Ponceau 4R induces aggregation in human serum albumin and morin acts as an anti-aggregating agent against dye induced aggregates

Aggregation of proteins occurs because of improper protein folding and is responsible for the development of multiple severe maladies such as Type II diabetes mellitus, Parkison's, Huntington's, and spongiform encephalopathy. In the current work, the interaction and aggregation of human serum albumin (HSA) in the presence of the food colorant Ponceau 4R at pH 2.0 was evaluated using multiple in-silico and multi-spectroscopic approaches. The UV-visible spectroscopy and steady state fluorescence spectroscopy confirmed the complex formation between Ponceau 4R and HSA. The binding of HSA to Ponceau 4R favored static mode of fluorescence quenching. The values of K_SV and K_b were found to be 1.905 × 10⁵ M^-1 and 1.43 × 10⁵ M^-1 respectively. Ponceau 4R leads to modification of HSA microenvironment, as proven by three-dimensional fluorescence spectroscopy. Ponceau 4R in the concentration range (60-200 µM) triggered the aggregation of HSA at pH 2.0 as confirmed by turbidity and Rayleigh light scattering (RLS). The secondary structure alteration of human serum albumin (α-helical structure to cross-β conformation) induced by 140 µM Ponceau 4R was evaluated utilizing far-UV CD spectroscopy. This concentration of Ponceau 4R (140 µM) will now be further used in aggregation inhibition studies. The production of β-rich aggregates of HSA is further established by a red shift in the absorption maxima of the Congo red spectra. The scanning electron microscopy (SEM) results showed that HSA aggregates induced by Ponceau 4R were amorphous in nature. Molecular docking analysis showed that electrostatic interactions, hydrophobic interactions and hydrogen bonding were responsible for the Ponceau 4R-induced aggregation of HSA. Furthermore, morin further inhibits the Ponceau 4R-induced aggregation of HSA. The results from turbidity, RLS, and SEM analysis confirmed that aggregates of HSA induced by 140 µM Ponceau 4R became soluble when human serum albumin was pre-incubated with various amounts of morin (0-600 µM). Thus, we conclude that Ponceau 4R triggers the aggregation of human serum albumin, whereas morin is responsible for the inhibition of aggregation of protein.

Aggregation; Circular dichroism; Human serum albumin; Molecular docking; Ponceau 4R; Scanning electron microscopy.