title: Disulfide-bond scrambling promotes amorphous aggregates in lysozyme and bovine serum albumin.
abstract: Disulfide bonds are naturally formed in more than 50% of amyloidogenic proteins, but the exact role of disulfide bonds in protein aggregation is still not well-understood. The intracellular reducing agents and/or improper use of antioxidants in extracellular environment can break proteins disulfide bonds, making them unstable and prone to misfolding and aggregation. In this study, we report the effect of disulfide-reducing agent dithiothreitol (DTT) on hen egg white lysozyme (lysozyme) and bovine serum albumin (BSA) aggregation at pH 7.2 and 37 °C. BSA and lysozyme proteins treated with disulfide-reducing agents form very distinct amorphous aggregates as observed by scanning electron microscope. However, proteins with intact disulfide bonds were stable and did not aggregate over time. BSA and lysozyme aggregates show unique but measurable differences in 8-anilino-1-naphthalenesulfonic acid (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence, suggesting a loose and flexible aggregate structure for lysozyme but a more compact aggregate structure for BSA. Scrambled disulfide-bonded protein aggregates were observed by nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for both proteins. Similar amorphous aggregates were also generated using a nonthiol-based reducing agent, tris(2-carboxyethyl)phosphine (TCEP), at pH 7.2 and 37 °C. In summary, formation of distinct amorphous aggregates by disulfide-reduced BSA and lysozyme suggests an alternate pathway for protein aggregation that may be relevant to several proteins.
authors.affiliation: Department of Chemistry, Michigan Technological University , Houghton, Michigan 49931, United States.
authors.forename: Mu
authors.initials: M
authors.lastname: Yang
authors.forename: Colina
authors.initials: C
authors.lastname: Dutta
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Tiwari
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002645:Chickens; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D008855:Microscopy, Electron, Scanning; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000817:Anilino Naphthalenesulfonates; D004220:Disulfides; D004229:Dithiothreitol; D009113:Muramidase; D010720:Phosphines; D012710:Serum Albumin, Bovine; C515594:8-anilino-1-naphthalenesulfonic acid; C080938:tris(2-carboxyethyl)phosphine; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); C421864:hen egg lysozyme