Human erythrocytes under stress. Spectroscopic fingerprints of known oxidative mechanisms and beyond

Abstract

In this work, we investigated the oxidative stress-related biochemical alterations in red blood cells (RBCs) and their membranes with the use of spectroscopic techniques. We aimed to show their great advantage for the in situ detection of lipid classes and secondary structures of proteins without the need for their extraction in the cellular environment. The exposition of the cells to peroxides, t-butyl hydroperoxide (tBOOH) or hydrogen peroxide (H₂O₂) led to different degradation processes encompassing the changes in the composition of membranes and structural modifications of hemoglobin (Hb). Our results indicated that tBOOH is generally a stronger oxidizing agent than H₂O₂ and this observation was congruent with the activity of superoxide and glutathione peroxidase. ATR-FTIR and Raman spectroscopies of membranes revealed that tBOOH caused primarily the partial loss and peroxidation of the lipids resulting in loss of the integrity of membranes. In turn, both peroxides induced several kinds of damage in the protein layer, including the partial decrease of their content and irreversible aggregation of spectrin, ankyrin, and membrane-bound globin. These changes were especially pronounced on the membrane surface where stress conditions induced the formation of β-sheets and intramolecular aggregates, particularly for tBOOH. Interestingly, nano-FTIR spectroscopy revealed the lipid peroxidative damage on the membrane surface in both cases. As far as hemoglobin was concerned, tBOOH and H₂O₂ caused the increase of the oxyhemoglobin species and conformational alterations of its polypeptide chain into β-sheets. Our findings confirm that applied spectroscopies effectively track the oxidative changes occurring in the structural components of red blood cells and the simplicity of conducting measurements and sample preparation can be readily applied to pharmacological and clinical studies.

Keywords: Fourier transform infrared spectroscopy–attenuated total reflectance (FTIR–ATR); Nano-FTIR spectroscopy (nano-FTIR); Oxidative stress; Raman spectroscopy (RS); Red blood cells (RBCs); Ultraviolet–visible (UV–Vis) spectroscopy.