Rational Mutagenesis to Engineer Heme Stability in Recombinant Human Hemoglobin to Design Potential Hemoglobin Based Oxygen Carrier

Abstract

Certain life-saving medical conditions like surgery, war wound and trauma necessitate blood transfusions. The transfusion process is associated with several risks and side effects. Heterologous expression of human hemoglobin for developing HBOCs seems to be an alternative to blood transfusion. However, such hemoglobins suffer quite a few disadvantages as described in Varnado et al. A major problem that prevents wider use of HBOCs is the dissociation of “heme” from its protein matrix. The released heme causes serious renal cytotoxicity which needs to be minimized. We successfully engineered heme stability into myoglobin by covalent linkage with a side chain from the apoprotein mimicking a cyanobacterial globin.Efforts are on in translating the heme stability in human hemoglobin. Based on rational mutagenesis, we were indeed able to engineer heme stability to an extent into recombinant human hemoglobin. Specific mutations in the α- and β- chains individually caused significant increase (∼3-4 fold) in the hemin retention. These mutations did not show any major influence on the structural stability and heme coordination of the protein. Moreover, engineered rHb mutants exhibited autooxidation rates similar to the wild type protein. Such preliminary success in engineering heme stability holds promise for the production of stable recombinant HBOCs.