Heparin is one of the members of the glycosaminoglycan (GAG) family, which has been associated with protein aggregation diseases including Alzheimer’s disease, Parkinson’s disease, and prion diseases. Here, we investigate heparin-induced aggregation of bovine serum albumin (BSA) using different spectroscopic techniques [absorption, 8-anilino-1-naphthalene sulfonic acid (ANS) and thioflavin T (ThT) fluorescence binding, and far- and near-UV circular dichroism]. Kinetic measurements revealed that heparin is involved in the significant enhancement of aggregation of BSA. The outcomes showed dearth of the lag phase and a considerable change in rate constant, which provides conclusive evidence, that is, heparin-induced BSA aggregation involves the pathway of the downhill polymerization mechanism. Heparin also causes enhancement of fluorescence intensity of BSA significantly. Moreover, heparin was observed to form amyloids and amorphous aggregates of BSA which were confirmed by ThT and ANS fluorescence, respectively. Circular dichroism measurements exhibit a considerable change in the secondary and tertiary structure of the protein due to heparin. In addition, binding studies of heparin with BSA to know the cause of aggregation, isothermal titration calorimetry measurements were exploited, from which heparin was observed to promote the aggregation of BSA by virtue of electrostatic interactions between positively charged amino acid residues of protein and negatively charged groups of GAG. The nature of binding of heparin with BSA is very much apparent with an appreciable heat of interaction and is largely exothermic in nature. Moreover, the Gibbs free energy change (ΔG) is negative, which indicates spontaneous nature of binding, and the enthalpy change (ΔH) and entropy change (ΔS) are also largely negative, which suggest that the interaction is driven by hydrogen bonding.

Protein aggregation and misfolding are some of the most challenging obstacles, customarily studied for their association with amyloid pathologies. The mechanism of amyloid fibrillation development is a dynamic phenomenon involving various factors such as the intrinsic properties of protein and the physical and chemical environmental conditions. The purpose of this study was to see the thermal aggregation profile of alpha-lactalbumin (α-LA) and to delineate the effect of trehalose on its aggregation profile. α-LA was subjected to thermal aggregation at high concentrations. UV-Vis spectroscopy, a turbidity assay, intrinsic fluorescence, Rayleigh scattering and a thioflavin T (ThT) assay explained the steady outcomes that 1 M trehalose repressed α-LA aggregation in the most effective way followed by 0.75 M and 0.5 M and to a significantly lesser degree by 0.25 M. Multi spectroscopic obser Sania Bashir ations were further entrenched by microscopy. Transmission electron microscopy confirmed that in the presence of its higher concentration, trehalose hinders fibril development in α-LA. In vitro studies were further validated by in silico studies. Molecular docking analysis indicated that trehalose occupied the binding pocket cavity of α-LA and offered several significant interactions, including H-bonds with important residues. This study provides a platform for trehalose in the therapeutic management of protein aggregation-related diseases.

GRAPHICAL ABSTRACT 1Kinetic measurements indicating that monosodium glutamate causes significant enhancement of aggregation of protein through nucleation-dependent polymerization mechanism. Even low concentration of MSG is involved in the unfolding of secondary structure of protein with the disappearance of original peaks (208 and 222 nm) and formation of unique peak (226 nm) in the far-UV CD.