OBJECTIVETo study the interactions between hemoglobin (Hb) and other proteins within human erythrocytes by using the electrophoresis release test (ERT) and co-immunoprecipitation.

METHODSFirst, the fresh normal adult anti-coagulated whole blood was washed to prepare packed RBCs, which were further prepared to erythrocyte suspension and hemolysate.The erythrocyte suspension and hemolysate were analyzed by the electrophoresis release test (ERT) at the same time, and then the band of HbA of erythrocyte sample (RA) and the corresponding band of hemolysate sample (HA) were cut out from the gel and were enriched by freeze-thaw method. Then, the samples were bound with hemoglobin β antibody (37-8) AC, the complexes were separated through 5%-12% SDS-PAGE followed by Q-TOF.

RESULTSFive bands were found in the gel, each of which was treated by hemoglobin β antibody (37-8) AC, the protein bands of 16,20,22,28 and 50 kD were emerged in RA, HA and RBC lysis.The bands were identified by MS, and the results showed that these bands were hemoglobin, band 3, peroxiredoxin2 (Prx2), band 3 and β-actin, band 3 respectively.

CONCLUSIONHbA may interact with Prx2, Band 3 and β-actin, and then the complexes are formed with each other within erythrocytes.