Herein, the interaction between caffeic acid (CA), a hydroxycinnamic acid with favorable health impacts through the prevention of degenerative pathologies, and bovine serum albumin (BSA) was investigated. The effect of CA on the conformation and thermal stability of BSA was studied using isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD) spectroscopy, and molecular docking. Thermodynamic parameters (ΔH, ΔS, and ΔG), number of site types (N), and binding constant (K) were determined by ITC. Competitive binding of ITC with warfarin (WAR) and Ibuprofen (IBP) suggested that one of the BSA binding sites for CA was missed in the fluorescence measurements. Molecular docking studies indicate that CA binds to BSA at two sites located in the IIIA and IIA subdomains of the protein native structure. This confirmed the ITC results of competitive binding. Comprehensive DSC and CD experiments revealed a protein stability enhancement in the presence of CA. At higher concentrations, CA induces a slight decrease in the α-helix content of BSA, as observed in the far-UV CD spectra, whereby a partial unfolding of the protein occurs, as proven via both the DSC and CD experiments. This extensive study provides substantial data regarding the ligand binding effect on the protein structure that is significant for understanding the biological activity of BSA in vivo.

A thermoanalytical approach reveals the dual action of GA on BSA thermal stability.

In this study, the effect of gamma irradiation (10 kGy) on proteins extracted from animal hide, scales, and wool was evidenced by calorimetric (μDSC) and spectroscopic (IR, circular dichroism, and EPR) methods. Keratin was obtained from sheep wool, collagen and bovine gelatin from bovine hide, and fish gelatin from fish scales. The μDSC experiments evidenced that gamma irradiation influences the thermal stability of these proteins differently. The thermal stability of keratin decreases, while a resistance to thermal denaturation was noticed for collagen and gelatins after gamma irradiation. The analysis of the IR spectra demonstrated that gamma irradiation determines changes in the vibrational modes of the amide groups that are associated with protein denaturation, most meaningfully in the case of keratin. As evidenced by circular dichroism for all proteins considered, exposure to gamma radiation produces changes in the secondary structure that are more significant than those produced by UV irradiation. Riboflavin has different effects on the secondary structure of the investigated proteins, a stabilizing effect for keratin and fish gelatin and a destabilizing effect for bovine gelatin, observed in both irradiated and non-irradiated samples. The EPR spectroscopy evidences the presence, in the gamma-irradiated samples, of free radicals centered on oxygen, and the increase in their EPR signals over time due to the presence of riboflavin.