Exploring the structural stability of hemoglobin in DBU-based ionic liquids: Insights from spectroscopic investigations

Abstract

Globular proteins like heme proteins have garnered significant attention owing to their multiple applications in biochemical as well as biophysical processes and are the major functionalities in various pharmaceuticals and health care products. Hemoglobin (Hb), is the widely studied protein in existence and is the most familiar constituent of vertebrate erythrocytes. Protein solubility and stability in aqueous solutions is a major concern to develop therapeutic protein products as the protein lose its activity under unfolded state. Academic and industrial communities have shown great interest in the selection of an apt solvent for biomolecules in suitable biocatalytic applications. Ionic liquids (ILs) have emerged as a suitable cosolvent for the protein molecules by virtue of its high thermal as well as chemical stabilities. Each IL-Protein interaction is specific and generalizing the behaviour is tedious because the same IL can act as both stabilizer and denaturant depending on the external parameters and IL concentration as well. Here in this study, we have synthesized 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) based ILs derived from heterocyclic bases like 1,2,4- triazole, imidazole and morpholine. DBU based ILs are least explored in biomolecule applications but are known for their high thermal stabilities. The interaction of these ILs with hemoglobin was analysed using various spectroscopic techniques including UV–Visible spectroscopy, steady-state fluorescence, Fluorescence Time-Correlated Single Photon Counting (TCSPC), and Dynamic Light Scattering (DLS) to gain better insight into the effects of these ionic liquids on Hb. These results revealed that all the ILs used here are showing Hb friendly behaviour at lower concentrations as it retains the absorbance and fluorescence behaviour of the protein molecule. DLS data explains the solubilization of the Hb molecule, as the ‘z’ average values decreased with periodic addition of the ILs to the system. Quantitative evaluation of the Hb-IL interactions through Benesi-Hildebrand equation suggested that the possibility of complex formations are minimal and this further substantiate that these ILs can act as better cosolvents for Hb in various biochemical reactions.