Haemoglobin Structure & Hemoglobinopathies

Haemoglobin is the iron-containing oxygen-carrying conjugated protein of the red cell, and a perennial favourite of NEET PG because it cuts across Biochemistry, Physiology, Pathology and Medicine. This note builds from molecular architecture to the oxygen-dissociation curve, then to the structural and quantitative hemoglobinopathies and methemoglobinemia.

Structure of Haemoglobin

Adult haemoglobin (HbA) is a tetramer of four polypeptide globin chains (α2β2), each folded around one haem group. Each subunit therefore carries one iron, so one Hb molecule binds a maximum of 4 O2 molecules.

• Globin chains: Each chain is largely α-helical (eight helices labelled A–H), an arrangement called the globin fold (a member of the all-α "myoglobin fold" family).
• Haem: Protoporphyrin IX + ferrous iron (Fe2+). Iron is six-coordinate: 4 bonds to porphyrin nitrogens, one to the proximal histidine (F8), and the sixth to O2. The distal histidine (E7) stabilises bound O2 and sterically discourages CO binding.
• Quaternary nature is the key contrast with myoglobin, a single-chain monomer that stores O2 in muscle.

High-yield: Iron in functional haemoglobin/myoglobin must remain ferrous (Fe2+). Oxidation to ferric (Fe3+) gives methaemoglobin, which cannot carry O2.

Haemoglobin vs Myoglobin

Feature	Haemoglobin (HbA)	Myoglobin
Structure	Tetramer (α2β2)	Monomer
O2 binding curve	Sigmoid (cooperative)	Hyperbolic
P50 (mmHg)	~26–27	~1–5
Function	O2 transport in blood	O2 storage in muscle
Allosteric regulation	Yes (2,3-BPG, H+, CO2)	No
Hill coefficient	~2.8 (cooperative)	~1

Cooperativity is the structural reason for the sigmoid curve: binding of O2 to one subunit increases the affinity of the remaining subunits. Haemoglobin oscillates between two quaternary states — the T (tense, deoxy, low-affinity) state and the R (relaxed, oxy, high-affinity) state. Oxygenation triggers the T → R transition.

High-yield: Sigmoid curve = cooperative binding = positive homotropic effect. Myoglobin is non-cooperative (hyperbolic), so it loads O2 even at low PO2, ideal for a storage molecule.

Normal Human Haemoglobins

Haemoglobin	Chains	Notes
HbA	α2β2	~96–97% of adult Hb
HbA2	α2δ2	~2.5%; raised in β-thalassaemia (>3.5%)
HbF (fetal)	α2γ2	Main fetal Hb; higher O2 affinity
Gower 1	ζ2ε2	Embryonic
Gower 2	α2ε2	Embryonic
Portland	ζ2γ2	Embryonic

HbF has higher O2 affinity because the γ chain binds 2,3-BPG poorly (lacks the cationic β-chain residues, notably His143). This left-shift lets the fetus extract O2 across the placenta. HbF is replaced by HbA over the first 6 months of life — which is also why β-chain disorders (sickle cell, β-thalassaemia) manifest after ~6 months, while α-chain disorders present even in utero.

High-yield: β-globin diseases declare themselves after 6 months of age, as HbF (no β chain) wanes and defective β chains take over.

The Oxygen–Haemoglobin Dissociation Curve (ODC)

P50 = the PO2 at which haemoglobin is 50% saturated (~26–27 mmHg). A change in P50 reflects a shift in affinity.

• Right shift = decreased affinity = ↑P50 = O2 released to tissues.
• Left shift = increased affinity = ↓P50 = O2 held by Hb.

Causes of RIGHT shift (think "exercising muscle / high metabolic demand"): ↑CO2 → ↑H+ (↓pH) → ↑temperature → ↑2,3-BPG → exercise/altitude/chronic hypoxia

Causes of LEFT shift: ↓CO2 → ↓H+ (↑pH/alkalosis) → ↓temperature → ↓2,3-BPG → HbF → carbon monoxide → methaemoglobin

Mnemonic for RIGHT shift — "CADET, face Right!"

CO2 ↑, Acid (H+) ↑, DPG (2,3-BPG) ↑, Exercise, Temperature ↑ → curve shifts Right.

Factor	Right shift (↑P50)	Left shift (↓P50)
pH	Low (acidosis)	High (alkalosis)
PCO2	High	Low
Temperature	High	Low
2,3-BPG	High	Low
Special	Altitude, anaemia, chronic hypoxia	HbF, CO, MetHb, stored blood

High-yield: Stored (banked) blood is 2,3-BPG depleted → left shift → poor tissue O2 unloading immediately after transfusion (regenerates within 24 hours).

The Bohr effect

Increased H+ and CO2 lower Hb's affinity for O2 (right shift), promoting O2 delivery to metabolically active, acidic, CO2-rich tissues. Molecularly, H+ and CO2 stabilise the T state. Conversely, in the lungs, off-loading of CO2/H+ raises affinity and promotes O2 uptake.

High-yield: The Bohr effect = effect of pH/CO2 on O2 binding. The reciprocal — effect of O2 on CO2 carriage — is the Haldane effect (deoxygenated Hb carries more CO2 and binds more H+).

Role of 2,3-BPG (2,3-bisphosphoglycerate)

• Product of the Rapoport–Luebering shunt of glycolysis in the RBC.
• Binds in the central cavity of deoxy-Hb (T state), between the two β chains, cross-linking them and stabilising the low-affinity state → right shift.
• Increased in chronic hypoxia, anaemia, high altitude, COPD → adaptive enhanced O2 delivery.
• Binds HbF poorly → HbF stays left-shifted.

Carbon dioxide transport

CO2 is carried as bicarbonate (~70%), carbaminohaemoglobin (~23%, bound to N-terminal amino groups of globin, NOT to iron), and dissolved (~7%).

Sickle Cell Disease (HbS)

A structural (qualitative) hemoglobinopathy.

• Molecular basis: Point mutation in the β-globin gene (HBB) — GAG → GTG, causing glutamate → valine at position 6 (β6 Glu→Val). (Adenine → Thymine, a transversion.)
• Mechanism: The hydrophobic valine allows deoxy-HbS to polymerise into rigid fibres → RBCs sickle → vaso-occlusion + chronic haemolysis.
• Trigger of sickling: Anything causing deoxygenation — hypoxia, acidosis, dehydration, infection, cold, high altitude.

Pathophysiology flow: Deoxygenation → HbS polymerisation → sickle-shaped rigid RBC → microvascular occlusion + membrane damage → vaso-occlusive crises + extravascular & intravascular haemolysis → chronic anaemia, organ infarcts, functional asplenia.

Clinical features:

• Vaso-occlusive (painful) crises, dactylitis (hand-foot syndrome — often first sign in infants), acute chest syndrome (leading cause of death), autosplenectomy → encapsulated-organism sepsis.
• Aplastic crisis classically from Parvovirus B19.
• Other: stroke, priapism, avascular necrosis of femoral head, leg ulcers, retinopathy, renal papillary necrosis / isosthenuria, gallstones (pigment).
• Sickle cell trait (HbAS) is largely asymptomatic but confers resistance to falciparum malaria (balanced polymorphism); may have painless haematuria from papillary necrosis.

Diagnosis:

• Haemoglobin electrophoresis / HPLC is the investigation of choice (confirmatory). HbS migrates slower than HbA on alkaline electrophoresis.
• Sickling test / solubility test (sodium metabisulphite): screening, positive in both trait and disease (cannot distinguish).
• Peripheral smear: sickle cells, target cells, Howell–Jolly bodies (hyposplenism).

Management:

• Hydroxyurea is the drug of choice for prophylaxis — increases HbF, reduces crises and acute chest syndrome.
• Crisis: hydration, oxygen, analgesia (opioids), treat precipitant.
• Penicillin prophylaxis + vaccination (pneumococcus, Hib, meningococcus) for functional asplenia.
• Newer: L-glutamine, voxelotor (HbS polymerisation inhibitor), crizanlizumab (anti-P-selectin).
• Allogeneic stem cell transplant = only established cure; gene therapy now approved in some settings.

High-yield: Sickle = β6 Glu→Val. Electrophoresis is confirmatory; metabisulphite/solubility is only a screen. Hydroxyurea works by raising HbF.

β-Thalassaemia

A quantitative hemoglobinopathy: reduced/absent synthesis of β-globin chains due to mostly point mutations in HBB (promoter, splice site, nonsense).

• β+ = reduced β synthesis; β0 = absent β synthesis.
• Excess unpaired α chains precipitate → ineffective erythropoiesis + haemolysis.

Type	Genotype	Severity
Thalassaemia minor (trait)	β/β+ or β/β0	Mild microcytic anaemia, often asymptomatic
Thalassaemia intermedia	variable	Moderate, transfusion-independent (mostly)
Thalassaemia major (Cooley anaemia)	β0/β0	Severe, transfusion-dependent from infancy

Clinical (major): Severe anaemia after 6 months, hepatosplenomegaly, expansion of marrow → "chipmunk/rodent facies", frontal bossing, "hair-on-end / crew-cut" skull X-ray, growth failure, and iron overload (from transfusions + increased absorption) causing cardiac, hepatic and endocrine damage.

Diagnosis:

• Microcytic hypochromic anaemia with a normal/raised RBC count and **low Mentzer index (MCV/RBC count <13)** — distinguishes from iron deficiency (Mentzer >13).
• HbA2 raised (>3.5%) and HbF raised on electrophoresis/HPLC — investigation of choice.
• Smear: target cells, basophilic stippling, nucleated RBCs.

Management:

• Regular transfusions + iron chelation (deferoxamine, deferasirox, deferiprone).
• Folic acid; splenectomy in selected cases; stem cell transplant is curative.

High-yield: β-thalassaemia trait — think a patient with microcytosis but normal/high RBC count who fails to respond to iron; check HbA2 (>3.5%). Mentzer index <13** suggests thalassaemia, **>13 iron deficiency.

α-Thalassaemia (quick contrast)

Due to deletions of the 4 α-globin genes: 1 gene = silent carrier; 2 genes = α-thal trait; 3 genes = HbH disease (β4 tetramers); 4 genes = Hb Barts (γ4) → hydrops fetalis, incompatible with life. Because α chains are needed by fetal Hb too, severe α-thalassaemia presents in utero.

Methaemoglobinaemia

• Definition: Haemoglobin with iron in the ferric (Fe3+) state, which cannot bind O2; also causes a left shift of the remaining ferrous haem (functional anaemia).
• Causes: Congenital (cytochrome b5 reductase / NADH-methaemoglobin reductase deficiency, HbM disease) or acquired — oxidant drugs/toxins: dapsone, nitrates/nitrites, local anaesthetics (benzocaine, prilocaine), sulphonamides, aniline dyes.
• Clinical: Chocolate-brown blood, cyanosis unresponsive to oxygen, normal PaO2 but low measured SpO2 (classically pulse oximeter reads ~85%); cyanosis appears at lower levels than seen with desaturation.

High-yield: Cyanosis + normal PaO2 + chocolate-brown blood + low SpO2 not corrected by O2 = methaemoglobinaemia.

Treatment flow:

1. Remove offending agent + supportive O2.
2. Methylene blue is the drug of choice (acts via NADPH-methaemoglobin reductase to reduce Fe3+ → Fe2+).
3. Caveat: Methylene blue is ineffective and may worsen haemolysis in G6PD deficiency (and in NADPH reductase deficiency) → use ascorbic acid / exchange transfusion instead.

Key Differentials

Microcytic anaemia	Distinguishing clue
Iron deficiency	↓Ferritin, ↑TIBC, Mentzer >13, low RBC count
β-thalassaemia trait	↑HbA2, Mentzer <13, normal/high RBC count
Anaemia of chronic disease	↑/normal ferritin, ↓TIBC
Sideroblastic	Ring sideroblasts, raised ferritin

High-yield: HbC = β6 Glu→Lys (target cells, crystals). HbS = β6 Glu→Val. Same codon, different amino acid — a classic exam trap.

Recently asked / exam angle

• Most repeated: factors causing right vs left shift of the ODC, and the precise definition of Bohr vs Haldane effect — expect a single-best-answer or assertion–reason.
• 2,3-BPG: where it binds (deoxy/T-state central cavity), and why HbF and stored blood are left-shifted.
• Molecular lesion of sickle cell: β6 Glu→Val, GAG→GTG — frequently asked at exact codon level.
• Investigation of choice: electrophoresis/HPLC for both sickle and thalassaemia; HbA2 >3.5% for β-thal trait.
• Methaemoglobinaemia: drug triggers (dapsone, nitrites, benzocaine), chocolate-brown blood, methylene blue (and its contraindication in G6PD deficiency).
• Mentzer index cut-off (13) to separate thalassaemia from iron deficiency.
• Hydroxyurea mechanism (↑HbF) in sickle cell.
• Aplastic crisis → Parvovirus B19; autosplenectomy → encapsulated organisms / Howell–Jolly bodies.

Rapid revision

1. HbA = α2β2; one tetramer binds 4 O2; iron must be Fe2+.
2. Haemoglobin curve = sigmoid (cooperative); myoglobin = hyperbolic; P50 ≈ 26–27 mmHg.
3. Right shift = ↑CO2, ↑H+, ↑temp, ↑2,3-BPG, exercise/altitude — "CADET face Right" → O2 delivered.
4. Left shift = HbF, CO, methaemoglobin, alkalosis, hypothermia, stored blood (low 2,3-BPG).
5. Bohr = pH/CO2 on O2 binding; Haldane = O2 on CO2 carriage.
6. 2,3-BPG binds deoxy (T-state) β-chain central cavity; HbF binds it poorly → left-shifted.
7. HbF = α2γ2, high affinity; β-chain diseases appear after 6 months.
8. Sickle = β6 Glu→Val (GAG→GTG); deoxy-HbS polymerises; hydroxyurea (↑HbF) is prophylaxis.
9. Sickle trait protects against falciparum malaria; acute chest syndrome = leading killer.
10. β-thal trait: ↑HbA2 (>3.5%), normal/high RBC, Mentzer <13; major = Cooley anaemia, hair-on-end skull, iron overload.
11. α-thal: gene deletions; 4-gene loss = Hb Barts → hydrops fetalis.
12. Methaemoglobin = Fe3+, chocolate-brown blood, cyanosis not corrected by O2, treat with methylene blue (avoid in G6PD deficiency).