The Study Of Hemoglobin's Interactions Within Erythrocytes

Part 1 Study the Interactions of Hemoglobin in Human Erythrocytes by Co-immunoprecipitation coupled with Hemoglobin Release TestObjective Couple the hemoglobin release test?HRT?with co-immunoprecipitatio-n to study the interactions between hemoglobin?Hb?and other proteins within human erythrocytes.Methods First,fresh normal adult anti-coagulated whole blood was washed to prepare packed erythrocytes,which were further prepared to erythrocytes suspension and hemolysate.The erythrocytes suspension and hemolysate were then electrophoresised with hemoglobin release test?HRT?at the same time,and HbA or HbA₂ bands of erythrocytes suspension?RA or RA₂?and hemolysate?HA or HA₂?were cut out from the gel and enriched with Sephadex G-25.Then RA,HA and erythrocyte lysis were co-immunoprecipitated with Hemoglobin ? antibody,and the co-immunoprecipitated complexes of RA,HA and erythrocyte lysis were separated with 5-12% SDS-PAGE followed by Q-TOF-MS ?IDQD and Western blot,respectively.Result Five main protein bands?about 16 k D,20 k D,22 k D,28 k D and 50 k D?were found in the co-immunoprecipitated complexes o f RA,HA and erythrocyte lysis.The bands were identified by Q-TOF MS and the results showed that the 16 k D and 22 k D bands were hemoglobin subunit beta and peroxiredoxin2?Prx2?.IDQD predicting results showed Prx2 was speculated to have interaction with HbA,but have no interaction with HbA₂.Finaly,Prx2 was proved to exit in the bands of HA,RA and RA₂,but not in HA₂ by Western blot.Conclusion Prx2 was speculated to have interaction with HbA,but not HbA₂ within human erythrocytes.Part 2 Study the Interaction of Hemoglobin in Human Erythrocytes by Hemoglobin Release Test coupled with 2D electrophoresisObjective Couple the hemoglobin release test?HRT?with 2D electrophoresis?2-DE?to study the interactions between hemoglobin?Hb?and other proteins within human erythrocytes.Methods First,the fresh normal adult anti-coagulated whole blood was washed to prepare packed erythrocytes,which were further prepared to erythrocytes suspension and hemolysate.The erythrocytes suspension and hemolysate were then electrophoresised with hemoglobin release test?HRT?at the same time,and then the bands of HbA,HbA₂,the part between HbA and HbA₂ and the re-released Hb of erythrocyte sample and the corresponding bands of hemolysate sample,which were cut out from the gel and were enriched by Sephadex G-25.Separated the complexes through 2-DE and then identified the proteins by Q-TOF-MS.Results HbA,HbA₂,the part between HbA and HbA₂,re-release Hb of erythrocytes suspension and the corresponding part of the hemolysate were divided into four group,A,B,C and D and then were electropho resised with 2D electrophoresis,respectively.?1?Compare each band of Hb?HbA,HbA₂,the part between HbA and HbA₂,re-release Hb?of the erythrocytes suspension and the corresponding of the hemolysate after gel image scanning,the number of the protein in different Hb part of the erythrocytes suspension are more than the corresponding part of the hemolysate,and different ial protein spots were found obviously.Twelve differential protein spots were found in group A,and 9,9,13 differential protein spots were respectively found in group B,C and D.?2?Prx2,Hsp 90,Glyceraldehyde-3-phosphate dehydrogenase,Actin and Catalasewere found in the part of HbA of erythrocytes suspension and hemolysate.But differential protein was not found.Conclusions Compare each band of Hb?HbA,HbA₂,the part between HbA and HbA₂,re-release Hb?of the erythrocytes suspension and the corresponding of the hemolysate,differential protein spots were found,but differential protein was not found yet.