Preparation of highly purified hemoglobin by affinity elution

The large scale production of recombinant hemoglobin (Hb) from microorganism or transgenic hosts for Hb-based blood substitutes places utmost emphasis on purity. In the present study, a high-resolution, convenient and inexpensive purification method is developed for purification of Hb from mixtures containing E. coli extract and bovine serum. This method is based on affinity elution by pyrophosphate (PPi) of Hb adsorbed on an FPLC column of the anion exchanger Toyopearl DEAE-650M. Compared to pH elution or NaCl elution, PPi elution makes possible the preparation of Hb of much higher purity. A procedure combining pH elution and PPi elution sequentially using a single column proves particularly valuable. The purification method is also applicable to the purification of cyanomet-Hb (CNHb+).