradation of the amyloid fibril came to be 1.3 hours for pepsin, 9.2 hours for the pH change for use of trypsin, and 14 hours for trypsin. This indicates that pepsin at pH 2 had catalyzed proteolysis of the fibril faster than trypsin or changes in pH. In vitro formation of amyloid fibril from hen egg white lysozyme at pH 2 was also studied using ThT fluorescence. The excitation and emission wavelengths were 440 and 480 nm for the lysozyme-ThT complex. Scanning electron microscopy (SEM) studies of gamma globulin at 0.24 mg/mL in pH 2 50mM phosphate buffer and HEWL at 0.1 mg/mL in pH 2 KCl saline at 65°C produced dendritic structures due to the air drying preparation for SEM. Future studies will expand the exploration of these degradation processes for amyloid fibril and applications for developing potential anti-amyloid agents along with efforts to further view the fibrils using the SEM.
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