Molecule to dissociation curve
Haemoglobin structure, cooperative binding and allostery
What you should already have
About 60 minutes
Plus the time it takes to redraw this lesson’s figures from memory, which is the fastest way to find out what you have not understood.
Where this shows up
Oxygen delivery is a product of three terms, and a normal saturation constrains only one of them. This lesson is where a reassuring monitor is separated from an adequately oxygenated patient — and where the dissociation curve stops being a shape to memorise and becomes a prediction about unloading.
Learning outcomes
By the end of this lesson you should be able to:
- Describe haemoglobin's structure and the T to R allosteric transition as a chain of events from the iron to the subunits.
- Explain cooperative binding and why it produces a sigmoid rather than a hyperbolic curve.
- Calculate arterial oxygen content and oxygen delivery, defining every symbol and its units.
- Draw the oxyhaemoglobin dissociation curve with both axes labelled, the P50 marked, and the arterial and mixed venous points placed.
- Name the factors that shift the curve in each direction, and explain why they shift it where oxygen is needed.
- Distinguish the Bohr from the Haldane effect, and describe the double Bohr effect at the placenta.
- Compare HbA, HbF and HbS, and state the conditions that precipitate sickling.
Together these settle 3 syllabus objectives: Haemoglobin structure and cooperativity; HbA, HbF and HbS; Arterial oxygen content and delivery and The oxyhaemoglobin dissociation curve and its shifts. Tick them on the Physiology objective list once you can do all of the above without notes.
1 · Structure
A globular conjugated metalloprotein of molecular weight about 64.5 kDa, built from four subunits. Adult haemoglobin is α₂β₂: two α chains of 141 amino acids and two β chains of 146. The subunits are held by non-covalent bonds and arranged as two αβ dimers that slide against one another when oxygen binds — that sliding is the allosteric transition.
| Component | What it is | Why it matters |
|---|---|---|
| Globin chains | Four polypeptides, each with a hydrophobic cleft holding one haem | The chain type defines the variant — β in HbA, γ in HbF, δ in HbA₂ |
| Haem | Protoporphyrin IX ring with one central ferrous (Fe²⁺) iron | The oxygen binding site. Four per molecule, so four O₂ per haemoglobin |
| Proximal histidine (F8) | Covalently bonded to iron on one face of the ring | The mechanical link: when iron moves, this histidine drags its whole helix with it |
| Distal histidine (E7) | On the opposite face, not bonded to iron | Sterically hinders binding, reducing carbon monoxide affinity roughly 200-fold from what it would otherwise be |
| Central cavity | Space between the β chains, lined with positively charged residues | The 2,3-DPG binding site — and the reason HbF, whose γ chains lack these residues, has a higher affinity |
2 · The T and R states
| T state (tense) | R state (relaxed) | |
|---|---|---|
| Oxygen affinity | Low | High — roughly 300 times greater |
| Salt bridges | Present, constraining the subunits | Broken |
| Central cavity | Open — 2,3-DPG can bind | Narrowed — 2,3-DPG expelled |
| Predominates in | Deoxygenated blood, tissue capillaries | Oxygenated blood, pulmonary capillaries |
| Stabilised by | H⁺, CO₂, 2,3-DPG, heat, chloride | Oxygen binding itself |
| Position on the ODC | Steep lower portion | Upper plateau |
3 · Cooperativity and the sigmoid shape
Because binding at one site raises the affinity of the others, the four sites do not behave independently. This is positive cooperativity, and it is the entire explanation for the sigmoid curve.
| Haemoglobin | Myoglobin | |
|---|---|---|
| Subunits | Four (α₂β₂) | One |
| Cooperativity | Present; Hill coefficient ≈2.8 | None; Hill coefficient 1 |
| Curve shape | Sigmoid | Hyperbolic |
| P50 | 26–27 mmHg (3.5 kPa) | ≈2.7 mmHg |
| Function | Transport — loads in the lung, unloads in tissue | Storage — accepts oxygen from haemoglobin, releases it only at very low tissue PO₂ |
The Hill coefficient is 2.8 rather than 4 because the four sites do not switch simultaneously. Physiologically the sigmoid shape does two useful things at once: the plateau guarantees near-complete loading in the lung even if alveolar PO₂ falls substantially, and the steep segment guarantees generous unloading for a modest fall in tissue PO₂.
From tense to relaxed haemoglobin
HbA · conceptual occupancy states · four haem sites
Oxygenation leaves iron in the ferrous state. Oxidation to Fe³⁺ produces methaemoglobin, which cannot bind O₂.
T state predominates · deoxyhaemoglobin
Deoxygenated haemoglobin favours the tense (T) quaternary conformation. The β chains are relatively far apart, the central cavity can accommodate 2,3-DPG, and strong inter-subunit electrostatic interactions stabilise the low-affinity ensemble.
- Affinity
- Lowest affinity for O₂
- Central cavity
- Wide central cavity; 2,3-DPG binding favoured
- Interfaces
- T-state electrostatic bonds and salt bridges retained
- ODC region
- Toe of the ODC; the first binding event is least favoured
Tense (T) conformation → low oxygen affinity → O₂ binds Fe²⁺ within the haem crevice → allosteric conformational change → electrostatic bonds weaken and interfaces rearrange → relaxed (R) conformation → affinity at remaining sites increases → positive cooperative binding → sigmoid ODC.
HbA is an α₂β₂ tetramer held together largely by non-covalent electrostatic interactions. Each globin chain contains one haem, and each haem binds one O₂ molecule at Fe²⁺.
Deoxyhaemoglobin favours the tense, lower-affinity conformation. Salt bridges and other ionic interactions stabilise the tetramer, while the wide β-chain cavity binds 2,3-DPG.
O₂ binding at one haem changes the tertiary structure of that subunit and the quaternary relationship between subunits. A binding event at one site therefore alters distant sites.
Each successive O₂ binding event increases affinity at the remaining unoccupied sites. O₂ is a homotropic positive allosteric effector; this interaction among equivalent sites produces the sigmoid curve.
H⁺, CO₂ and 2,3-DPG bind away from the haem oxygen site and preferentially stabilise the T ensemble. They are heterotropic allosteric modulators. Temperature changes affinity but is not a binding ligand.
Oxygenated haemoglobin favours the relaxed, higher-affinity conformation, with a narrowed central cavity. In tissues, falling PO₂ plus H⁺, CO₂, heat and 2,3-DPG shift the equilibrium back toward T and promote unloading.
Cooperativity describes interaction among the four O₂-binding sites. Allostery is the wider principle by which binding or modulation at one site changes behaviour at another site. Cooperative oxygen binding is therefore a form of allosteric regulation.
“Tense” does not mean mechanically stretched, and “relaxed” does not mean oxygen is released more easily. T is the lower-affinity ensemble; R is the higher-affinity ensemble. Both are conformational states rather than rigid sequential boxes.
HbA, HbF and HbS
Compare composition first, then connect the molecular difference to affinity, solubility, oxygen transfer and clinical behaviour.
| Feature | HbA | HbF | HbS |
|---|---|---|---|
| Physiological status | Major normal adult haemoglobin | Predominant fetal haemoglobin; falls after birth | Abnormal structural β-globin variant |
| Globin composition | α₂β₂ | α₂γ₂ | α₂βˢ₂ |
| Defining molecular feature | Normal adult β chains | γ chains replace β chains | Valine replaces glutamic acid at β-chain position 6: β6 Glu→Val |
| 2,3-DPG interaction | Binds the central cavity between β chains and stabilises T-state deoxyHb | γ chains bind 2,3-DPG less avidly, so T-state stabilisation is reduced | βˢ chains remain subject to erythrocyte allosteric modifiers; 2,3-DPG is not the defining abnormality |
| O₂ affinity and P50 | Adult reference: P50 about 26.3 mmHg (3.5 kPa) | Higher affinity, lower P50 and left-shifted ODC | Do not assign one universal shift without conditions; pH, temperature, 2,3-DPG, anaemia and HbF fraction modify the measured curve |
| Defining functional consequence | Efficient pulmonary loading and tissue unloading through cooperativity and allostery | Facilitates maternal-to-fetal oxygen transfer at the placenta | Deoxy-HbS loses solubility, polymerises and distorts red cells into rigid sickle forms |
| Clinical significance | Reference for oxygen-content and ODC calculations | Higher HbF reduces deoxy-HbS polymerisation and usually ameliorates sickling | Haemolysis, vaso-occlusion, tissue ischaemia and progressive organ injury |
HbA · normal major adult haemoglobin
HbA is the reference adult tetramer. Each of its four globin subunits carries one haem containing Fe²⁺, so one HbA molecule can reversibly bind four O₂ molecules.
It exhibits T↔R allosteric transition and positive cooperative binding. Its β-chain central cavity binds 2,3-DPG, which preferentially stabilises deoxyhaemoglobin in the T state and lowers oxygen affinity.
- α₂β₂
- four haem groups
- Fe²⁺
- positive cooperativity
- T and R conformations
- 2,3-DPG binding
4 · Allosteric modulators
| Modulator | Mechanism | Effect on affinity |
|---|---|---|
| H⁺ (low pH) | Protonation of histidine residues stabilises the T-state salt bridges — the Bohr effect | Reduced: curve shifts right |
| CO₂ | Carbamino formation with terminal amino groups stabilises T, plus an indirect effect through carbonic acid and H⁺ | Reduced: curve shifts right |
| 2,3-DPG | Binds the central cavity and cross-links the β chains in the T conformation. A product of the Rapoport–Luebering shunt; rises in chronic hypoxaemia, anaemia and at altitude, falls in stored blood | Reduced: curve shifts right |
| Temperature | Heat destabilises the oxygen–haem bond | Reduced: curve shifts right |
| Chloride | Binds preferentially to the T state | Reduced, modestly |
| Carbon monoxide | Binds one or more haem sites and holds the molecule in R, so the remaining sites release oxygen unwillingly | Increased: curve shifts left — and capacity is lost as well |
5 · Variants and abnormal haemoglobins
| Haemoglobin | Chains or defect | Behaviour | Clinical significance |
|---|---|---|---|
| HbA | α₂β₂ — about 97% of adult haemoglobin | P50 26–27 mmHg (3.5 kPa) | The reference against which everything else is described |
| HbA₂ | α₂δ₂ — about 2% | Similar to HbA | Raised proportion in β-thalassaemia trait |
| HbF | α₂γ₂; γ chains lack the residues that bind 2,3-DPG | P50 ≈19 mmHg (2.5 kPa) — left-shifted | Allows the fetus to load oxygen from a placental PO₂ of only 30–35 mmHg. Replaced by HbA over the first 6 months — hence physiological anaemia of infancy |
| HbS | Point mutation: valine for glutamate at position 6 of the β chain | Right-shifted; polymerises when deoxygenated | Sickling precipitated by hypoxia, acidosis, hypothermia, dehydration and stasis — the five things anaesthesia must avoid |
| Methaemoglobin | Iron oxidised to ferric (Fe³⁺) | Cannot bind oxygen; left-shifts the remaining normal haem groups | Chocolate-brown blood, cyanosis unresponsive to oxygen, oximeter drawn towards 85%. Causes include prilocaine, nitrates, sulphonamides. Treat with methylene blue 1–2 mg/kg |
| Carboxyhaemoglobin | Carbon monoxide bound in place of oxygen | Affinity 200–250× that of oxygen; marked left shift | Capacity and unloading both impaired. The pulse oximeter over-reads. Half-life ≈4–5 h on air, 40–80 min on 100% oxygen |
Blood to tissue
Oxygen content, delivery and extraction
| Symbol and units | What it describes | What it does not tell you | |
|---|---|---|---|
| Tension | PaO₂, mmHg or kPa | The pressure driving diffusion into tissue; set by the lung | Nothing about how much oxygen is present — it reflects the dissolved fraction alone |
| Saturation | SaO₂, % | The percentage of available binding sites occupied | Nothing about how many binding sites exist. 100% of very few is still very few |
| Content | CaO₂, mL/dL | The oxygen carried per 100 mL of blood | Nothing about flow — content without cardiac output delivers nothing |
1 · The three equations
| Symbol | Units | Note |
|---|---|---|
| CaO₂ | mL O₂ per dL blood | Hb in g/dL, SaO₂ as a fraction |
| 1.34 | mL O₂ per g Hb | Hüfner’s constant. The theoretical value from molecular weight is 1.39; 1.31–1.34 is measured in vivo because some haemoglobin is always MetHb or COHb |
| 0.0031 | mL O₂ per dL per mmHg | Dissolved oxygen, from Henry’s law. Use ≈0.023 if PaO₂ is in kPa |
| ḊO₂ | mL/min | The ×10 converts dL to L so it matches cardiac output |
| Extraction ratio | Dimensionless | V̇O₂/ḊO₂ = (CaO₂ − CvO₂)/CaO₂; normally about 0.25 |
2 · The normal numbers, in sequence
| Variable | Normal value | Derivation |
|---|---|---|
| CaO₂ | ≈ 20 mL/dL | (1.34 × 15 × 0.98) + 0.3 |
| CvO₂ | ≈ 15 mL/dL | At SvO₂ 75%, PvO₂ 40 mmHg |
| Cardiac output | 5 L/min | 70 mL × 72 beats/min |
| Oxygen delivery, ḊO₂ | ≈ 1000 mL/min | 5 × 20 × 10 |
| Oxygen consumption, V̇O₂ | ≈ 250 mL/min | 5 × (20 − 15) × 10 |
| Extraction ratio | ≈ 25% | 250 / 1000 |
| Mixed venous saturation | 70–75% | The residue after extraction |
The last two lines carry the message: at rest the body uses only a quarter of the oxygen delivered to it, so there is a fourfold reserve. That reserve is what lets extraction rise before consumption is compromised, and why a falling delivery is tolerated for some distance before anything visible happens.
3 · The delivery–consumption relationship
Plot V̇O₂ against ḊO₂ and the relationship is biphasic. As delivery falls, extraction rises to compensate and consumption is held constant — the supply-independent region. Below a threshold, extraction can rise no further and consumption falls with delivery: the supply-dependent region. That threshold is critical oxygen delivery, around 330 mL/min/m² (roughly 8–10 mL/kg/min), beyond which anaerobic metabolism begins and lactate rises. Every resuscitation target — haemoglobin, cardiac output, saturation — is an attempt to stay on the safe side of that point.
4 · The four types of tissue hypoxia
| Type | Which term falls | Signature | Example |
|---|---|---|---|
| Hypoxic | SaO₂, and so CaO₂ | Low PaO₂ and low SpO₂ | V/Q mismatch, shunt, hypoventilation, altitude |
| Anaemic | Hb, and so CaO₂ | Normal PaO₂ and normal SpO₂ — the trap | Haemorrhage, chronic anaemia, carbon monoxide poisoning, methaemoglobinaemia |
| Stagnant | Cardiac output | Normal CaO₂; widened arteriovenous difference and low SvO₂ | Cardiogenic shock, hypovolaemia, aortic cross-clamp, local occlusion |
| Histotoxic | None — delivery is normal | High SvO₂ with lactic acidosis: oxygen arrives but cannot be used | Cyanide, which inhibits cytochrome oxidase |
5 · Compensation for a falling delivery
| Timescale | Response | Mechanism |
|---|---|---|
| Immediate | Increased extraction | SvO₂ falls; the arteriovenous content difference widens |
| Seconds to minutes | Increased cardiac output | Chiefly tachycardia; in anaemia, reduced viscosity assists venous return |
| Hours to days | Right-shifted dissociation curve | 2,3-DPG synthesis rises, raising P50 and improving unloading at the same tissue PO₂ |
| Days to weeks | Increased haemoglobin | Erythropoietin from the renal peritubular cells restores carrying capacity |
Affinity and unloading
The oxyhaemoglobin dissociation curve
The curve plots percentage saturation of haemoglobin on the y-axis against PO₂ on the x-axis. Label both axes with units before anything else — it is the commonest reason a correct curve scores poorly.
Oxyhaemoglobin dissociation curve laboratory
Preparing the interactive graph…
1 · The three points that let you draw it from memory
| PO₂ | Saturation | Physiological identity |
|---|---|---|
| 27 mmHg (3.5 kPa) | 50% | P50 — the definition of affinity |
| 40 mmHg (5.3 kPa) | 75% | Normal mixed venous point |
| 100 mmHg (13.3 kPa) | ≈ 97% | Normal arterial point |
Plot those three, join them smoothly, and the shape is unavoidable. Add 60 mmHg / 90% for a fourth: it marks the knee of the curve, and it is why a saturation of 90% is treated as a clinical floor — below it the curve is steep, and a small further fall in PO₂ produces a large fall in saturation.
2 · The two regions and what each is for
| Plateau (above ≈60 mmHg) | Steep portion (below ≈60 mmHg) | |
|---|---|---|
| Where it operates | Pulmonary capillary | Systemic tissue capillary |
| Functional benefit | Saturation is well maintained even if alveolar PO₂ falls substantially — a safety margin for loading | A modest fall in PO₂ releases a large quantity of oxygen at a maintained diffusion gradient |
| Consequence | Raising PaO₂ above 100 mmHg adds almost no content — which is why a high FiO₂ cannot compensate for shunt or anaemia | Small changes in tissue PO₂ cause large changes in saturation, so venous saturation is a sensitive index of extraction |
3 · Shifts
| Right shift — lower affinity, higher P50 | Left shift — higher affinity, lower P50 |
|---|---|
| ↑ H⁺ (acidosis) | ↓ H⁺ (alkalosis) |
| ↑ PCO₂ | ↓ PCO₂ |
| ↑ Temperature | ↓ Temperature, including therapeutic hypothermia |
| ↑ 2,3-DPG — chronic anaemia, chronic hypoxaemia, altitude, pregnancy, hyperthyroidism | ↓ 2,3-DPG — stored blood, hypophosphataemia, septic shock |
| HbS | HbF, methaemoglobin, carboxyhaemoglobin |
| Net effect: favours unloading in the tissues | Net effect: favours loading in the lung, impairs unloading in the tissues |
4 · The Bohr and Haldane effects
Two halves of one reciprocal arrangement, and routinely confused. The reliable way to keep them apart is by asking which gas is being affected.
| Bohr effect | Haldane effect | |
|---|---|---|
| Definition | CO₂ and H⁺ reduce the affinity of haemoglobin for oxygen | Deoxygenated haemoglobin carries more carbon dioxide than oxygenated haemoglobin |
| Gas affected | Oxygen | Carbon dioxide |
| Mechanism | Protonation of histidine residues stabilises T-state salt bridges; carbamino formation does the same | Deoxyhaemoglobin is a better proton buffer and forms carbamino compounds more readily |
| Where it helps | In the tissues: local acidosis and CO₂ promote oxygen release | In the tissues, oxygen release enhances CO₂ uptake; in the lung, oxygen uptake drives CO₂ off |
| Magnitude | P50 rises about 2–3 mmHg for each 0.1 fall in pH | Accounts for roughly a quarter to a third of arteriovenous CO₂ transport |
5 · The double effects at the placenta
The double Bohr effect operates on both sides of the placental membrane at once. Carbon dioxide passes from fetus to mother, so maternal blood becomes more acidic and its curve shifts right, releasing oxygen; simultaneously fetal blood loses carbon dioxide, becomes more alkaline, and its curve shifts further left, taking oxygen up. One exchange of carbon dioxide therefore improves transfer at both ends of the gradient. The double Haldane effect is its mirror image: fetal haemoglobin becoming oxygenated releases carbon dioxide, while maternal haemoglobin becoming deoxygenated takes it up.
| Factor | Value or effect | Contribution |
|---|---|---|
| HbF | α₂γ₂, binds 2,3-DPG poorly; P50 ≈19 mmHg | Loads at a PO₂ that would be alarming in an adult |
| Fetal haemoglobin concentration | ≈17 g/dL | Raises content at any given saturation |
| Double Bohr effect | Both curves shift favourably at once | Improves transfer at both ends of the gradient |
| Umbilical blood flow | High relative to fetal mass | Maintains delivery despite the low tension |
From understanding to performance
Draw, explain, calculate, retrieve
The drawing checklists
- Axes labelled, PO₂ in mmHg
- Every stage in sequence, none omitted
- Normal value at each stage
- Mechanism named at each fall
- Alveolar gas equation written out
- Axes: saturation % against PO₂, with units
- Plot 27/50, 40/75 and 100/97
- P50, arterial and mixed venous marked
- Plateau and steep portion identified
- A shifted curve alongside, with its causes
- Lung height against flow per unit volume
- Both lines rising towards the base
- Perfusion the steeper of the two
- Crossing point marked at V/Q = 1
- Ratio curve added: 3.3 at apex to 0.6 at base
- α₂β₂ with four haem Fe²⁺ sites
- T as low affinity, R as high affinity
- Iron into the plane, F helix, salt bridges broken
- Positive cooperativity named
- H⁺, CO₂ and 2,3-DPG as T-state stabilisers
Integrated clinical reasoning
| Clinical situation | The chain of reasoning expected |
|---|---|
| Carbon monoxide poisoning with SpO₂ of 99% | CO binds haemoglobin 200–250× more avidly than oxygen, so capacity falls; it also left-shifts the curve, so what remains is unloaded poorly. PaO₂ and therefore conventional SpO₂ are unchanged — the reading is meaningless. Measure carboxyhaemoglobin by co-oximetry and give 100% oxygen to shorten its half-life |
| Desaturation during one-lung ventilation | The non-dependent lung is perfused but unventilated: true shunt with V/Q = 0. Hypoxic pulmonary vasoconstriction reduces it over about 15 minutes, but volatile agents blunt that response. Oxygen corrects it poorly because shunted blood never meets alveolar gas |
| Lactate rising despite SpO₂ 100% and Hb 14 g/dL | Content is adequate, so the failure is in flow or utilisation. Consider stagnant hypoxia — check cardiac output and the arteriovenous difference — or histotoxic hypoxia, in which the mixed venous saturation is characteristically high |
| PaO₂ of 60 mmHg in a 75-year-old on air | Compute PAO₂ from the alveolar gas equation, then the gradient, then compare it against (age/4) + 4 rather than a fixed 10 mmHg. An apparently low PaO₂ may be entirely age-appropriate |
| Massive transfusion with a falling core temperature | Stored blood is 2,3-DPG depleted, hypothermia shifts the curve left, and citrate-derived alkalosis shifts it further. Content may be restored while unloading remains impaired |
| Sudden fall in end-tidal CO₂ with unchanged ventilation | Pulmonary blood flow has fallen — embolism, low cardiac output, haemorrhage or arrest. The alveolar unit is now ventilated but not perfused, which is alveolar dead space at the V/Q = ∞ end of the spectrum |