Abstract

Under in vitro conditions that destabilize the native state, proteins often undergo structural changes leading to aggregation, implicated in disorders such as Alzheimer’s disease. Human Serum Albumin (HSA), a major plasma protein, serves as a model for studying such aggregation-related changes. HSA was incubated at 65°C for 0–256 hours to monitor time-dependent structural alterations. Spectrophotometric analyses, including intrinsic fluorescence, UV and Congo Red absorbance, ThT fluorescence, and CD spectroscopy, were employed. The protective effect with varying concentrations of curcumin (15-60 μM), a known anti-aggregatory agent, was investigated in the presence of curcumin. Thermal treatment of HSA resulted in increased ThT fluorescence at 128 hours and UV absorbance up to 5-fold, indicating protein unfolding and consequently aggregation. Red-shifted CR absorbance of 5 nm and the appearance of non-native β-sheet minima in the CD spectra at 218 nm. In the presence of curcumin, a marked reduction in UV and CR absorbance, along with decreased ThT and intrinsic fluorescence, suggested a partial restoration of native structure. CD analysis confirmed, curcumin mitigated thermally induced secondary structural changes by reappearance of peaks at 208 nm and 222 nm. Thermal incubation of HSA for 128 hours leads to time-dependent unfolding and aggregation, mimicking pathogenic protein aggregation. TEM imaging proves curcumin (45 μM) exhibits a protective effect by inhibiting aggregation and preserving native protein structure, highlighting its potential therapeutic relevance in aggregation-related disorders and as a structural stabilizer in heat-challenged biopharmaceutical systems as well.

Key words: Protein aggregation, Curcumin, Human Serum Albumin (HSA), Thermal Stress, Thioflavin T (ThT) fluorescence, Structural stabilization