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Human Fetal Hemoglobin : Biochemical Characterization and Recombinant Production

Ratanasopa, Khuanpiroon LU (2015)
Abstract (Swedish)
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... (More)
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. (Less)
Abstract
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.... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Dr. Silaghi-Dumitrescu, Radu, Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University, Romania
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Blood substitutes, HBOCs, HbF, recombinant hemoglobin, pseudoperoxidase, ITC, haptoglobin
categories
Higher Education
pages
82 pages
publisher
Division of Pure and Applied Biochemistry
defense location
Lecture Hall B , Center for Chemistry and Chemical Engineering, Lund University Faculty of Engineering (LTH), Getingevägen 60, Lund
defense date
2015-02-09 10:15
ISBN
978-91-7422-387-3
language
English
LU publication?
yes
id
9f6b12cc-ae72-443a-b570-ab649ce07150 (old id 4934645)
date added to LUP
2015-01-15 07:38:38
date last changed
2016-09-19 08:45:09
@misc{9f6b12cc-ae72-443a-b570-ab649ce07150,
  abstract     = {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. <br/><br>
 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.<br/><br>
 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.},
  author       = {Ratanasopa, Khuanpiroon},
  isbn         = {978-91-7422-387-3},
  keyword      = {Blood substitutes,HBOCs,HbF,recombinant hemoglobin,pseudoperoxidase,ITC,haptoglobin},
  language     = {eng},
  pages        = {82},
  publisher    = {ARRAY(0x8f522c8)},
  title        = {Human Fetal Hemoglobin : Biochemical Characterization and Recombinant Production},
  year         = {2015},
}