Progress Toward an O2-carrying Therapeutic Based on a Single-chain Hemoglobin Framework

Our research is focused on the production of a hemoglobin based oxygen carrier (HBOC) that can be used as a therapeutic in the event of acute blood loss. In addition, HBOCs have been shown to increase the efficacy of ionizing radiation during cancer treatment. To achieve this goal, the subunits of the cell-free HBOC must not dissociate when transfused. Ideally, the HBOC would have the oxygen carrying characteristics (binding affinity and cooperativity) of whole blood. Our initial design implemented the co-expression of circular permutated β-globin subunits (cpβ) with α-globin subunits (α-cpβ). Subsequent constructs incorporated the covalent linkage of α and β-globin subunits to generate a single-chain α-cpβ heterodimers (sc-α-cpβ). Preliminary experimentation indicated that our permuteins preferred the high affinity R-state conformation over the low affinity T state. This led to the addition of T-state stabilizing point mutations and the covalent linkage between sc-α-cpβ dimers (scHb). Preliminary ligand binding kinetics data for scHb indicate a preference for the T-state. We are currently working on the structural characterization of scHb as well as inclusion of further T-state stabilizing mutations