Molecular interactions of re-released proteins in electrophoresis of human erythrocytes.
Recently, we found that hemoglobin (Hb) could be re-released from live erythrocytes during electrophoresis release test (ERT). The re-released Hb displays single-band and multiple-band re-release types, but its exact mechanism is not well understood. In this article, the protein components of the single-band re-released Hb were examined. First, the re-released band of erythrocytes and the corresponding band of hemolysate, which was used as control, were cut out from starch-agarose mixed gel. Next, proteins were recovered from the starch-agarose mixed gel by freeze-thaw method. After condensing in a vacuum freeze drier, the samples were loaded onto a 5-12% SDS-PAGE. After electrophoresis, three protein bands (16, 28.9, and 29.3 kDa) emerged from the erythrocytes re-released Hb single-band (R-R), but only one band (29.3 kDa) emerged from the corresponding hemolysate control band (H-R). Finally, these bands were analyzed by MALDI-TOF MS. The results showed that these proteins were beta-globin (16 kDa), carbonic anhydrase 1 (CA1, 28.9 kDa), and carbonic anhydrase 2 (CA2, 29.3 kDa). Because CA2 exists in both erythrocytes re-released band and hemolysate control band, we conclude that the single-band re-released Hb is mainly composed of HbA and CA1. Studying the possible interaction between HbA and CA1 will help us further understand the in vivo function of Hb.
Department of Biochemistry and Molecular Biology, Baotou Medical College, Baotou, China.
Yan
Y
Su
Jing
J
Shen
Lijun
L
Gao
Huifang
H
Tian
Zhihua
Z
Tian
Wenbin
W
Qin
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis*; D024401:Carbonic Anhydrase I / Q000097:blood; D024402:Carbonic Anhydrase II / Q000097:blood; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004912:Erythrocytes / Q000737:chemistry*; D006441:Hemoglobin A / Q000032:analysis; D006461:Hemolysis; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001798:Blood Proteins; D006441:Hemoglobin A; D024401:Carbonic Anhydrase I; D024402:Carbonic Anhydrase II