The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity

In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta- subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.