Purification and characterization of human hemoglobin: effect of the hemolysis conditions.
Human hemoglobin was isolated and purified by anion exchange chromatography. To isolate hemoglobin, outdated red blood cells (RBC) were transformed into carbonylhemoglobin, by reaction with carbon monoxide, and submitted to washing/centrifugation procedures, to eliminate other plasma proteins. Albumin was quantified in each supernatant, by the bromcresol green method. Hemolysis was performed in three different hypotonic media (water, 0.01 M NaCl and 5 mM Tris/HCl buffer at pH 7.4), at 8 degrees C for 24 h. Sonication for 5 min was also used to lyse RBC. After isolation of hemoglobin, additional purification was carried out by anion exchange chromatography on AG MP-1, Q-SFF and both exchangers. Hemoglobin concentration of hemolysates and of purified solutions were determined by the hemiglobincyanide method. Residual phospholipids were extracted from the four different hemolysates, as well as from the purified hemoglobin solutions, and were analyzed by high performance liquid chromatography. Native and SDS-polyacrylamide gel electrophoresis experiments were performed on purified hemoglobin samples to verify the presence of proteins other than hemoglobin. According to the results, the hemolysis conditions have influence on the purification of hemoglobin.
Andrade
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Instituto de Macromoléculas Professora Eloisa Mano, Universidade Federal do Rio de Janeiro, P.O. Box 68525, 21945-970 Rio de Janeiro, RJ, Brazil. ctandrade@ima.ufrj.br
Barros
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Lima
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Azero
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D000418:Albumins / Q000737:chemistry; D001769:Blood / Q000378:metabolism; D002498:Centrifugation; D002624:Chemistry, Clinical / Q000379:methods*; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes; D006454:Hemoglobins / Q000737:chemistry* / Q000302:isolation & purification*; D006461:Hemolysis; D006801:Humans; D010100:Oxygen / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry; D013997:Time Factors
D000418:Albumins; D006454:Hemoglobins; D010743:Phospholipids; D010100:Oxygen