Comparison of hemoglobin Koln erythrocyte membranes with malondialdehyde-reacted normal erythrocyte membranes

Splenectomized patients with hemoglobin (Hb) Koln have rigid RBCs with membrane polypeptide aggregates that are not dissociable with disulfide-reducing agents. Malondialdehyde (MDA) action on normal RBCs produced rigid RBCs with similar nondissociable aggregates. To test the hypothesis that Hb Koln RBC aggregates contained unsaturated MDA-type bonds, we reduced normal control RBC membranes, Hb Koln RBC membranes, and MDA-reacted membranes with [³H]NaBH₄. Hb Koln RBC membranes and MDA-reacted membranes both had significantly more ³H incorporation than control membranes. Furthermore, ³H incorporation in both Hb Koln and MDA-treated membranes was located in the membrane polypeptide aggregates, presumably saturating the crosslinking bonds. After reaction of RBCs with [¹⁴C]MDA, the MDA label was similarly concentrated in the membrane polypeptide aggregates. Normal RBC membranes incubated with MDA were analyzed with and without reduction by NaBH₄ prior to amino acid determination by high-performance liquid chromatography (HPLC). Reduction with NaBH₄ after MDA treatment decreased the lysyl residues by 33% and the serine by 7% and increased by 10% the methionyl residues, but did not affect 12 other amino acids. Similar changes could be detected in NaBH₄-reduced Hb Koln aggregates in methionine and serine content. MDA may also alter protein configuration, as evidenced by an increase in the protease susceptibility of membrane proteins from MDA-treated and Hb Koln RBCs. We conclude that Hb Koln RBC membranes, like MDA-treated membranes, have similar high molecular weight aggregates conferring decreased membrane deformability, [³H]NaBH₄-reducible unsaturated bonds, changes in amino acid composition upon reduction, and protease-sensitive configurational changes.