Food Additive Hexametaphosphate Promotes Amyloid Formation in Human Serum Albumin: A Molecular Insight

ABSTRACT This study investigates the aggregation behavior of human serum albumin (HSA) in its cationic (pH 2.0) and anionic (pH 8.0) states upon exposure to hexametaphosphate (HMP), a polyanionic compound. UV–Vis turbidity measurements revealed that cationic HSA aggregated in a concentration‐dependent manner starting at 0.01 mM HMP and plateaued beyond 0.05 mM, while anionic HSA remained soluble even at 15 mM HMP. Intrinsic fluorescence analysis showed a blue shift in the emission maximum of cationic HSA, indicating conformational changes associated with aggregation, whereas no shift was observed in anionic HSA. Far‐UV circular dichroism (CD) spectroscopy demonstrated that cationic HSA lost its alpha‐helical structure and adopted cross‐beta sheet conformations at HMP concentrations ≥ 0.05 mM, consistent with amyloid formation, which was further supported by increased Thioflavin T (ThT) fluorescence. Rayleigh light scattering (RLS) and ThT kinetic studies confirmed rapid, saturation‐limited aggregation without a lag phase. Transmission electron microscopy (TEM) further verified the presence of amyloid‐like fibrils in cationic HSA treated with HMP. In contrast, anionic HSA showed no structural or aggregation changes under identical conditions. These findings highlight the pH‐dependent, amyloidogenic potential of HSA in the presence of HMP and underscore the role of electrostatic interactions in protein aggregation.