Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test

To elucidate the protein-protein interactions of hemoglobin (Hb) variants A and A₂, HbA was first shown to bind with HbA₂ in live red blood cells (RBCs) by diagonal electrophoresis and then the interaction between HbA and HbA₂ outside the RBC was shown by cross electrophoresis. The starch-agarose ge...

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Bibliographic Details
Published in:Electrophoresis 2010-09, Vol.31 (17), p.2913-2920
Main Authors: Su, Yan, Gao, Lijun, Ma, Qiang, Zhou, Lishe, Qin, Liangyi, Han, Lihong, Qin, Wenbin
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Biomedical materials
Blood cells
Cell Extracts - chemistry
Electrophoresis
Electrophoresis - methods
Erythrocyte Membrane - metabolism
Erythrocytes - chemistry
Erythrocytes - metabolism
Hemoglobin
Hemoglobin A - chemistry
Hemoglobin A - metabolism
Hemoglobin A2 - chemistry
Hemoglobin A2 - metabolism
Humans
In vivo testing
In vivo tests
Interaction
Molecular Sequence Data
Peroxiredoxin
Protein Binding
Protein Interaction Mapping
Proteins
Red blood cell
Red blood cells
Tandem Mass Spectrometry
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Summary:To elucidate the protein-protein interactions of hemoglobin (Hb) variants A and A₂, HbA was first shown to bind with HbA₂ in live red blood cells (RBCs) by diagonal electrophoresis and then the interaction between HbA and HbA₂ 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 HbA₂ was affected by membrane integrity. To identify the proteins involved in the interaction, protein components located between HbA and HbA₂ in RBCs (RBC HbA-HbA₂) and hemolysate (hemolysate HbA-HbA₂) were isolated from the starch-agarose gel and separated by 5-12% SDS-PAGE. The results showed that there was a [almost equal to]22 kDa protein band located in the RBC HbA-HbA₂ but not in hemolysate HbA-HbA₂. 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.
ISSN:0173-0835
1522-2683
1522-2683
DOI:10.1002/elps.201000034