Monitoring the Time-Dependency of Multi-Stage Events in Thermal BSA Aggregation-Fibrillation: Electrochemical Admittance Approach

Bovine serum albumin (BSA) thermal aggregation follows hyperbolic behavior, lacking a distinct nucleation process, suggesting spontaneous unfolding and assembly above the melting point. Common methods like thioflavin T (ThT) assay may not effectively discern initial BSA aggregation events. To overcome this limitation, advanced electrochemical approaches have been used to clarify the mechanisms of conformational changes and the overall kinetics of the process. Time domain monitoring of the BSA conformational changes under thermal aggregation using fast Fourier transform admittance measurement revealed four preliminary stages of the BSA aggregation process. These four stages were further investigated using various spectroscopic techniques, electron microscopy, ζ -potential analysis, and surface tension measurements. Temperature, as the main driving force, has different effects across different temperature ranges. Key findings included the onset of unfolding post-42 °C (0–1.25 min), assembly near the melting point (3.5 min), and the formation of aggregates and β -sheets at thermal equilibrium (after 3.5 min) based on data obtained. MD simulation of BSA showed that high temperatures accelerate the unfolding process and correspond to β -sheet formation. Electrochemical admittance findings provided valuable insights into BSA conformational changes, showcasing its promising potential to study protein misfolding-related diseases.