Amyloid detecting dye "Congo Red" induces amorphous aggregates in hen egg white lysozyme at physiological pH

Congo Red (CR) is an azo dye commonly used for amyloid detection and histological staining. However, its potential influence on protein aggregation remains unclear. In this study, we investigated the effect of CR on the aggregation of Hen Egg White Lysozyme (HEWL) under physiological and variable environmental conditions. At pH 7.4, HEWL remained soluble in the absence of CR; however, the addition of CR at concentrations ≥0.05 mM induced rapid and visible aggregation without a detectable lag phase, suggesting a non-nucleated aggregation mechanism. SDS-PAGE confirmed a marked reduction in soluble HEWL at CR concentrations above 0.05 mM. Circular dichroism spectroscopy indicated that the protein retained its α-helical structure but exhibited a progressive loss in ellipticity, consistent with the formation of amorphous aggregates. Transmission electron microscopy further verified the presence of amorphous aggregates. The presence of salts, particularly (NH₄)₂SO₄, strongly enhanced aggregation, while acidic pH (2.0) promoted aggregation, whereas alkaline pH reduced it. Polyethylene glycol 4000 (PEG 4000) was ineffective at solubilizing preformed aggregates. Molecular docking and MD simulations showed that CR binds stably to HEWL, with interactions involving catalytic residues Asp52 and Arg114 via hydrogen bonding and electrostatic interactions. The complex remained conformationally stable for 100 ns, suggesting strong and persistent interactions. Overall, our findings demonstrate that CR can directly promote lysozyme aggregation and emphasize caution when using CR in amyloid research.