Human Fetal Hemoglobin : Biochemical Characterization and Recombinant Production

Blood transfusion is a common medical procedure when oxygen supply in the body is impaired. However, blood transfusion is not always without risks. The uncontrolled spreading of blood borne pathogens such as HIV or hepatitis B and C virus has urged researchers to look for “blood substitutes”. Many research groups have put their focuses on hemoglobin-based products (HBOCs). The major source of haemoglobin previous developed products is outdated human blood or bovine blood. However, these haemoglobin (Hb) sources have theirs limit. Therefore, alternative hemoglobin sources for HBOC development have been considered. In this thesis, human fetal hemoglobin (HbF) is introduced as an alternative hemoglobin source for HBOC development. The study of the reaction between ferricHb and H2O2 has indicated that HbF exhibits a better pseudoperoxidase activity compared to that of human adult hemoglobin (HbA). The reaction between oxyHb and nitrite also supports the notion that HbF is a more reactive molecule than HbA. Moreover, the study of the reaction between Hb and Hp using isothermal titration calorimetry (ITC) revealed a high affinity between ferric HbF and Hp 1-1. The results from differential scanning calorimetry (DSC) also revealed that ferricHb is partly inactive at a near body temperature (35˚C) and that may contribute to a lower in Hp binding affinity compared to the binding affinity at 25 ˚C. The enhanced pseudo-peroxidase activity and the higher structural stability of HbF are considered as advantages when considering HBOC development. Furthermore, the observation on a production of recombinant Hb in E. coli supports the original thought that HbF is a suitable platform for recombinant Hb for HBOC development regarding its stability and reasonable production yields in E. coli under well-optimized conditions. Moreover, modified HbF by fusion of α- and γ-subunit (fHbF) can improve a production yield to about 3-fold. The O2 and CO kinetic binding studies have revealed that fHbF can bind small ligands reversibly. Together with alkaline stability and pseudo peroxidative activity tests, we conclude that this fusion globin is a functional protein that may be used as a starting material for further HBOC development.