Haemoglobin and Evolution of Circulatory Systems
Haemoglobin is the oxygen carrying component of human blood. It is a tetrameric protein found within erythrocytes (red blood cells) that allows the transport of oxygen (O2) from the oxygen rich lungs to oxygen dependent body systems. The oxygen binding capacity of haemoglobin also allows the transport of carbon dioxide (CO2) from respiring body systems to the lungs where it is expired.
The evolution of an oxygen carrying circulatory system enabled oxygen delivery to areas beyond the restraints of diffusional distance. This allowed an indefinite increase in organism size from small multi-cellular organisms to the great blue whale with oxygen availability to the furthest extremities.
The circulatory systems of larger organisms relied on the evolution of a molecule able to take up oxygen from areas of high concentration and release it to areas of low concentration. This molecule in mammals is haemoglobin which is characteristically ideal for the job (Figure 1):
• Reversibly binds oxygen
• Carries oxygen with high efficiency (4 molecules oxygen per haemoglobin)
• Has high oxygen binding affinity in high oxygen, high pH environment as found in the lungs
• Releases oxygen in low oxygen, low pH environments, as found in respiring tissues with high CO2
The effect of pH on oxygen-haemoglobin loading and unloading is known as the Bohr effect.
The Oxygen-Haemoglobin Dissociation Curve
The percentage of haemoglobin oxygen saturation at different partial pressures of oxygen is known as the oxygen-haemoglobin dissociation curve (Figure 2). It represents the oxygen tension at which haemoglobin binds and dissociates oxygen. The graph is sigmoidal, representing difficulty with binding of the first oxygen molecule, but this facilitates the binding of subsequent molecules. However, full saturation is also difficult due to the reversible nature of the binding. The partial pressure of oxygen at which 50% of haemoglobin is bound to oxygen, the P50, is typically 26.6 mmHg in a healthy adult
Structure of Haemoglobin:
Haemoglobin is an aggregation of four globular proteins (typically two alpha and two beta subunits), each with an embedded haeme group. Each haeme group allows the binding of an oxygen molecule. Haeme derives from an organic porphyrin ring with an inorganic iron molecule (Fe) (Figure 3). It is this molecule that provides that characteristic red blood colour when bound to oxygen (oxyhaemoglobin), and blue/red blood colour when deoxygenated (deoxyhaemoglobin). This haemoglobin is encased in erythrocytes; nucleus free disk shaped cells known commonly as red blood cells (RBC).
Adult vs Fetal Haemoglobin
Adult haemoglobin is made up of four subunits. The most common form, Hb A consists of two alpha and two beta subunits (α2 β2). However a second similar but less abundant form is also found, Hb A2, with two delta subunits replacing the beta (α2 δ2) (Table 1).
During pregnancy, the mother is responsible for delivering oxygen to the baby, and removing CO2. In order for this to happen, maternal and fetal circulations come into very close proximity in the placenta. It is important that this system preferentially moves oxygen into fetal circulation and CO2 into maternal circulation. This would not happen if both circulations had the same oxygen transporter with the same binding characteristics as the gases would bind equally between the two circulations.
To overcome this, mammals have evolved a unique form of haemoglobin for fetuses, known as fetal haemoglobin (Hb F) consisting of two alpha and two gamma subunits (α2 γ2) (Table 1). This allows Hb F to bind oxygen with a higher affinity than adult haemoglobin (Hb A), allowing extraction of oxygen from maternal circulation. This results in a left-shifted haemoglobin-oxygen dissociation curve compared to adult haemoglobin (Figure 4). This means oxygen loading occurs at lower oxygen concentrations.
Overcoming Higher Oxygen Affinity
A higher affinity for oxygen allows higher concentrations of oxygen into fetal circulation, however this also inhibits oxygen dissociation into fetal tissue where the oxygen is needed. To overcome this, other mechanisms are in place to ensure oxygen delivery to fetal tissue:
• Increased hematocrit (Hct) – higher number of red blood cells per blood volume. This is a common reaction to reduced oxygen availability.
• Exacerbated Bohr effect – acidic pH has a greater effect on oxygen unloading in fetal tissues allowing better oxygen delivery. Acidic pH shifts the fetal oxygen-haemoglobin dissociation curve to the right, so that oxygen unloading can occur at higher oxygen partial pressures.
Fetal to Adult Haemoglobin switchover
Adult haemoglobin starts to be produced in utero, at around the 13th week of gestation. Initially in small amounts, its concentration gradually increases until 20-30% of the babies’ haemoglobin is the Hb A form. The switch is not completed until around 6 months of age (although the precise timing is not known), when Hb F levels dramatically decrease and Hb A predominates. The different globin isomers, genes and expression are summarised in Table 2, including embryonic globins ζ and ε, precursors to fetal haemoglobin.
Adult and fetal erythrocytes are not different cells, rather modifications or isoforms of an erythrocyte and therefore are derived from the same progenitors. The switchover is not determinant of different cells being produced, but genetic modification that results in different haemoglobin isoforms being contained in the erythrocyte.
Expression of the α-globin subunit is continuous as it comprises half of both fetal and adult tetrameric haemoglobins. The gene is split into two sequences, both residing on chromosome 16, along with two ζ-genes that are the embryonic version of the α-subunit (Figure 5). The γ, β and δ globin genes (and the embryonic equivalent, ε) are all located on the same chromosome, chromosome 11 (Figure 5).
Proteins Regulating Genetic Switchover
BLC11A: BCL11A is among one of the first proteins found to developmentally regulate expression of β-globulin genes on chromosome 11. It is a zinc-finger transcription factor that is involved in gene switching and silencing of Hb F.
SOX6: Represses β- like globins β and δ during embryonic and fetal stages of development in mice. SOX6 deficient mice have up-regulated β- containing haemoglobin at the fetal stage of development. In humans, the roles are uncertain, but known to act as an activator and repressor of proteins associated with the haemoglobin switch.
GATA1: GATA1 is a zinc-finger transcription factor that binds to β-globin loci and aids maturation of erythroid cells. GATA1 zinc finger mutations result in elevated Hb F in some congenital erythrpoietic syndromes.
Other proteins that also play a role in the fetal to adult haemoglobin switch include: NF-E4, COUP-TF DRED, MBD2 and Ikaros-PYR complex. However their mechanisms are not fully understood.
DNA modifications also play a role in regulating β-like globin expression in the fetal to adult haemoglobin switchover. Histone acetylation allows transcription factors to bind to β-globin genes. Histone acetylase enzymes or ATP-driven systems e.g. SWI/SNF are important for transcriptional activation by disrupting histone-DNA interactions and allowing binding of transcription factors. Conversely, histone deacetylation inhibits transcription factor binding to γ-globins to inhibit fetal haemoglobin production. Together, all of these proteins and complexes silence production of Hb F and encourage Hb A production, gradually resulting in Hb A as the predominant oxygen-carrying molecule.
Hb F production can still happen in adults when stimulated by hypoxic conditions, such as high altitude, causing stress erythropoiesis (production on RBCs). This is likely because Hb F is better at extracting oxygen due to its higher affinity. This is achieved by an increase in γ –globulin expression by removing silencing complexes (such as BCL11A) and allowing binding of transcription complexes.
Hereditary persistence of fetal haemoglobin (HPFH) – A condition in which fetal haemoglobin continues into adult life caused by a genetic mutation on β-globin gene cluster. The proportion of Hb F can range from 10% to 100% depending on severity or the condition. It is mostly harmless and symptomless, usually only discovered when searching for other haemoglobin pathologies. HPFH can reduce severity of β-globin haemoglobinopathies and is therefore more prevalent in populations where incidence of these conditions is high, such as African populations with high incidence of β-thalassemia. However, high Hb F may increase the risk of SIDS (see below).
Sudden Infant Death Syndrome (SIDS) - Low levels of adult haemoglobin, expressed as percentage of total haemoglobin, soon after birth have been associated with increased risk of SIDS. This could be due to a delay or faulty transition from fetal to adult haemoglobin. Persistently high levels of fetal haemoglobin as a neonate may affect oxygen delivery to tissues due to its high affinity for oxygen.
Sickle cell disease (SS) – An autosomal recessive disorder in the β-globin gene resulting in production and accumulation of βS-globin forming haemoglobin S (HbS, α2βS2) at fetal to adult haemoglobin switchover. This mutation creates characteristic sickle-shaped red blood cells which have reduced oxygen carrying capacity. Life expectancy with SS is 40-50 years old and presents in childhood. This disease is prevalent in tropical malaria hotspots as carriers of the disease are immune to malaria.
β Thalassemia – A group of inherited genetic blood disorders leading to a decreased or absent β-globin production necessary for Hb A. This results in an accumulation of α-globin leading to ineffective erythropoiesis that is treated with regular blood transfusions. Symptoms include severe anaemia and poor growth during infancy.
Hb F as potential therapies - BCL11A silences HBG1 and HBG2 genes that express the γ-globin components of Hb F. Removing this silencing by inhibiting BCL11A expression could act as a potential therapy for β-haemoglobinopathies by producing β-globin free haemoglobin and replacing with γ-globin containing Hb F.
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