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Haemoglobin is the single most studied protein in biochemistry. It is the molecule that makes vertebrate life at our size possible: a globular oxygen-binding pigment housed inside red blood cells, capable of loading oxygen efficiently at the pulmonary capillary, holding it tightly during transit through the systemic arteries, and releasing it selectively in the tissues where it is most needed. The molecular behaviour of haemoglobin is not a passive carrier story but a sophisticated piece of allosteric engineering — its four polypeptide chains communicate, its oxygen affinity changes with local pH, CO₂, temperature and 2,3-bisphosphoglycerate, and a single amino acid substitution can convert it into the agent of a lethal genetic disease. This lesson examines haemoglobin's quaternary architecture, the sigmoidal oxygen dissociation curve, the Bohr shift, foetal and myoglobin variants, high-altitude adaptation, the three pathways of CO₂ transport, and the molecular basis of sickle-cell disease and its heterozygote advantage. Together these are the central exam-relevant content of AQA 7402 Section 3.3.4 mass transport in animals.
This lesson maps to AQA 7402 Section 3.3.4 — Mass transport in animals (haemoglobin) (refer to the official AQA specification document for exact wording). The specification requires that students understand the role of haemoglobins as a group of chemically similar molecules with quaternary structure, the cooperative loading and unloading of oxygen, the significance of the oxygen dissociation curve, the Bohr effect, and adaptations of haemoglobins to environments with different oxygen availability.
Adult haemoglobin (HbA) is a tetramer of four polypeptide subunits: two α (alpha) chains of 141 amino acids each and two β (beta) chains of 146 amino acids each. The four chains are held together by non-covalent interactions — hydrogen bonds, ionic bonds, hydrophobic interactions — in a near-spherical assembly with two-fold symmetry. Each subunit folds into the globin fold of eight α-helices arranged around a central pocket. Inside that pocket sits a haem prosthetic group — a flat porphyrin ring with a single Fe²⁺ ion at its centre, anchored to the globin via a histidine residue (the proximal histidine, F8). Each tetramer therefore carries four haem groups and binds four molecules of O₂ at full saturation.
The Fe²⁺ ion is essential. The lone pair of electrons on iron's 4s orbital allows reversible donation to molecular oxygen — the iron forms a weak coordinate bond with O₂ that breaks on demand. If the iron is oxidised to Fe³⁺, the resulting protein is called methaemoglobin and either cannot bind O₂ or, in some abnormal variants, binds it irreversibly tightly. Methaemoglobinaemia (Fe³⁺ haemoglobin > ~10 % of total) produces tissue hypoxia despite normal arterial PO₂ — congenital from cytochrome b5 reductase deficiency, or acquired from nitrites, sulphonamides or some local anaesthetics. Normal red blood cells continuously reduce stray Fe³⁺ back to Fe²⁺ via NADH-dependent cytochrome b5 reductase; this is a synoptic link to Section 3.5 respiration and reduced coenzymes.
The plural "haemoglobins" is deliberate. The AQA specification treats haemoglobin as a family of chemically similar proteins, each with the four-chain, four-haem quaternary structure, but with different chain compositions and oxygen affinities suited to different organisms or different life stages of the same organism. Foetal HbF replaces α₂β₂ with α₂γ₂ (gamma chains). Embryonic haemoglobins use ζ (zeta) and ε (epsilon) chains during the first weeks of gestation. Llamas, vicuñas and bar-headed geese — high-altitude mammals and birds — have α chains with single amino acid substitutions that raise oxygen affinity. Crocodilians and some diving turtles have haemoglobins regulated by bicarbonate rather than 2,3-BPG, releasing more O₂ as CO₂ accumulates during prolonged dives. The variation is a beautiful illustration of how a single protein scaffold has been tuned by natural selection to environmental oxygen availability.
Plot the percentage saturation of haemoglobin (y-axis) against the partial pressure of oxygen (PO₂, x-axis) and the result is a sigmoidal (S-shaped) curve that is the defining graphical object of A-Level Biology Section 3.3.
flowchart LR
A[Low PO2 ~2 kPa<br/>Hb ~10% saturated<br/>steep lower limb<br/>resting tissue range]
B[Mid PO2 ~5 kPa<br/>Hb ~50% saturated<br/>active tissue range<br/>P50 reference point]
C[Knee PO2 ~8 kPa<br/>Hb ~85% saturated<br/>curve flattens]
D[Plateau PO2 ~13 kPa<br/>Hb ~98% saturated<br/>pulmonary capillary<br/>full loading]
A --> B --> C --> D
The sigmoidal shape is the consequence of cooperative binding between the four haem groups. When the first O₂ binds to one haem, the resulting movement of Fe²⁺ into the plane of the porphyrin ring drags the proximal histidine and the F-helix with it. This small movement is transmitted through the protein's quaternary contacts to the other three subunits, causing the whole tetramer to switch from a tense (T) state with low oxygen affinity to a relaxed (R) state with much higher affinity. The second, third and fourth O₂ molecules therefore bind progressively more easily. This cooperativity is the molecular basis of the curve's S-shape: at low PO₂ binding is sluggish (the lower limb), at moderate PO₂ binding accelerates as more subunits flip to R-state (the steep middle region), and at high PO₂ binding plateaus (the upper limb) as all four sites approach saturation.
Three frameworks describe this mathematically: Adair's 1925 stepwise equilibrium constants for sequential O₂ binding; Hill's empirical equation with the Hill coefficient n (n ≈ 2.8 for adult Hb, indicating strong cooperativity); and the Monod-Wyman-Changeux (MWC) two-state allosteric model. AQA does not require these names, but the concept of an allosteric T → R transition is genuine A-Level depth and frames the more advanced extension reading.
The curve is shaped exactly right for vertebrate physiology. The flat upper limb at high PO₂ means that small reductions in alveolar PO₂ — at altitude, in lung disease, in airway obstruction — barely affect the saturation of arterial blood: haemoglobin is a robust loader. The steep middle limb at tissue PO₂ means that small reductions in tissue PO₂ — as cells consume O₂ — produce large reductions in saturation: haemoglobin is a sensitive unloader. The same molecule manages to be a tenacious carrier in the lungs and a generous donor in the tissues. No simple linear binding curve could do this.
P50 is the PO₂ at which haemoglobin is 50 % saturated. For adult HbA at pH 7.4 and 37 °C, P50 ≈ 3.5 kPa (~26 mmHg). P50 is a single-number summary of oxygen affinity: a lower P50 means a left-shifted curve and higher affinity (Hb holds onto O₂ more tightly — good for loading, bad for unloading); a higher P50 means a right-shifted curve and lower affinity (the opposite). P50 is therefore the key tunable parameter on which the variations between haemoglobins are organised.
Air at sea level has PO₂ ≈ 21 kPa. After mixing with residual gas in the alveoli and being humidified, alveolar PO₂ falls to ~13 kPa. Pulmonary capillary blood equilibrates with alveolar gas, so arterial PO₂ ≈ 13 kPa and haemoglobin saturation ≈ 98 %. The blood is then pumped through the systemic circulation to tissues. Resting tissue PO₂ ≈ 5 kPa; haemoglobin re-equilibrates and unloads to ~70 % saturation, donating ~28 % of its bound O₂. Active tissue PO₂ falls to ~2 kPa; haemoglobin unloads to ~25 %, donating a much larger fraction. This is the core demand-led delivery mechanism: tissues consuming most O₂ create the largest concentration gradient and extract the most O₂ from passing blood.
| Site | PO₂ (kPa) | Hb saturation (HbA) | Notes |
|---|---|---|---|
| Alveolus | 13 | ~98 % | full loading |
| Systemic arteries | 12 | ~97 % | minor pulmonary venous admixture |
| Resting tissue capillary | 5 | ~70 % | ~28 % unloaded |
| Active muscle capillary | 2 | ~25 % | majority unloaded |
| Mixed venous blood | 5–6 | ~75 % | returns to right heart |
The mathematics of this is the reason vertebrate oxygen delivery works at any tissue size. Haemoglobin carries ~20 mL O₂ per 100 mL blood at full saturation, ~70 times the solubility of O₂ in plasma alone. Without haemoglobin, cardiac output would have to be ~70-fold higher to deliver the same flux — impossible.
The Danish physiologist Christian Bohr (1904) showed that the oxygen dissociation curve shifts to the right when blood pH falls or PCO₂ rises. The biological logic is exact. Active tissues consume O₂ and produce CO₂; the CO₂ enters red blood cells and is converted to H⁺ and bicarbonate (see below). Both rising CO₂ and rising H⁺ stabilise the T-state of haemoglobin and lower its oxygen affinity. The right-shifted curve means that at any given tissue PO₂, haemoglobin unloads more O₂ than it would in the absence of the Bohr shift.
The mechanism is twofold. CO₂ binds directly to terminal amino groups on the globin chains, forming carbamino compounds that favour T-state. H⁺ binds to specific histidine residues whose pKa is poised near physiological pH, again favouring T-state. The two effects reinforce each other (the Haldane effect at the same protein describes how deoxygenated Hb binds H⁺ and CO₂ more readily than oxygenated Hb, completing a self-reinforcing cycle of oxygen unloading and CO₂ loading at tissues, and the reverse at lungs).
flowchart LR
T[Active tissue<br/>CO2 produced<br/>H+ produced<br/>temperature rises] -->|right shift| C1[Hb affinity falls<br/>P50 rises]
C1 --> O[More O2 unloaded<br/>to active tissue]
O --> RBC[CO2 enters RBC<br/>H+ and HCO3- form]
RBC --> V[Venous blood returns]
V --> LU[Lung capillary<br/>CO2 unloaded<br/>H+ falls] -->|left shift| C2[Hb affinity rises<br/>P50 falls]
C2 --> L[Maximal O2 loading]
Three other factors shift the curve rightward: temperature (a fevered or exercising muscle is warmer and unloads more O₂); 2,3-bisphosphoglycerate (2,3-BPG), a glycolytic intermediate that accumulates in red blood cells and binds between the β chains in T-state, stabilising T and lowering affinity; and chronic hypoxia, which up-regulates 2,3-BPG synthesis. The opposite shifts (alkalosis, low CO₂, low temperature, low 2,3-BPG, foetal Hb, carbon monoxide) move the curve leftward and impair tissue unloading.
The foetus must extract oxygen from maternal blood across the placenta. Maternal arterial blood arriving at the placenta is already partially desaturated (PO₂ ~5–6 kPa); foetal blood must load O₂ from this against a relatively shallow gradient. The solution is foetal haemoglobin (HbF), a tetramer of two α chains and two γ chains in place of the adult β chains. HbF has a left-shifted dissociation curve relative to HbA (P50 ~2.5 kPa vs ~3.5 kPa). At any placental PO₂, foetal blood is therefore more saturated than maternal blood, and net O₂ transfer from mother to foetus is sustained.
The molecular reason for the shift is that γ chains bind 2,3-BPG less tightly than β chains, removing one of the major rightward shifters. HbF is gradually replaced by HbA over the first six months of postnatal life; the switch is regulated at the transcriptional level by repressors that recognise the γ-globin promoter. Reactivation of HbF in adulthood would mitigate sickle-cell disease, and pharmacological induction of HbF (e.g. by hydroxyurea) is a recognised treatment.
Myoglobin is the muscle oxygen-binding pigment. It is a single polypeptide of 153 amino acids with a single haem group — essentially a monomeric ancestor of a haemoglobin subunit. Because it is monomeric, there is no cooperativity and its oxygen dissociation curve is hyperbolic, not sigmoidal, with a very low P50 (~0.3 kPa). Myoglobin therefore holds O₂ tightly at virtually all tissue PO₂ values and releases it only when PO₂ falls to very low values — under sustained heavy exercise or transient ischaemia. It acts as an emergency O₂ store for working muscle. Diving mammals (seals, whales) have spectacularly elevated myoglobin concentrations supporting prolonged apnoea. The release of myoglobin from crushed muscle (rhabdomyolysis) into plasma can precipitate in renal tubules and cause acute kidney injury — a synoptic link to Section 3.6.
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