Research on the secondary structure and hydration water around human serum albumin induced by ethanol with infrared and near-infrared spectroscopy

Given the role of ethanol played in the precipitation of various proteins, it is of great significance for the refined purification of proteins to understand the mechanism of protein secondary structure and hy-dration effect under the ethanol perturbation. Infrared (IR) spectroscopy and near-infrared (NIR) spec-troscopy combined with chemometrics and aquaphotomics were utilized to get a generalized picture of the second structure of human serum albumin (HSA) and the surrounding hydrogen bond network of water after perturbation with different concentrations of ethanol ranging from 0% to 45% (v/v). The two-dimensional correlation (2DCOS) and moving window two-dimensional correlation analysis (MW-2DCOS) of IR spectroscopy provide evidence that the native structure of HSA was stabilized by the formation of alpha-helical structure at a low concentration of ethanol. However, the formation of intermolecular beta- sheet structure by increasing ethanol is an important reason for the aggregation and precipitation of HSA. The analysis of NIR spectroscopy with the McCabe-Fisher method and aquaphotomics showed that free hydrogen bonded water was dynamic and fluctuating, 0%-20% ethanol could enhance the hydrogen bonded water cluster while the increasement of ethanol from 20% to 45% would lead to the formation of weak hydrogen bonded water cluster. These measurements provide a novel perspective on the relation-ship between ethanol and HSA, which is useful for maintaining activity and improving purity during the purification of proteins or other biological macromolecules.(c) 2022 Elsevier B.V. All rights reserved.