Junctional sites of erythrocyte skeletal proteins are specific targets of tert-butylhydroperoxide oxidative damage.

The oxidative denaturation of the erythrocyte membrane, which is considered a major cause of the haemolytic process, was evaluated upon 'in vitro' oxidative stress with tertbutylhydroperoxide. Biochemical and ultrastructural analyses were performed to point out the effect of this substance on the skeletal network, which is mainly responsible for red cell shape and viability. Moreover, cell morphology was observed by scanning electron microscopy and membrane rigidity assessed by EPR measurements. The most relevant features of the membrane denaturation were, (i) lipid peroxidation, as assessed by malonidialdehyde production, (ii) spectrin and ankyrin degradation with simultaneous globin binding to the membrane, as evidenced by electrophoretic pattern of red cell ghosts. These phenomena were related to the drug concentration in the incubation medium, and accompanied by depletion of intracellular reduced glutathione. The denaturation of protein components hindered the release of spectrin in a hypotonic extraction medium and could be only partially reversed by dithiothreitol. The extensive membrane protein and lipid degradation, at high drug concentration, was coherent with a marked increase of membrane order (membrane 'rigidity'). No clustering of intramembrane proteins was shown by the transmission electron microscopy images. At the same time scanning electron microscopy demonstrated shrinking and disco-stomatocytic deformation of erythrocytes. Ultrastructural analysis of the membrane skeleton by fluorescence-labelling of spectrin and actin, allowed to point out that exposure to t-BHP caused the marginalization of spectrin and the rearrangement of actin molecules with formation of micro aggregates, so that a detachment of actin from the spectrin network was suggested. In addition to the generalized damage of red cell membrane, tertbutylhydroperoxide was found to induce a specific alteration of the skeletal network at the horizontal junction sites involving spectrin, actin, and protein 4.1 and thus to modify the cytoskeletal assembly. This effect on the membrane skeletal components was consistent with the hypothesis that oxidative stress plays a key role in the haemolytic process.