Abnormal redox status of membrane-protein XXXX in sickle erythrocytes.

@entity1433 @entity3208 @entity1432 @entity2975 @entity1520
Although sickle erythrocytes (RBC) undergo excessive autooxidation, investigators have not found evidence for abnormal oxidation of protein @entity1433 in sickle RBC membranes (e.g., protein aggregates linked by intermolecular @entity1520 bonds). However, the conventional techniques heretofore used cannot detect more subtle changes in @entity1432 status such as abnormal intramolecular @entity1520 bonds. We examined RBC membranes using @entity1432 - @entity1520 exchange chromatography which partitions @entity3208 -solubilized proteins on the basis of reactive @entity1433 , yielding gel-bound (reduced- @entity1432 ) and filtrate (oxidized/blocked- @entity1432 ) fractions. Membranes from normal RBC partition so that only 13.6 +/- 1.4% of all membrane protein is found in the filtrate fraction. An abnormally increased amount of membrane protein from sickle RBC (21.5 +/- 4.3%) partitions into the filtrate fraction (P less than 0.001). Since sickle RBC do not have high molecular weight aggregates of membrane protein, this indicates abnormal intramolecular @entity1432 oxidation in sickle RBC membranes. Treatment of normal RBC with @entity1432 blockers and oxidants simulates this shift of membrane protein into the filtrate fraction. Analysis using @entity2975 gel electrophoresis reveals that the filtrate fraction derived from normal RBC consists mostly of band 7 and glycophorins, with only trace amounts of other membrane proteins. Superimposed upon this normal background, sickle RBC filtrates are enriched with all proteins (including cytoskeletal protein bands 1, 2, 2.1, and 4.1), suggesting a generalized oxidative perturbation of sickle RBC membranes. These observations support the concept that excessive RBC autooxidation may play a role in sickle disease pathophysiology, and they perhaps help explain the development of those membrane abnormalities that may reflect cytoskeletal dysfunction in sickle erythrocytes.
