Membrane Skeletal Protein S-Glutathionylation in Human Red Blood Cells as Index of Oxidative Stress

Glutathione (GSH) is one of the most well-studied biomarkers of oxidative stress. Under oxidizing conditions, GSH is transformed into its disulfide forms, glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG), which are considered to be reliable biomarkers of oxidative stress. In red blood cells (RBCs), the main targets of S-glutathionylation are hemoglobin and membrane-associated skeletal proteins, but S-glutathionylated hemoglobin (HbSSG) has been more thoroughly studied as a biomarker of oxidative stress than S-glutahionylated RBC membrane skeletal proteins. Here, we have investigated whether and how all these biomarkers are altered in human RBCs treated with a slow and cyclically intermittent flux of the oxidant tert-butyl hydroperoxide. To this aim, a new device for sample treatment and collection was developed. During and at the end of the treatment, GSH, GSSG, and PSSG (discriminating between HbSSG and membrane PSSG) were measured by the use of spectrophotometer (for GSSG) and HPLC (for GSH, HbSSG, and membrane PSSG). The main results of our study are as follows: (i) GSH decreased and GSSG increased, but only in the presence of the oxidant, and recovered their initial values at the end of the infusion; (ii) the increase in total PSSG concentration was lower than that of GSSG, but it kept on throughout the experiments; (iii) membrane skeletal proteins did not recover their initial values, whereas HbSSG levels recovered their initial values similarly to GSH and GSSG; (d) membrane skeletal PSSG were more stable and also more abundant than HbSSG. Western blot analysis indicated spectrin, ankyrin, and bands 3, 4.1, and 4.2 as the proteins most susceptible to S-glutathionylation in RBC membrane. These results suggest that S-glutathionylated membrane skeletal proteins can be considered as a suitable biomarker of oxidative stress. Mostly when the oxidant insult is slight and intermittent, PSSG in RBC membranes are worth measuring in addition to GSSG by virtue of their greater stability.