Human Fetal Hemoglobin : Biochemical Characterization and Recombinant Production

Abstract: Popular Abstract in English Blood, a deep red fluid, circulates in vessels throughout our body. It consists of two main components, a liquid part and a cell part. The liquid portion, which is also called ‘plasma’, takes a main function in transporting water, nutrients, and immune substances. The cell components, which are suspended in the plasma, consist of three main cell types including platelets, white blood cells (WBCs) and red blood cells (RBCs). Platelets have a main function to stop bleeding in case of injuries. WBCs involve mainly in defending our body from attacks of foreign organisms. RBCs, which are filled with hemoglobin molecules (substances responsible for the red color of the blood), have a major role in transportation of oxygen. Hemoglobin can pick up oxygen (O2) from the lungs and deliver it to all parts of the body, then take up carbon dioxide (CO2) and transport it to the lungs where it can be removed from the body by the breathing process. Significant blood loss (>30 % of circulating blood volume) by any mean can cause a drop in blood pressure and impair oxygen delivery to the tissues. These can lead to serious complications or death. Therefore, blood transfusion is a common medical procedure to retain both the plasma volume and oxygen supply. In modern healthcare, blood is always needed, and current healthy donors are not able to fulfill this demand. Moreover, the uncontrolled spreading of blood borne pathogens such as HIV or hepatitis C and B is another important issue. Every unit of blood has to be tested for the evidence of pathogens before it can be given to recipients. To respond to this trend, the development of blood substitutes has emerged. Many research groups have been looking for suitable substitutes that can deliver oxygen as efficient as blood. Ideally, these substitutes should also be stable at higher temperatures so that special facilities for storage are not required. Moreover, they could be given to recipients directly without worrying of incompatibility between different blood groups. The term “blood substitute” may not properly describe the development products since the main function of these products is to deliver oxygen. Therefore, a more specific term, hemoglobin-based oxygen carriers (HBOCs) is used instead. For development of HBOC many different sources of hemoglobin have been used, including the hemoglobin from outdated human blood and bovine blood. However, these available sources have its limit. In case of bovine hemoglobin, contamination of a virus causing a mad cow disease which can transmit to human beings is a major concern. Therefore, other alternative sources of hemoglobin are needed. Human fetal hemoglobin (HbF) is known to be a main hemoglobin during fetus development and to have properties suitable for HBOC development. Compared to that of adult hemoglobin (HbA), HbF has a higher oxygen affinity and it is more stable at a broad pH range. In this thesis hemoglobin properties, which should be considered regarding HBOC development have been studied. This involves the study of an ability of hemoglobin to break down a small molecule, hydrogen peroxide (H2O2). This ability sometimes is referred to as ‘pseudoperoxidase’ activity of hemoglobin. H2O2 is produced normally in our body. However, this small molecule can cause damages to cells and tissues. Therefore removing or converting it to a harmless molecule is essential. The results indicate that HbF reacts rapidly to H2O2 compared to that HbA suggesting that HbF is better in pseudo-peroxidase activity, enables HbF to remove H2O2 rapidly compared to HbA. The interaction between hemoglobin and haptoglobin (Hp) has also been studied due to the fact that forming the complex with Hp is one of the clearance mechanisms of cell-free hemoglobin from the body. The results have demonstrated that HbF can bind firmly to Hp, which indicates that HbF can be removed from the body by a natural process. This thesis has also emphasized on using recombinant hemoglobin as a starting material in HBOC development. Therefore, a production of hemoglobin in bacteria (Escherichia coli or E. coli) is explained. The focus has been placed on the production of HbF. Both a wild type HbF (the recombinant hemoglobin that has the same structure as the hemoglobin found in our body), and a modified hemoglobin HbF have been produced. Many observations done on these two recombinant hemoglobins demonstrate that the wild type and the modified HbF may be further used as a starting material for HBOC development. Additionally, the modified hemoglobin has improved a production yield in E. coli about 3-fold higher compared to that wild type HbF production yield. Considering HBOC development, a large amount of Hb is needed. Therefore, increasing of the production yield is contributing towards the idea that the production of recombinant hemoglobin may be an economically viable method.