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.

Thiol disulfide exchange reactions in human serum albumin: the apparent paradox of the redox transitions of Cys34

“…Only in a recent study, the presence of albumin with mixed disulfides involving other protein cysteines like Cys 90 , and Cys 112 in hyper-lipidemic patients was discovered [23]; indeed, even partial disruption of its disulfide bridges could represent a dramatic event as such albumin undergoes deleterious aggregation and amyloid polymerization [24]. Only in a recent study, the presence of albumin with mixed disulfides involving other protein cysteines like Cys 90 , and Cys 112 in hyper-lipidemic patients was discovered [23]; indeed, even partial disruption of its disulfide bridges could represent a dramatic event as such albumin undergoes deleterious aggregation and amyloid polymerization [24].…”

Amyloid formation kinetics of hen egg white lysozyme under heat and acidic conditions revealed by Raman spectroscopy

“…Dynamic light scattering and atomic force microscopy (AFM) experiments further suggested that the amyloid fibril assembly of lysozyme followed a strict hierarchical aggregation routine, in a so‐called on‐pathway from the amyloid monomers, oligomers, protofibrils, and more complex structures . In addition, the very distinct amorphous aggregates of HEWL were found to be formed following disulfide‐reducing treatment, implying an alternate pathway for protein aggregation …”

Effect of crowding on several stages of protein aggregation in test systems in the presence of α-crystallin

“…Dithiothreitol (DTT)-induced aggregation was demonstrated, for example, for ␣-lactalbumin, insulin, lysozyme and bovine serum albumin [1][2][3][4]. The study of the kinetics of DTT-induced aggregation of ␣-lactalbumin and insulin showed that the formation of start aggregates including the hundreds of molecules of denatured protein occurred at the initial stages of aggregation [4][5][6][7]. Start aggregates emerge on allor-none principle without accumulation of intermediates between the non-aggregated protein and start aggregates.…”