Amyloid-like Fibril Formation by Trypsin in Aqueous Ethanol. Inhibition of Fibrillation by PEG

Abstract

Abstract: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 o C. 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.

Inhibition of Amyloid-like Fibril Formation of Trypsin by Red Wines

“…Light dispersion of the sample was significantly decreased by the addition of 50-fold diluted Egri bikavér wine, suggesting that a lower amount of aggregates is present compared to the sample not containing the additive. It was shown previously, that high concentrations of ethanol promote structural transitions of PMS-trypsin, resulting in an increase of β-sheet structure of the protein [24]. Such structural features were not changed remarkably by the addition of diluted red wine.…”

“…Earlier we have demonstrated that phenylmethylsulfonyl-trypsin (PMS-trypsin) forms amyloid-like fibrils in 60% (v/v) ethanol/10 mM phosphate buffer (PBS) at pH = 7.0 [24]. Here, we report that various red wines inhibit the formation of amyloid-like fibrils of PMS-trypsin dosedependently.…”

“…PMS-trypsin amyloid-like fibrils were prepared in aqueous ethanol according to our previous report [24]. To determine whether red wines affect fibril formation of PMStrypsin, samples were incubated in aqueous ethanol at pH 7.0, in the presence and absence of different red wines.…”

The Anti-amyloidogenic effect of natural product extracts on amyloid-like fibril formation of trypsin in aqueous organic solvents

“…The chemically modified enzyme is catalytically inactive, thus there is no autolysis at pH 7.0. Amyloid-like fibrils were prepared using PMS-trypsin as previously reported by the incubation of the soluble protein in the presence of 60% ethanol at 24 o C at pH 7.0 (Kotormán et al, 2015). The turbidity measurement is a procedure for detecting aggregates (Zhao et al, 2016).…”

“…It is known from our research group's previous experiments that in the presence of 10 mM phosphate buffer at pH 7.0 in the structure of the PMS-trypsin, α-helix is present with the dominance of the βsheet. The ECD spectrum obtained in the presence of 60% ethanol was characteristic of amyloid fibrils (Kotormán et al, 2015). Differences between the measured spectra arise from the differences in size distribution of the aggregates evolving gradually.…”