Abnormal redox status of membrane-protein thiols in sickle erythrocytes.
Although sickle erythrocytes (RBC) undergo excessive autooxidation, investigators have not found evidence for abnormal oxidation of protein thiols in sickle RBC membranes (e.g., protein aggregates linked by intermolecular disulfide bonds). However, the conventional techniques heretofore used cannot detect more subtle changes in thiol status such as abnormal intramolecular disulfide bonds. We examined RBC membranes using thiol-disulfide exchange chromatography which partitions sodium dodecyl sulfate-solubilized proteins on the basis of reactive thiols, yielding gel-bound (reduced-thiol) and filtrate (oxidized/blocked-thiol) 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 thiol oxidation in sickle RBC membranes. Treatment of normal RBC with thiol blockers and oxidants simulates this shift of membrane protein into the filtrate fraction. Analysis using polyacrylamide 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.
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives; D000328:Adult; D000755:Anemia, Sickle Cell / Q000097:blood*; D005591:Chemical Fractionation; D004220:Disulfides; D004229:Dithiothreitol; D004910:Erythrocyte Membrane / Q000032:analysis / Q000378:metabolism; D004913:Erythrocytes, Abnormal / Q000032:analysis*; D005374:Filtration; D006801:Humans; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000097:blood* / Q000302:isolation & purification
D004220:Disulfides; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; C001105:2,2'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; D004229:Dithiothreitol