The formation of amyloid-like fibrils was studied by using the well-known serine protease
trypsin as a model protein in the presence of ethanol as organic solvent. Trypsin forms amyloid-like
fibrils in aqueous ethanol at pH = 7.0. The dye Congo red (CR) was used to detect the presence of
amyloid-like fibrils in the samples. The binding of CR to fibrils led to an increase in absorption intensity
and a red shift in the absorption band of CR. Thioflavin T (ThT) and 8-anilino-1-
naphthalenesulfonic acid (ANS) binding assays were employed to characterize amyloid-like fibril formation. The ThT
binding assay revealed that the protein exhibited maximum aggregation in 60% (v/v) ethanol after incubation for 24 h at
24 oC. The ANS binding results indicated that the hydrophobic residues were more exposed to the solvent in the aggregated
form of the protein. The effects of polyethylene glycol (PEG) on the formation of amyloid-like fibrils was studied in
vitro. The aggregation of trypsin was followed via the kinetics of aggregation, the far-UV circular dichroism (CD) and
transmission electron microscopy (TEM) in the presence and absence of PEG. The CD measurements indicated that the
protein aggregates have a cross-beta structure in 60% ethanol. TEM revealed that trypsin forms fibrils with a thread-like
structure. The inhibitory effect of PEG on the aggregation of trypsin increased with rising PEG concentration. PEG therefore
inhibits the formation of amyloid-like fibrils of trypsin in aqueous ethanol.
Keywords: Amyloid-like fibril, trypsin, organic solvent, polyethylene glycol, circular dichroism, Congo red.
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