Respiratory Biochemistry

Lecture 13: Structure of Haemoglobin

• Need for circulation and haemoglobin: due to the size of human body, diffusion of oxygen is insufficient to provide for cells. A transport system is required and this is in the form of blood circulation. Furthermore solubility of O2 is poor in blood (3mL/L) and inadequate to provide so you require an oxygen carrying molecule such as haemoglobin
• Features of circulation:
- A surface for efficient gas exchange
- A system for efficient moving solutions/suspensions
- Efficient oxygen bind/transport protein (utilize iron as the transition metal ion)
• Erythrocytes: bi-concave cell with no nucleus and mitochondria, responsible for housing haemoglobins that transported oxygen.
- Structure: shape in between flat disk and sphere for rapid oxygen uptake and strength.
- Hemocrit value: percentage of volume of blood occupied by red blood cell
- Features: have a half life of 120 days; contains haemoglobin which would otherwise be toxic and highly viscous in blood
• Haemoglobin: quaternary protein consisting of 4 subunits, 2 alpha and 2 beta groups
- Haeme: ring-like structure at the center of each subunits that binds to iron (Fe2+) with 4 surrounding nitrogen groups
- Iron state: bounded iron must be in ferrous state to be functional
- Subunits: the alpha and beta chains are coded on chromosome 16 and 11 and are 141 and 146 amino acid each respectively. Each subunit is mainly alpha helix.
- Synthesis: occurs in marrow of flat bones such as pelvis and ribs from precursor reticulocytes.
• Histidine: important amino acid in maintaining the functionality and structure of haemoglobin
- Proximal histidine: forms co-ordination bond with iron (as a ligand) to hold the heme group in place
- Distal histine: acts indirectly on the iron-oxygen bond as its location prevent oxygen from binding perpendicularly to the iron and prevent Fe2+ from becoming Fe3+
• Homotropism: co-operative nature of the haemoglobin in which binding of one O2 will change the conformation of other binding sites and improve affinity for further oxygen binding.
- Graphic interpretation: graph of partial pressure with relative oxygen is sigmoid as at the beginning, most haemoglobin only has one O2 molecule bound so “non-optimal binding” but with increase content of O2, more oxygen will bind and allosterically enhance binding of other subunit active sites.
- Hill co-efficient: expression for the level of homotropism or co-operativity between the haemoglobin chains.
- P50: partial pressure of oxygen (content) at which the blood or haemoglobin binding sites are half saturated
• Transition of resting and exercise: during resting, oxygen unloading capacity is minute, i.e. 100% to 70% saturation but during exercise, unloading reaches as low as 10%. Hence the human body is adapted to strenuous activities
• Heterotropism: binding nature of oxygen is influenced by other substances (not competitive) that bind to allosteric sites on the haemoglobin protein and lower oxygen affinity. Example include H+ and Cl+ ions (effectors)
• Bohr effect: the phenomenon in which decreasing pH leads to decreased oxygen affinity as H+ is a non-competitive allosteric inhibitor (for acute conditions).

H-Hb + O2 Hb-O2 + H+

- The effect can be demonstrated on the saturation graph as a right shift. At around pH of 6.8, 100 mmHg of lung oxygen pressure produce 90% saturation but at pH of 7.6, saturation drops to 80% (not significant)
- Near muscles the effect becomes more apparent with 40 mmHg PO2, basic conditions confer 80% saturation while acidic 40% saturation. This has important implications, as anaerobic muscles produce lactic acids and so oxygenated haemoglobin will deliver more oxygen to support aerobic conditions.
• 2,3-diphosphoglycerate: a side product of glycolysis in RBC and is the most important allosteric effector (regulator) of haemoglobin function. The level of this substance is able to be internally regulated (for chronic reasons) to compensation changes in O2 binding due to anemia, mutations or environments.
- In fetal blood, DPG steals oxygen from mother. This is because, 2,3 DPG binding site is less charged in fetal haemoglobin so binds DPG less tightly and as a result of decrease allosteric inhibition, fetal haemoglobin binds to O2 with a higher affinity.
- Concentration of intracellular DPG rise in situations such as hypoxia as a result of obstructive pulmonary emphysema, anemia, congestive heart failure to maximize O2 unloading. While at the lungs, DPG is metabolized to improve O2 binding.
- Enzyme defects such as hexokinase deficiency will lead to lower rate of glycolysis and thus DPG while pyruvate kinase deficiency will increase DPG
- DPG can be used to counter effects of pH as a glycolytic enzymes phosphofructokinase is pH dependent (increase activity with low pH).

Deoxy Hb – DPG + O2 Hb-O2 + DPG

• Temperature: rise in temperature lowers the oxygen affinity of haemoglobin
• Chloride effect: Cl- ions also bind preferentially to deoxy haemoglobin and lower its oxygen affinity.
• Transport of CO2: there are two methods of transferring CO2 in blood
- Carbamino formation
- Bicarbonate formation
• Carbamino formation: CO2 react directly with amine functional groups on deoxygenated haemoglobin while lowering O2 affinity. 15% of CO2 is transported this way

Hb – NH2 + CO2 Hb-NHCO2 + H+

- The hydrogen ion produced acts to further enhance oxygen unloading effect and aerobic environment
• Bicarbonate formation:
- Near the tissue within a RBC, carbon dioxide produced is rapidly converted to H2CO3 by combining with water. The catalyst is carbonic anhydrase.
- H2CO3 spontaneously decomposes into HCO3- and H+ (where the H+ aid oxygen delivery
- HCO3-/Cl- exchanger, for every Cl- that passes in, a bicarbonate ion is pumped out.
- Since bicarbonate is extremely soluble in blood, majority of CO2 is present in blood
- At the lung however, the exchanger reverses with pumping out Cl- outwards and HCO3- inwards. CO2 is produced and released from the cell to the lung

Lecture 16: Defects in Haemoglobin

• Haemoglobinopathy: defects in the haemoglobin proteins. There are more than 500 different mutations identified with a portion causing a compromise in the function of the protein. This can be divided into three categories:
- Surface residues: affects interaction between proteins as structural changes occurs on the surface
- Haeme cavity: alterations in the haeme location that will affect oxygen affinity or iron oxidation
- Subunit interface: the rarest form of mutations occurring within the subunits that will affect dissociation or cooperativity.
• Polycythaemia: excessive number of red blood cells in the blood. This condition is brought about through non-functional haemoglobin (due to mutations) and the body signals to produce extra in order to compensate.
• Fetal and adult haemoglobin:
- Adult have 1 copies of the alpha subunit gene and 2 copies of the beta
- Fetal haemoglobin consists of 2 alpha and 2 gamma subunits with the gamma chain used to “steal” O2¬ from the mother through the placenta.
- Development: from the start of the zygote, alpha subunits are constantly produced. Conversely beta subunits is only produced after birth with the degradation of all gamma heme as the newborn acquire its own oxygen through respiration.
• Sickle cell haemoglobin: condition in which a point mutation of the beta gene changes the 6th glutamic acid residue (GTG) into a valine amino acid (GAG).
- Background: haemoglobin have bound and unbound forms. When the subunit is bounded with O2, the glutamic acid residue is buried inside. During deoxygenation, the glutamic acid residue is exposed and due to its negative charge, haemoglobin will repel and remain stable.
- Pathology: having glutamic acid replaced with valine, a hydrophobic amino acid, during deoxygenation, the exposed residues will attract instead of repel. The result is the hydrophobic patch on the mutant haemoglobin (termed HbS) causes irreversible polymerization. Consequently long strands of HbS fibers stretch the erythrocyte giving a sickle appearance.
- Manifestation: vascular occlusion by sickle cells produce “painful crisis” (episodes of extreme pain) and organs damage, eventually leading to death.
• Balanced polymorphism: while mutation of both beta gene (homozygous) will inevitably lead to the death but the fact that more than 100 million people in the world possess this mutations demonstrate the idea of balanced polymorphism. This is a condition in which the heterozygous form actually confers a survival advantage.
- Protection against malaria: malaria caused by a parasite that colonizes red blood cells in order to proliferate and later erupt out of the cell to spread thereby killing the host. However in heterozygotes, the sickle cell condition is still partially expressed. Some red blood cells may precipitate during deoxygenation (hydrophobic nature) and these are detected by the spleen and removed, along with any parasites that inhabited it.
• Metaemoglobinaemia: mutation of the distal His to Tyr leads to oxidation of the haem iron to ferric (methaemoglobin). Oxidised iron is no longer able to carry oxygen.
- Etiology: arise from environment factors such as drug taking. The intrinsic reducing system of the body can not reduce the ferric ion back as tyrosine is always present to oxidize it.
- Consequences: increasing subunit affected leads to increased chance of chance with more than two chains mutated almost lethal
• Production of haemoglobin: expression of the alpha and beta gene is independent to each other but coincidentally matches. Production occurs in bone marrows stem cells producing reticulocytes. After transcription, the haeme group is added to the chains forming globulins and so haemoglobin. Reticulocytes lose its nucleus and become red blood cells.
• Thalassaemia: imbalance in the number of different type of subunits produced. This can leads to excess chains precipitating within the red blood cells as brown deposits known as Heinz bodies and lead to premature RBC destruction and iron depots in internal organs.
- splenomegaly: rapid turnover of red blood cells causes overworking of the spleen and enlargement
- long bones and fingers: loss of haemoglobin stimulates further production in the body and all long bone marrows are employed for production (polycythaemia)
• Alpha thalassaemia: a lack of alpha chain synthesis due to a gene deletion in the gene. Since our body have 4 copies of alpha gene, variable number of the genes mutated will lead to different discrete types of disease
- Alpha thalassaemia 2: 3 genes present normal and expressing the protein. Conditions are mild and no major red blood cells defects
- Alpha thalassaemia 1: 2 genes present and expressing protein. There will be red blood cell defects and some problem with O2 transport.
- Haemoglobin H disease: 1 gene present and expressing alpha protein with serious blood defects and oxygen transport problems. Majority of haemoglobin presents as HbH4 (4-beta globin)
- Hydrio fetalis: no alpha gene is present and death occur in utero as no viable haemoglobin can be made for oxygen transport. (alpha chain important in all stage of life)
• Beta thalassaemia: less than normal synthesis of the beta chain due mutation and also fault in the gene expression, i.e. gene splicing or translation but DNA itself is normal along with correct quantity synthesized.
- Unlike alpha chain which has fixed levels of severity depending on the number of alpha gene lost, beta thalassaemia is produced from variable mutations of the gene. The consequence is a wider continuous range of effects
- Beta chain is only produced in adults, so defective/absent beta subunits leads to immediate death at birth. At times the fetal haemoglobin is preserved to adapt.
- Manifestation: common presentation similar to alpha.
• Types of beta thalassaemia: many different classifications exist for beta thalassaemia.
- Clinical: based on trait, split into thalassaemia intermedia and major
- Molecular: based on type of mutation, split into betao which is nonsense frame shift mutations producing no viable mRNA or beta+ which involves transcription or intron processing, and promoter/regulator defect that leads to low level of viable mRNA.

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