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