(a)(i) Explain sigmoidal nature of oxygen dissociation curve for hemoglobin. (10 marks) (a)(ii) Describe differences between…

(a)

(i) Sigmoidal nature of the oxygen dissociation curve of haemoglobin

The oxygen dissociation curve plots the percentage saturation of haemoglobin (Hb) against the partial pressure of oxygen (pO₂). For Hb it is S-shaped (sigmoidal), whereas for single-chain myoglobin it is a rectangular hyperbola.

• Cooperative binding: Hb is an α₂β₂ tetramer with four haem groups. Deoxy-Hb is in the tense (T) state, held by salt bridges between subunits and with low O₂ affinity, so the first O₂ binds with difficulty. Binding pulls the haem iron into the plane of the porphyrin ring, moving the proximal histidine (F8) and helix F; this strain breaks salt bridges and shifts the α₁β₂ interface, favouring the relaxed (R) state. Each O₂ bound makes binding of the next easier, so affinity for the fourth O₂ is roughly a hundred times that for the first.
• Shape: the flat lower part reflects low initial affinity, the steep middle part reflects cooperative switching, and the upper plateau reflects near-saturation. The Hill coefficient is about 2.8 (1.0 for myoglobin), and the P₅₀ of adult blood is about 26–27 mm Hg.
• Physiological significance: in the lungs (pO₂ about 100 mm Hg) Hb is about 97–98% saturated, and even a fall to 60 mm Hg barely lowers saturation, a safety margin at altitude or in lung disease. In the tissues (pO₂ about 40 mm Hg at rest, 20 mm Hg or less in exercising muscle) the curve is steep, so a small fall in pO₂ releases a large amount of O₂: about 25% at rest and up to 75% in exercise.
• Modulators: raised CO₂, H⁺ (the Bohr effect), temperature and 2,3-bisphosphoglycerate (2,3-BPG) stabilise the T state and shift the curve to the right, promoting unloading in active tissues; 2,3-BPG binds in the central cavity between the β chains of deoxy-Hb. The opposite changes in the lungs shift it to the left and promote loading.

(ii) Adult and fetal haemoglobin and their physiological significance

• Chains: adult haemoglobin HbA is α₂β₂ (about 97% of adult Hb, with HbA₂, α₂δ₂, about 2–3%); fetal haemoglobin HbF is α₂γ₂. The γ chain differs from β at 39 of 146 residues; the two γ genes differ at position 136 (glycine in ᴳγ, alanine in ᴬγ).
• 2,3-BPG binding: at position 143 the β chain has histidine, which helps bind 2,3-BPG, while the γ chain has serine. HbF therefore binds 2,3-BPG weakly, its T state is less stabilised, and it has a higher O₂ affinity.
• Oxygen affinity: the HbF curve lies to the left of HbA, with a P₅₀ of about 19–20 mm Hg compared with about 26–27 mm Hg.
• Significance for the fetus: in the placenta, where pO₂ is low, maternal HbA gives up O₂ (helped by a Bohr shift as fetal CO₂ enters maternal blood) while fetal HbF takes it up. Fetal blood also has a higher Hb concentration (about 17 g/dL), so it carries enough O₂ despite low pO₂.
• Developmental switch: γ-globin synthesis falls and β-globin rises around birth, so HbF falls to below 1% of total Hb by about one year of age. The transcription factor BCL11A represses γ-globin in adults.
• Clinical significance: persistence or reactivation of HbF lessens disease in β-thalassaemia (it replaces missing β chains) and in sickle-cell disease (it does not join HbS polymers). Hydroxyurea raises HbF, and the CRISPR-based therapy exa-cel (Casgevy, approved in 2023) disrupts the BCL11A enhancer for the same purpose. Hereditary persistence of fetal haemoglobin is a benign condition.

(b) Mechanism of action of cytotoxic T cells

Cytotoxic T lymphocytes (CTLs) are CD8⁺ T cells that kill virus-infected cells, tumour cells and foreign graft cells.

• Activation: a naive CD8⁺ T cell is activated in a lymph node when its T-cell receptor (TCR) recognises a foreign peptide on MHC class I of a dendritic cell, with CD8 binding the MHC molecule, co-stimulation (B7 on the dendritic cell binding CD28) and help from CD4⁺ T cells through IL-2. It proliferates clonally and differentiates into effector CTLs with cytoplasmic granules.
• Recognition of the target: effector CTLs leave the node and bind any cell displaying the same peptide–MHC I complex; adhesion is strengthened by LFA-1 binding ICAM-1.
• Immunological synapse: a ring of adhesion molecules forms around the central TCR cluster, and the microtubule-organising centre and lytic granules move towards the contact site, so killing is precise and bystander cells are spared.
• Perforin–granzyme pathway: granules release perforin, which polymerises in the target membrane to form pores about 16 nm wide, and granzymes (serine proteases, mainly granzyme B), which enter the cell. Granzyme B activates caspase-3 and cleaves Bid, releasing cytochrome c from mitochondria; the caspase cascade then causes apoptosis with DNA fragmentation. Granulysin also damages microbial membranes.
• Fas–FasL pathway: Fas ligand on the CTL binds Fas (CD95) on the target, recruiting FADD and caspase-8 and triggering apoptosis; this pathway also removes activated lymphocytes.
• Cytokines: CTLs secrete IFN-γ, which increases MHC I expression and activates macrophages, and TNF-α, which can kill some tumour cells and promotes inflammation.
• Serial killing and memory: the CTL detaches unharmed (protected by cathepsin B on its own membrane) and can kill several targets in succession; some become long-lived memory CD8⁺ cells that respond rapidly on re-infection.

Apoptosis dismantles the target cell and its viral contents without releasing them, limiting spread and inflammation. CTLs are central to antiviral and antitumour immunity (the basis of CAR-T and checkpoint-inhibitor therapies) and to graft rejection.

(c) Homeotic genes and their role in body axis formation in the chick

Homeotic genes are master regulatory (selector) genes that specify the identity of body segments or regions; their mutation transforms one body part into another, as in Antennapedia (legs in place of antennae) and bithorax in Drosophila. Most encode transcription factors with a 60-amino-acid homeodomain, encoded by a 180-bp homeobox, which binds DNA and switches on or off the downstream genes that build region-specific structures.

• Hox clusters: Drosophila has one Hox complex (the Antennapedia and bithorax complexes), whereas the chick, like other amniotes, has four clusters (HoxA–HoxD) with 39 genes in 13 paralogous groups.
• Colinearity: genes lie on the chromosome in the same order as their expression domains along the anterior–posterior axis (spatial colinearity); 3′ genes are expressed earlier and more anteriorly and 5′ genes later and more posteriorly (temporal colinearity). In the chick embryo, cells in the epiblast next to the primitive streak switch on Hox genes in sequence as the streak regresses, so cells that ingress later acquire more posterior identities.
• Hox code: each region is defined by the combination of Hox genes expressed there, with nested, overlapping domains in the neural tube, paraxial mesoderm (somites) and lateral plate mesoderm.
• Hindbrain: Hox paralogous groups 1–4 pattern the rhombomeres; for example Hoxb1 is expressed in rhombomere 4 and specifies its facial motor neurons.
• Vertebral identity: in somites, the anterior limit of Hoxc6 marks the transition from cervical to thoracic vertebrae (the first rib-bearing vertebra). In the chick, with 14 cervical vertebrae, this boundary lies much further back than in the mouse, with 7, showing that axial differences between species arise by shifting Hox boundaries. Hox10 paralogues suppress rib formation in the lumbar region.
• Limbs: Hox expression in the lateral plate mesoderm positions the limbs; the wing arises at the level of the Hoxc6 boundary, and 5′ Hoxa and Hoxd genes (groups 9–13) pattern the limb from upper arm to digits.
• Regulation: retinoic acid, FGF and Wnt gradients from the node and the posterior embryo set the timing and extent of Hox expression; excess retinoic acid shifts Hox expression domains and causes homeotic transformations of vertebrae.

Thus Hox genes do not create the axis itself, which is established by the primitive streak and Hensen's node, but give each level along it its regional identity.