describe the structure of a molecule of haemoglobin as an example of a globular protein, including the formation of its quaternary structure from two alpha (α) chains (α–globin), two beta (β) chains (β–globin) and a haem group

Proteins – Haemoglobin as a Model Globular Protein

Learning objectives (Cambridge syllabus)

• AO1: Describe the structure of haemoglobin, showing how its quaternary structure is built from two α‑chains, two β‑chains and four haem groups.
• AO2: Explain how the structure of haemoglobin determines its function, especially cooperative oxygen binding and the influence of physiological regulators.
• AO3: Identify practical techniques used to study haemoglobin (e.g. SDS‑PAGE, spectrophotometry, microscopy).

Context – why haemoglobin?

Haemoglobin is a classic example used in the Cambridge syllabus to illustrate the four levels of protein structure, the contrast between globular and fibrous proteins, and the link between structure and function. Understanding haemoglobin therefore helps students answer a wide range of exam questions on proteins in general.

Key vocabulary (AO1)

• subunit
• prosthetic group
• haem
• tetramer
• quaternary structure
• co‑operative binding
• allosteric
• T‑state (tense)
• R‑state (relaxed)

1. Levels of protein structure (exact syllabus wording)

• Primary structure: linear sequence of amino‑acid residues linked by peptide bonds.
• Secondary structure: regular folding patterns (α‑helix, β‑sheet) stabilised by hydrogen bonds.
• Tertiary structure: three‑dimensional shape of a single polypeptide chain, produced by interactions among side‑chains (hydrophobic interactions, hydrogen bonds, ionic bonds and covalent/disulphide bonds).
• Quaternary structure: association of two or more polypeptide subunits into a functional protein complex, held together by the same types of non‑covalent interactions as in tertiary structure.

2. Globular vs. fibrous proteins (AO1)

• Globular proteins – soluble, roughly spherical, perform functions such as transport, catalysis and immune defence. Example: haemoglobin.
• Fibrous proteins – insoluble, elongated, provide structural support. Example: collagen, which consists of three polypeptide chains wound into a triple helix with a repeating Gly‑X‑Y motif (X and Y are often proline or hydroxy‑proline).

3. Overall architecture of human haemoglobin (HbA)

Haemoglobin is a tetrameric globular protein found in red blood cells. Its quaternary structure consists of:

• Two α‑globin chains (≈ 140 amino‑acid residues each).
• Two β‑globin chains (≈ 145 amino‑acid residues each).
• Four non‑covalently bound haem prosthetic groups, one in each subunit.

4. Structure of a single globin subunit

• Globin fold: eight α‑helices (named A–H) arranged around a central hydrophobic core.
• Haem pocket: formed mainly by helices E and F; the iron ion is coordinated by the proximal histidine (His F8) and can bind one O₂ molecule.
• Stabilising interactions within the subunit:

  • Hydrophobic core interactions between non‑polar side‑chains.
  • Intra‑chain hydrogen bonds that maintain the α‑helices.
  • Salt bridges (ionic interactions) between oppositely charged residues.

5. The haem prosthetic group

Haem is a planar porphyrin ring (protoporphyrin IX) with an Fe²⁺ ion at its centre. It is held tightly in the hydrophobic pocket but is not covalently attached to the protein.

• Formula (approx.): C34H32N4O4Fe
• Each haem binds one molecule of O₂ via the iron ion.
• Strong absorption peak at ~410 nm (the Soret band) is used for spectrophotometric identification.

6. Quaternary assembly – “dimer of dimers” (AO1)

• α₁β₁ forms one dimer; α₂β₂ forms the second dimer.
• The two dimers associate through non‑covalent interactions (hydrophobic contacts, hydrogen bonds, salt bridges) to give the functional tetramer α₂β₂.
• This arrangement creates two distinct conformational states:

  • T‑state (tense): low affinity for O₂; stabilised by inter‑subunit contacts.
  • R‑state (relaxed): high affinity for O₂; produced after the first O₂ molecule binds.

7. Cooperative oxygen binding and physiological regulators (AO2)

The transition from T → R after one haem binds O₂ causes a small shift in the relative positions of the α‑ and β‑chains. This shift weakens the contacts that stabilise the T‑state, increasing the O₂ affinity of the remaining haem groups – the basis of co‑operative (allosteric) binding. The equilibrium between T and R is modulated by several physiological effectors listed in the syllabus:

• 2,3‑Bisphosphoglycerate (2,3‑BPG): binds in the central cavity of the T‑state, stabilising it and lowering O₂ affinity.
• H⁺ ions (Bohr effect) and CO₂: bind to specific residues, stabilising the T‑state and promoting O₂ release in metabolically active tissues.
• Temperature: higher temperature favours the T‑state, reducing O₂ affinity.

These regulators allow haemoglobin to load O₂ efficiently in the lungs (high O₂, low 2,3‑BPG, neutral pH) and release it in tissues where O₂ demand is high (low O₂, high 2,3‑BPG, acidic pH, elevated CO₂).

8. Functional implications of the quaternary structure (AO1)

1. Binding of the first O₂ molecule triggers the T → R transition.
2. The conformational change propagates to the other three subunits, raising their O₂ affinity.
3. The resulting sigmoidal O₂‑dissociation curve enables rapid loading in the lungs and efficient unloading in peripheral tissues.

9. Subunit and haem content – summary table

10. Practical techniques (AO3)

• SDS‑PAGE: separates the four subunits; on a denaturing gel haemoglobin appears as a single band of ~64 kDa (combined mass).
• Spectrophotometry: the strong Soret band at 410 nm confirms the presence of the haem prosthetic group.
• Light microscopy of blood smears: red cells appear uniformly pink because of the high concentration of haemoglobin.

11. Cross‑topic links (AO2)

• Cellular respiration: the haem group of haemoglobin is chemically identical to the haem in cytochromes of the electron‑transport chain, linking oxygen transport to aerobic metabolism.
• Genetic mutations: a single point mutation in the β‑globin gene (Glu→Val) produces sickle‑cell disease (HbS), illustrating how a change in primary structure can disrupt quaternary assembly and function.
• Allosteric effectors: 2,3‑BPG, H⁺, CO₂ and temperature shift the T ↔ R equilibrium, providing physiological regulation of haemoglobin activity.

12. Summary (AO1)

1. Primary structure – linear amino‑acid sequences of the α‑ and β‑chains.
2. Secondary structure – α‑helices stabilised by hydrogen bonds.
3. Tertiary structure – globin fold with a hydrophobic core, held together by hydrophobic interactions, hydrogen bonds and ionic bonds.
4. Quaternary structure – tetramer (α₂β₂) with four haem groups; the “dimer‑of‑dimers” arrangement enables the T ↔ R transition that underpins cooperative, allosteric oxygen binding.
5. The structure‑function relationship of haemoglobin exemplifies how hierarchical protein organisation determines biological role and how physiological regulators fine‑tune that role.