Multispectroscopic and computational insights into amyloid fibril formation of alpha lactalbumin induced by sodium hexametaphosphate

Abstract

Abstract The impact of sodium hexametaphosphate (SHMP) on the aggregation behavior of α-lactalbumin (α-LA) was studied at pH 7.4 and 2.0. Turbidity measurements showed a concentration-dependent aggregation of α-LA at pH 2.0 in the presence of SHMP, while no aggregation was observed at pH 7.4. Light scattering (LS) and Thioflavin-T (ThT) data revealed that the aggregation was rapid, following nucleation-independent pathways. In other kinetics experiments such as turbidity and ThT confirmed that SHMP-induced α-LA aggregation was dependent on SHMP concentration rather than incubation time. Once formed, the aggregates remained unchanged for up to five days. Intrinsic fluorescence studies indicated conformational changes in α-LA upon SHMP addition, and dye-binding assays with ThT and Congo Red demonstrated the formation of amyloid-like aggregates. Far-UV circular dichroism (CD) data suggested a structural transition from α-helical to β-structures in α-LA in the presence of SHMP at pH 2.0. Molecular docking studies confirmed stronger interactions between α-LA and SHMP at pH 2.0 (ΔG = −6.2 kcal/mol) compared to pH 7.4 (ΔG = −5.3 kcal/mol), driven by electrostatic forces and hydrogen bonding. These results suggest that SHMP induces amyloid-like aggregation of α-LA, particularly at acidic pH.

Keywords

• Amyloid fibril
• Protein conformation
• Stability
• Alpha-lactalbumin
• Sodium hexametaphosphate
• ThT