OxyFile #132

Biochimica et Biophysica Acta, 691 (1982) 300-308
Elsevier Biomedical Press

Alterations of Red Cell Membrane Proteins and Hemoglobin Under 
Natural and Experimental Oxidant Stress

N. Alloisio, D. Michelon, E. Bannier, A. Revol, Y. Beuzard, and J. 

We compared on red cell membrane proteins and hemoglobin (Hb) the 
effects of (i) natural oxidant stress that has been suggested to 
occur in a variety of oxidative hemolytic anemias, and (ii) 
experimental stress induced by hydrogen peroxide.  SDS-polyacrylamide 
gel electrophoresis was used for protein analysis.  Under natural 
conditions (thalassemias, hemoglobinopathies with Hb unstability), a 
high molecular weight polymer (HMWP) and variable amounts of globin 
mono- and dimers became apparent.  Furthermore, a major 12 kDa 
polypeptide, its dimer, and conspicuous spectrin degradation products 
in the band 2.2-2.6 region occurred in a patient carrying the highly 
unstable Hb Hammersmith.  Under experimental conditions, incubation 
of erythrocyte ghosts with H2O2 in the presence of minimal 
concentration (25 uM) of Hb generated a HMWP at the expense of 
membrane proteins, mainly spectrin.  Incubation of a diluted (200 uM) 
membrane-free hemolysate with H2O2 induced a HMWP, an array of globin 
oligomers and a 12 kDa polypeptide similar to that mentionned above.  
Therefore, the damage to the red cell membrane present in various 
oxidative hemolytic anemias, including polypeptide polymerisation and 
breakdown, can be produced by experimental oxidant stress.  These 
observations support the view that the alterations described in the 
patients result directly from oxidative reactions.  However, we did 
not observe in the patient the sharp breakdown of polyunsaturated 
fatty acids that was triggered in vitro by H2O2 in the presence of Hb 
acting as a catalyst.  In most cases, oligo- and polymers were 
resistant to B-mercaptoethanol, and the chemical nature of the 
underlying cross-links is discussed.  To our knowledge, the 12 kDa 
polypeptide, that we consider as arising from globin proteolysis, has 
never been reported under pathological conditions.

Received September 14, 1981.
Revised manuscript received March 12, 1982

Key words: Erythrocyte membrane protein; Hemoglobin; Hemolytic 
           anemia; Oxidative stress