Spectrin-haemoglobin crosslinkages associated with in vitro oxidant hypersensitivity in pathologic and artificially dehydrated red cells

Abstract

Protein crosslinkages are apparent at 215,000-250,000 daltons in electrophoregrams of membranes from H2O2 treated erythrocytes. H2O2 is also capable of inducing crosslinkages of identical MW in stage I and II (red) ghosts and in a mixture of purified spectrin and Hb, but not in white ghosts or in either spectrin or Hb alone. Autoradiographic studies using 14C-methemoglobin and 32P-spectrin confirm the involvement of spectrin and Hb in this reaction. The .alpha. chains of both proteins are more reactive than the corresponding .beta. chains: 3 times more reactive in the case of spectrin and 10 times more reactive in Hb. The reaction is almost totally inhibited by NaCN and partially inhibited by N-ethylmaleimide. Direct addition of malondialdehyde to a spectrin-Hb mixture does not result in protein crosslinkage. Metabolic depletion (40 h), in vivo aging and sucrose dehydration of fresh, normal cells enhance the reaction considerably, whereas in vitro rehydration of xerocytes normalizes their H2O2 sensitivity.