title: Purification of hemoglobin from red blood cells using tangential flow filtration and immobilized metal ion affinity chromatography.
abstract: Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter.
authors.affiliation: William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Elmer
authors.forename: David
authors.initials: D
authors.lastname: Harris
authors.forename: Andre F
authors.initials: AF
authors.lastname: Palmer
mesh_terms: D000818:Animals; D002417:Cattle; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000737:chemistry*; D005374:Filtration / Q000379:methods*; D006454:Hemoglobins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006454:Hemoglobins; D010100:Oxygen