Investigation of protective effect of ethanol on the natural structure of protein with infrared spectroscopy

Abstract

The stability of biological drugs with protein as an active substance depends heavily on the retention of natural protein structure during freeze-drying. Stabilizers have become important substances in the process of protein freeze-drying. In order to further understand the mechanism of the interaction between protein and stabilizers, human serum albumin (HSA) and simple hydroxyl compound ethanol were used as models. Infrared (IR) spectroscopy combined with chemometrics was implemented to investigate the changes of secondary structure and hydration of HSA when different concentrations of ethanol were considered as interference. Through the analysis of the protein secondary structure and hydrated layer, we found that the addition of ethanol-d6 increased the α-helix of HSA and reduced the disordered structure. The hydrogen bond structure around HSA was enhanced and intermolecular aggregation was reduced through the action of the water molecules. The hypothesis was verified by circular dichroism (CD) and transmission electron microscopy (TEM) observation by adding different concentrations of ethanol-d6. It was found that a small amount of ethanol could protect the native conformation of HSA. In conclusion, this study revealed the mechanism of ethanol as a protein protector, provided a new idea for protein purification process and a theoretical basis for biomolecular interaction.