title: Amyloid-like Fibril Formation by Trypsin in Aqueous Ethanol. Inhibition of Fibrillation by PEG.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Science and Informatics, University of Szeged, H-6726 Szeged, Középfasor 52, Hungary. kotorman@expbio.bio.u-szeged.hu.
authors.forename: Márta
authors.initials: M
authors.lastname: Kotormán
authors.forename: Mária L
authors.initials: ML
authors.lastname: Simon
authors.forename: Attila
authors.initials: A
authors.lastname: Borics
authors.forename: Márton Richárd
authors.initials: MR
authors.lastname: Szabó
authors.forename: Kitti
authors.initials: K
authors.lastname: Szabó
authors.forename: Titanilla
authors.initials: T
authors.lastname: Szögi
authors.forename: Lívia
authors.initials: L
authors.lastname: Fülöp
mesh_terms: D000682:Amyloid / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D002942:Circular Dichroism; D003224:Congo Red; D000431:Ethanol / Q000737:chemistry*; D007700:Kinetics; D009391:Nephelometry and Turbidimetry; D011092:Polyethylene Glycols / Q000494:pharmacology*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000682:Amyloid; D000431:Ethanol; D003224:Congo Red; D011092:Polyethylene Glycols; D014357:Trypsin