Haemoglobin Structure & Function
Physiology · Blood · lean revision notes
Haemoglobin Structure & Function
Haemoglobin (Hb) is the iron-containing oxygen-transport metalloprotein of red blood cells, and arguably the single most exam-relevant molecule in physiology and haematology. Its quaternary architecture, cooperative oxygen binding, and the variants/derivatives that arise from it underpin everything from the sigmoid dissociation curve to thalassaemia, sickle cell disease, and carbon-monoxide poisoning.
High-yield: One gram of haemoglobin carries 1.34 mL of oxygen (Hüfner's constant). Each haemoglobin molecule binds a maximum of 4 O₂ molecules.
Structure of Haemoglobin
Haemoglobin is a tetramer — a quaternary protein built from four polypeptide (globin) chains, each folded around one haem prosthetic group. Therefore every haemoglobin molecule contains 4 globin chains + 4 haem groups + 4 iron atoms and can bind 4 O₂.
The Globin Chains
- Globin chains are predominantly alpha-helical (~75%), with eight helices labelled A–H.
- Each chain encloses a haem in a hydrophobic cleft between helices E and F.
- Adult tetramer = two α-chains + two non-α chains (α₂β₂ for HbA).
The Haem Group
- Haem = protoporphyrin IX ring + ferrous iron (Fe²⁺) at its centre.
- Iron forms six coordination bonds:
- 4 to the nitrogen atoms of the porphyrin ring (in the plane).
- 1 to the proximal histidine (F8) of the globin chain.
- 1 (the sixth) to molecular oxygen — stabilised by the distal histidine (E7).
High-yield: Iron must be in the ferrous (Fe²⁺) state to bind O₂. Oxidation to ferric (Fe³⁺) produces methaemoglobin, which cannot carry oxygen.
High-yield: Oxygen binding to haem is oxygenation, NOT oxidation — iron remains Fe²⁺ throughout. This is a classic MCQ trap.
Globin Gene Locations
| Globin chain | Chromosome | Cluster |
|---|---|---|
| α (alpha) | Chromosome 16 | 4 α-genes total (αα/αα) |
| β, γ, δ, ε | Chromosome 11 | β-globin gene cluster |
High-yield: Alpha = 16 (4 genes), Beta = 11 (2 genes). This is why α-thalassaemia has four clinical grades (silent → Hb Bart's hydrops fetalis) while β-thalassaemia has fewer.
Normal Haemoglobin Variants
The combination of globin chains changes across development, giving physiological variants.
| Haemoglobin | Chain composition | Where/When | Normal adult % |
|---|---|---|---|
| HbA | α₂β₂ | Main adult Hb | ~96–97% |
| HbA₂ | α₂δ₂ | Minor adult Hb | ~1.5–3.5% (↑ in β-thalassaemia trait) |
| HbF | α₂γ₂ | Fetal/newborn | <1% adult; ~70–80% at birth |
| Gower 1 | ζ₂ε₂ | Early embryo | — |
| Gower 2 | α₂ε₂ | Early embryo | — |
| Portland | ζ₂γ₂ | Early embryo | — |
High-yield: HbF (α₂γ₂) has higher oxygen affinity than HbA because γ-chains bind 2,3-DPG poorly — this lets the fetus extract O₂ from maternal blood across the placenta.
High-yield: HbA₂ is raised in β-thalassaemia trait (>3.5%) — the single most useful screening clue for beta-thalassaemia minor.
Developmental switch flow: Embryonic (Gower/Portland) → Fetal HbF → Adult HbA. The γ→β switch completes by ~6 months of age, which is why β-thalassaemia major and sickle cell disease present after the first 6 months, not at birth.
Pathological variant: HbS
- HbS arises from a point mutation in the β-globin gene: Glutamate → Valine at position 6 of the β-chain (β6 Glu→Val).
- This is a missense (single base substitution: GAG→GTG) mutation.
- Deoxygenated HbS polymerises into rigid fibres → sickling of RBCs.
High-yield: Mnemonic for sickle cell mutation — "Glutamate to Valine, position 6, beta chain." HbC is Glu→Lysine at the same β6 position.
Oxygen Binding & the Dissociation Curve
Cooperative (Allosteric) Binding
Haemoglobin binds oxygen cooperatively — binding of the first O₂ increases affinity for subsequent O₂. This produces the characteristic sigmoid (S-shaped) oxygen–haemoglobin dissociation curve. (Myoglobin, a monomer, has a hyperbolic curve and higher affinity — it stores rather than transports.)
R and T States (Allostery)
| Feature | T state (Tense) | R state (Relaxed) |
|---|---|---|
| Oxygen affinity | Low | High |
| Form | Deoxyhaemoglobin | Oxyhaemoglobin |
| 2,3-DPG binding | Binds, stabilises T | Expelled |
| Iron position | Out of porphyrin plane | Pulled into plane |
The mechanism (Perutz mechanism): O₂ binding pulls the Fe²⁺ into the porphyrin plane, dragging the proximal histidine and triggering the T→R conformational shift that raises affinity of the remaining subunits.
High-yield: P₅₀ = partial pressure of O₂ at which haemoglobin is 50% saturated. Normal adult P₅₀ ≈ 26–27 mmHg. A higher P₅₀ = lower affinity = right shift; lower P₅₀ = higher affinity = left shift.
Key landmarks on the curve
- At PaO₂ 100 mmHg (arterial) → ~97–98% saturated.
- At PaO₂ 40 mmHg (mixed venous) → ~75% saturated.
- At PaO₂ 26–27 mmHg → 50% (P₅₀).
Factors Shifting the Curve
Right shift (↓ affinity, O₂ released to tissues) — "CADET, face Right": ↑ CO₂, ↑ Acid (↓ pH), ↑ DPG (2,3-DPG), ↑ Exercise/Temperature.
| Right shift (unloading ↑) | Left shift (loading ↑) |
|---|---|
| ↑ 2,3-DPG | ↓ 2,3-DPG (stored blood) |
| ↑ H⁺ (↓ pH, acidosis) | ↑ pH (alkalosis) |
| ↑ CO₂ (Bohr effect) | ↓ CO₂ |
| ↑ Temperature | ↓ Temperature |
| HbS, high altitude (chronic) | HbF, CO-Hb, methaemoglobin |
High-yield: The Bohr effect = CO₂/H⁺ lower Hb's O₂ affinity (right shift), favouring O₂ release in metabolically active tissues. The Haldane effect = deoxygenated Hb carries more CO₂ (and binds H⁺ better), favouring CO₂ loading in tissues and unloading in lungs.
2,3-DPG (2,3-Bisphosphoglycerate)
- Produced in the RBC via the Rapoport–Luebering shunt (a side branch of glycolysis).
- Binds in the central cavity between the two β-chains, stabilising the T (deoxy) state → right shift.
- ↑ in: chronic hypoxia, high altitude, anaemia, COPD.
- ↓ in: stored (banked) blood → left shift → poor tissue O₂ delivery after massive transfusion.
High-yield: HbF resists 2,3-DPG binding (γ-chains lack the key residues), so HbF sits left of HbA — higher affinity, lower P₅₀.
Abnormal Haemoglobin Derivatives
| Derivative | Iron state / cause | Colour | O₂ carriage | Key facts |
|---|---|---|---|---|
| Oxyhaemoglobin | Fe²⁺ + O₂ | Bright red | Yes | Normal |
| Deoxyhaemoglobin | Fe²⁺, no O₂ | Dark red/blue | — | Causes cyanosis when >5 g/dL |
| Methaemoglobin | Fe³⁺ | Chocolate-brown | No | Causes functional anaemia; left-shifts curve |
| Carboxyhaemoglobin | Fe²⁺ + CO | Cherry-red | No | CO affinity ~240× O₂ |
| Sulfhaemoglobin | Sulphur-modified | Greenish | No | Irreversible; drugs (sulphonamides) |
Methaemoglobinaemia
- Iron oxidised to Fe³⁺ → cannot bind O₂ and left-shifts the curve for the remaining normal subunits (worsening tissue delivery).
- Causes: nitrites, nitrates, dapsone, primaquine, local anaesthetics (benzocaine, prilocaine), aniline dyes; congenital NADH-methaemoglobin reductase (cytochrome b5 reductase) deficiency, or HbM.
- Clinical clue: cyanosis NOT responding to oxygen, chocolate-brown blood, normal PaO₂ but low SpO₂, saturation gap.
- Drug of choice: Methylene blue IV (1–2 mg/kg). Ascorbic acid in mild/chronic cases. Avoid methylene blue in G6PD deficiency (can worsen haemolysis) — use ascorbic acid/exchange transfusion.
High-yield: Methaemoglobinaemia → cyanosis unresponsive to O₂ + chocolate-brown blood; treat with methylene blue. Pulse oximetry reads ~85% plateau regardless of true saturation.
Carbon Monoxide (Carboxyhaemoglobin)
- CO binds Hb with ~200–250× the affinity of O₂, forming carboxyhaemoglobin and left-shifting the residual curve → double hit on tissue O₂ delivery.
- Cherry-red skin/mucosa; SpO₂ falsely normal (pulse oximeter cannot distinguish HbCO from HbO₂).
- Diagnosis: co-oximetry (measures HbCO directly).
- Treatment: 100% oxygen (reduces half-life of HbCO from ~4–5 h to ~1 h); hyperbaric O₂ for severe cases/pregnancy/neuro signs.
High-yield: In both CO poisoning and methaemoglobinaemia, PaO₂ is normal but oxygen content is low — pulse oximetry is unreliable; use co-oximetry.
Clinical Correlations: Disorders Hinging on Hb Structure
Sickle Cell Disease (HbS)
- β6 Glu→Val → deoxy-HbS polymerises → sickling, vaso-occlusion, chronic haemolysis.
- Diagnosis: Hb electrophoresis / HPLC (HbS band); sickling test; solubility test (turbidity). Definitive — HPLC/electrophoresis.
- Right-shifted curve; HbF (and hydroxyurea, which raises HbF) reduces sickling.
Thalassaemias
- Reduced/absent globin chain synthesis (a quantitative defect; sickle cell is qualitative).
- β-thalassaemia: ↓β chains → excess α chains precipitate → ineffective erythropoiesis. HbA₂ and HbF raised.
- α-thalassaemia: depends on number of α-genes deleted (1=silent, 2=trait, 3=HbH disease β₄, 4=Hb Bart's γ₄ → hydrops fetalis).
| Feature | Sickle cell disease | Thalassaemia |
|---|---|---|
| Defect type | Qualitative (abnormal Hb) | Quantitative (↓ synthesis) |
| Mutation | Point (β6 Glu→Val) | Deletion (α) / point (β) |
| Diagnostic | HbS on HPLC | ↑HbA₂/HbF (β); chain analysis |
Diagnosis & Investigation of Choice
- Haemoglobin variant identification → HPLC (high-performance liquid chromatography) is now the investigation of choice, superseding alkaline electrophoresis (cellulose acetate, pH 8.6).
- Methaemoglobin / carboxyhaemoglobin → co-oximetry.
- Quantifying HbA₂/HbF → HPLC (key for β-thalassaemia trait).
- Sickle cell → solubility/sickling test for screening; HPLC/electrophoresis to confirm.
Management / Drug of Choice (Quick Map)
- Methaemoglobinaemia → Methylene blue (ascorbic acid if G6PD deficient).
- CO poisoning → 100% O₂ → hyperbaric O₂ if severe.
- Sickle cell → Hydroxyurea (↑ HbF), hydration, analgesia, transfusion in crises.
- β-thalassaemia major → transfusion + iron chelation (deferasirox/deferoxamine).
Complications (of Hb-related disease)
- Sickle cell: vaso-occlusive crises, acute chest syndrome, stroke, splenic sequestration, aplastic crisis (parvovirus B19), priapism, autosplenectomy.
- Thalassaemia: iron overload (transfusion → cardiac/hepatic siderosis), extramedullary haematopoiesis, "hair-on-end" skull, chipmunk facies.
- Methaemoglobinaemia: tissue hypoxia, seizures, death at high levels (>70%).
Key Differentials (Cyanosis with Normal PaO₂)
Central cyanosis unresponsive to O₂ → think: Methaemoglobinaemia (chocolate-brown blood) vs Sulfhaemoglobinaemia (greenish) vs right-to-left cardiac shunt. CO poisoning gives cherry-red, not cyanosis.
Recently asked / exam angle
- Cooperative binding & sigmoid curve vs myoglobin's hyperbolic curve — repeatedly asked.
- Right vs left shift factors (CADET face Right; HbF, CO, metHb, stored blood = left).
- P₅₀ value (26–27 mmHg) and its interpretation.
- 2,3-DPG site of action (β-chain central cavity), Rapoport–Luebering shunt, ↑ at altitude.
- Methaemoglobin — Fe³⁺, chocolate-brown blood, methylene blue; avoid in G6PD.
- CO affinity (~240×) and falsely normal pulse oximetry — co-oximetry is the answer.
- HbA₂ raised in β-thalassaemia trait; HbF composition (α₂γ₂) and high affinity.
- Sickle mutation (β6 Glu→Val, GAG→GTG, missense).
- Globin gene loci: α on chromosome 16, β on chromosome 11.
- Bohr vs Haldane effect distinction.
Rapid revision
- Hb = tetramer: 4 globin + 4 haem + 4 Fe²⁺, binds 4 O₂; 1 g Hb = 1.34 mL O₂.
- HbA = α₂β₂, HbA₂ = α₂δ₂, HbF = α₂γ₂.
- Iron must be Fe²⁺; Fe³⁺ = methaemoglobin (can't carry O₂).
- Proximal His (F8) binds iron; distal His (E7) stabilises O₂.
- α-globin = chromosome 16 (4 genes), β-globin = chromosome 11.
- Sickle mutation = β6 Glutamate → Valine (missense, GAG→GTG).
- Sigmoid curve (Hb, cooperative) vs hyperbolic (myoglobin); P₅₀ ≈ 26–27 mmHg.
- Right shift (CADET): ↑CO₂, ↑Acid, ↑DPG, ↑Exercise/Temp → O₂ released.
- Left shift: HbF, CO-Hb, metHb, stored blood, alkalosis, ↓DPG, ↓temp.
- 2,3-DPG binds β-chain central cavity → stabilises T state → right shift; ↑ at altitude.
- Bohr = CO₂/H⁺ unload O₂; Haldane = deoxy-Hb carries more CO₂.
- CO: cherry-red, ~240× affinity, false-normal SpO₂ → treat with 100% O₂/HBO; metHb → methylene blue.