Abstract
BackgroundHemoglobin C differs from normal hemoglobin A by a glutamate-to-lysine substitution at position 6 of beta globin and is oxidatively unstable. Compared to homozygous AA erythrocytes, homozygous CC erythrocytes contain higher levels of membrane-associated hemichromes and more extensively clustered band 3 proteins. These findings suggest that CC erythrocytes have a different membrane matrix than AA erythrocytes.Methodology and FindingsWe found that AA and CC erythrocytes differ in their membrane lipid composition, and that a subset of CC erythrocytes expresses increased levels of externalized phosphatidylserine. Detergent membrane analyses for raft marker proteins indicated that CC erythrocyte membranes are more resistant to detergent solubilization. These data suggest that membrane raft organization is modified in CC erythrocytes. In addition, the average zeta potential (a measure of surface electrochemical potential) of CC erythrocytes was ≈2 mV lower than that of AA erythrocytes, indicating that substantial rearrangements occur in the membrane matrix of CC erythrocytes. We were able to recapitulate this low zeta potential phenotype in AA erythrocytes by treating them with NaNO2 to oxidize hemoglobin A molecules and increase levels of membrane-associated hemichromes.ConclusionOur data support the possibility that increased hemichrome deposition and altered lipid composition induce molecular rearrangements in CC erythrocyte membranes, resulting in a unique membrane structure.
Highlights
Unstable hemoglobin (Hb) variants, such as HbC, sickle HbS, and unpaired beta globin chains present in a-thalassemic states, impart a greatly increased level of oxidative stress on erythrocytes that enhances the oxidative denaturation of Hb [1,2,3,4,5]
Lipid profile analysis Erythrocyte membranes contain most of the major lipids that are present in other biological membranes, including phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidyl-inositol (PI), and cholesterol
Extracted lipids were separated by HPLC and analyzed by a semi-quantitative method based on internal standards introduced in the sample
Summary
Unstable hemoglobin (Hb) variants, such as HbC, sickle HbS, and unpaired beta globin chains present in a-thalassemic states, impart a greatly increased level of oxidative stress on erythrocytes that enhances the oxidative denaturation of Hb [1,2,3,4,5]. HbC associates with erythrocyte membranes at a 5-fold greater rate than normal HbA [9] and binds more tightly to the inner leaflet, where it is believed to cause more extensive clustering of band 3 [10]. These changes in membrane structure, as well as dehydration-induced HbC crystallization and increased internal viscosity, are believed to play some role in the mild anemia that homozygous CC individuals experience as a result of accelerated erythrocyte turnover [11,12,13]. These findings suggest that CC erythrocytes have a different membrane matrix than AA erythrocytes
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