title: Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test.
abstract: To elucidate the protein-protein interactions of hemoglobin (Hb) variants A and A(2), HbA was first shown to bind with HbA(2) in live red blood cells (RBCs) by diagonal electrophoresis and then the interaction between HbA and HbA(2) outside the RBC was shown by cross electrophoresis. The starch-agarose gel electrophoresis of hemolysate, RBCs, freeze-thawed RBCs and the supernatant of freeze-thawed RBCs showed that the interaction between HbA and HbA(2) was affected by membrane integrity. To identify the proteins involved in the interaction, protein components located between HbA and HbA(2) in RBCs (RBC HbA-HbA(2)) and hemolysate (hemolysate HbA-HbA(2)) were isolated from the starch-agarose gel and separated by 5-12% SDS-PAGE. The results showed that there was a ≈22 kDa protein band located in the RBC HbA-HbA(2) but not in hemolysate HbA-HbA(2). Sequencing by LC/MS/MS showed that this band was a protein complex that included mainly thioredoxin peroxidase B, α-globin, δ-globin and β-globin. Thus, using our unique in vivo whole blood cell electrophoresis release test, Hbs were proven for the first time to interact with other proteins in the live RBC.
authors.affiliation: Laboratory of Hemoglobin, Baotou Medical College, Baotou, Inner Mongolia, P. R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Su
authors.forename: Lijun
authors.initials: L
authors.lastname: Gao
authors.forename: Qiang
authors.initials: Q
authors.lastname: Ma
authors.forename: Lishe
authors.initials: L
authors.lastname: Zhou
authors.forename: Liangyi
authors.initials: L
authors.lastname: Qin
authors.forename: Lihong
authors.initials: L
authors.lastname: Han
authors.forename: Wenbin
authors.initials: W
authors.lastname: Qin
mesh_terms: D000595:Amino Acid Sequence; D002457:Cell Extracts / Q000737:chemistry; D004586:Electrophoresis / Q000379:methods*; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D006441:Hemoglobin A / Q000737:chemistry / Q000378:metabolism*; D006443:Hemoglobin A2 / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping; D053719:Tandem Mass Spectrometry
chemical_list: D002457:Cell Extracts; D006441:Hemoglobin A; D006443:Hemoglobin A2