title: Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography.
abstract: Bovine and human hemoglobin (Hb) form the basis for many different types of Hb-based O(2) carriers (HBOCs) ranging from chemically modified Hbs to particle encapsulated Hbs. Hence, the development of a facile purification method for preparing ultrapure Hb is essential for the reliable synthesis and formulation of HBOCs. In this work, we describe a simple process for purifying ultrapure solutions of bovine and human Hb. Bovine and human red blood cells (RBCs) were lyzed, and Hb was purified from the cell lysate by anion exchange chromatography. The initial purity of Hb fractions was analyzed by SDS-PAGE. Pure Hb fractions (corresponding to a single band on the SDS-PAGE gel) were pooled together and the overall purity and identity assessed by LC-MS. LC-MS analysis yielded two peaks corresponding to the calculated theoretical molecular weight of the alpha and beta chains of Hb. The activity of HPLC pure Hb was assessed by measuring its oxygen affinity, cooperativity and methemoglobin level. These measures of activity were comparable to values in the literature. Taken together, our results demonstrate that ultrapure Hb (electrophoresis and HPLC pure) can be easily prepared via anion exchange chromatography. In general, this method can be more broadly applied to purify hemoglobin from any source of RBC. This work is significant, since it outlines a simple method for generating ultrapure Hb for synthesis and/or formulation of HBOCs.
authors: Department of Chemical and Biomolecular Engineering, The Ohio State University, 231A Koffolt Laboratories, 140 West 19th Avenue, Columbus, OH 43210, United States.
authors: Guoyong
authors: G
authors: Sun
authors: Andre F
authors: AF
authors: Palmer
mesh_terms: D000818:Animals; D000837:Anion Exchange Resins; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000302:isolation & purification*; D013058:Mass Spectrometry
chemical_list: D000837:Anion Exchange Resins; D006454:Hemoglobins