Biology › Animal transport and cardiovascular biology › Haemoglobin: an S-shaped curve and everything it explains

Haemoglobin: an S-shaped curve and everything it explains

One protein, four subunits, four oxygen molecules, and a curve whose shape does the work. Cooperative binding makes haemoglobin greedy at the lungs and generous at the tissues, and a shift of that curve left or right is how species and situations differ.

Before this Quaternary structure and prosthetic groups in proteins · Partial pressure as a measure of how much of a gas is present · Gas exchange at the alveolus

What you should be able to do

• Describe haemoglobin as a conjugated globular protein and say how many oxygen molecules one molecule carries.
• Explain cooperative binding and use it to account for the sigmoid shape of the dissociation curve.
• Read percentage saturation off a dissociation curve and calculate how much oxygen is unloaded between two partial pressures.
• Explain the Bohr shift and why it is useful in exercising muscle.
• Interpret a curve that lies to the left or to the right of the human adult curve, including foetal, high-altitude and small-mammal haemoglobins.
• Describe how carbon dioxide is carried, including the role of carbonic anhydrase and the chloride shift.

What haemoglobin is, precisely

Haemoglobin is a globular protein with quaternary structure: four polypeptide chains, two α and two β in an adult human, folded so that hydrophilic side chains face outwards, which is what makes it soluble enough to sit at high concentration inside a red blood cell.

Each of the four chains is wrapped around a haem group, which is not made of amino acids at all. A non-protein group permanently attached to a protein is a prosthetic group, and a protein carrying one is described as conjugated. At the centre of each haem group is an iron(II) ion, and each iron(II) ion binds one molecule of oxygen. Four chains, four haem groups, four Fe²⁺, four O₂ per haemoglobin molecule — those numbers are worth quoting exactly.

Oxygen binding is association and gives oxyhaemoglobin; oxygen release is dissociation. Neither is a covalent reaction: the oxygen is held reversibly, which is the entire point of a transport molecule. A red blood cell holds about 250 million haemoglobin molecules and has no nucleus and no mitochondria, so none of the oxygen it carries is used up in transit.

Conjugated protein

A protein with a non-protein prosthetic group permanently associated with it.

Prosthetic group

The non-protein component; in haemoglobin, the haem group with its iron(II) ion.

Partial pressure of oxygen

The contribution oxygen makes to the total pressure of a gas mixture, measured in kPa; a measure of how much oxygen is present.

Percentage saturation

The proportion of the oxygen-binding sites on haemoglobin that are actually occupied.

Why the curve is S-shaped and not a straight line

Plot percentage saturation against partial pressure of oxygen and you get a sigmoid curve: shallow at first, steep through the middle, levelling off at the top. That shape comes from a single mechanism.

The first oxygen molecule has a hard time binding, because the haemoglobin molecule is folded in a way that leaves its binding sites awkwardly presented. When it does bind, it changes the shape of the whole quaternary structure slightly, and that change makes the remaining sites easier to reach. The second oxygen binds more readily than the first, the third more readily still. That is cooperative binding, and it is why the curve is shallow at low partial pressures and then suddenly steep.

Number four is slightly harder again, simply because there is only one site left to find, and that is part of why the curve flattens at the top. The other part is that above about 90 per cent saturation there is hardly any spare capacity: no matter how much more oxygen you offer, there is almost nowhere to put it.

The shape is not a curiosity, it is the adaptation. At the alveoli, where the partial pressure of oxygen is around 12 to 13 kPa, haemoglobin sits on the plateau and loads to about 97 per cent saturation. In respiring tissue, where the partial pressure is around 4 kPa at rest, it sits on the steep part of the curve, where a small fall in partial pressure produces a large fall in saturation — that is, a large release of oxygen. Loading is reliable; unloading is sensitive. A straight-line relationship could not do both.

Reading loading and unloading off the graph

Almost every exam question on this curve is one of three things: read a saturation, calculate a difference, or explain a shift. The first two are arithmetic, so do them carefully and say what they mean.

To find how much oxygen is unloaded between two places, read the saturation at each and subtract. At 12 kPa the normal curve gives about 97 per cent; at 4 kPa it gives about 59 per cent; so the blood gives up about 38 per cent of its capacity to a resting tissue. In hard-working muscle the partial pressure may fall to 2 kPa, where saturation is only about 17 per cent, so four fifths of the oxygen is handed over. That is the steep part of the curve doing its work: halving the partial pressure from 4 kPa to 2 kPa costs the blood another 42 percentage points of saturation.

Watch the axis units too. Most papers use kPa; some older material and some medical sources use mmHg, where the lungs are around 100 mmHg rather than 13 kPa. The shape and the argument are identical, but do not read one scale against the other.

The Bohr shift

Respiring tissue produces carbon dioxide. Carbon dioxide dissolving in the blood plasma and in red blood cells forms carbonic acid, which releases hydrogen ions, which lower the pH. Those hydrogen ions bind to haemoglobin and alter its tertiary and quaternary structure slightly, and the altered shape holds oxygen less tightly.

The consequence on the graph is a shift to the right: at any given partial pressure of oxygen, haemoglobin in carbon-dioxide-rich blood is less saturated than it would otherwise be. Read that as it is meant: oxygen is released more readily.

Now put the tissue back into the picture. The tissues producing the most carbon dioxide are the ones respiring hardest, and they are precisely the ones needing the most oxygen. The very waste gas that signals high demand is the thing that makes haemoglobin let go. During sprinting, an exercising muscle may take 60 to 70 per cent of the oxygen out of the blood passing through it rather than the usual 25 to 30 per cent, and a large part of that increase is the Bohr shift.

And at the lungs? The partial pressure there is high enough that the shifted curve has already flattened out — better than 90 per cent saturation either way. Blowing off carbon dioxide at the alveoli also raises the pH locally, pushing the curve back towards normal. So the shift costs almost nothing where the blood is loading and gains a great deal where it is unloading.

Bohr shift

The rightward shift of the oxygen dissociation curve caused by an increased partial pressure of carbon dioxide and the fall in pH that goes with it, so that oxygen dissociates more readily at any given partial pressure.

Loading

Association of oxygen with haemoglobin, at the gas exchange surface.

Unloading

Dissociation of oxygen from haemoglobin, at respiring tissue.

Comparing haemoglobins, and what left and right mean

Different species and different life stages carry haemoglobins with different affinities for oxygen, and every one of them can be read off the position of the curve. Left of the human adult curve means a higher affinity: the molecule binds oxygen at lower partial pressures and holds on to it harder. Right means a lower affinity: it releases oxygen more easily.

Foetal haemoglobin sits to the left of adult haemoglobin, and it has to. A fetus receives oxygen at the placenta from its mother's blood, which by then has already given some up, so the partial pressure available is low. Because the foetal curve lies to the left, foetal haemoglobin is more saturated than maternal haemoglobin at that same low partial pressure, so oxygen moves from mother to fetus. Two chains differ from the adult protein, and that difference in primary structure is what changes the affinity.

Llamas and other animals living at high altitude have curves to the left as well, for a related reason: at 4000 m the partial pressure of oxygen in the air is roughly two thirds of the sea-level value, so a haemoglobin that only loads properly at 13 kPa would be loading badly. Higher affinity restores saturation at the lungs. The same logic covers the lugworm in a burrow and the diving seal.

Small mammals go the other way. A mouse has an enormous surface area to volume ratio, loses heat fast, and has a very high metabolic rate per gram — its heart beats around 600 times a minute. Its tissues need oxygen delivered quickly, so its haemoglobin has a curve to the right of ours: lower affinity, more readily unloaded. It loads a little less well at the lungs and it does not matter, because it lives in air at normal pressure and unloading is the constraint.

One warning about the word 'better'. None of these is a better haemoglobin than another. Each is a compromise between loading at whatever partial pressure the animal's lungs or gills or placenta provides and unloading at whatever partial pressure its tissues reach.

Carbon dioxide going the other way

Blood also carries carbon dioxide back, in three ways, and the proportions are worth remembering because questions ask for them: about 85 per cent as hydrogencarbonate ions in the plasma, about 10 per cent bound to haemoglobin as carbaminohaemoglobin, and about 5 per cent simply dissolved.

The main route runs like this. Carbon dioxide diffuses from respiring tissue into the plasma and on into red blood cells. There, the enzyme carbonic anhydrase catalyses its reaction with water to form carbonic acid, H₂CO₃. Carbonic acid dissociates into hydrogen ions and hydrogencarbonate ions, HCO₃⁻.

Those two ions then go separate ways. The hydrogencarbonate ions diffuse out of the red blood cell into the plasma, where most of the carbon dioxide is therefore carried. The hydrogen ions stay inside and are taken up by haemoglobin, which acts as a buffer and stops the pH of the cell crashing; haemoglobin holding hydrogen ions is called haemoglobinic acid. Note what has just happened: hydrogen ions binding to haemoglobin is the very thing that causes the Bohr shift, so carbon dioxide transport and oxygen release are the same event described twice.

That leaves an electrical problem. Negatively charged hydrogencarbonate ions have left the cell, so the inside would become relatively positive and further loss would stop. Chloride ions move in from the plasma to compensate — one negative ion in for one negative ion out — and the cell stays electrically neutral. This is the chloride shift.

At the lungs the whole sequence runs backwards. The partial pressure of carbon dioxide is low, so hydrogencarbonate ions move back into the red blood cells, recombine with hydrogen ions released from haemoglobin, and carbonic anhydrase — which works both ways — regenerates carbon dioxide and water. Carbon dioxide diffuses into the alveoli and is breathed out, the pH rises, haemoglobin's affinity for oxygen goes back up, and the loading you saw at the start of this lesson happens all over again.

Carbonic anhydrase

The enzyme in red blood cells catalysing the reversible reaction between carbon dioxide and water to form carbonic acid.

Chloride shift

The movement of chloride ions into a red blood cell as hydrogencarbonate ions move out, maintaining electrical neutrality.

Haemoglobinic acid

Haemoglobin that has taken up hydrogen ions, buffering the contents of the red blood cell.

In the exam

• Quote the numbers for the molecule: four polypeptide chains, four haem groups, four iron(II) ions, four oxygen molecules carried.
• For the sigmoid shape, the mark is for cooperative binding — the first oxygen changes the shape of the molecule and makes the next bind more easily. 'Because it is a curve' is not an explanation.
• A shift to the right means oxygen is released more readily at a given partial pressure. Write the direction and the consequence together, because half the marks are for the consequence.
• Foetal haemoglobin sits to the LEFT of adult haemoglobin. If your answer has it on the right, you have said the fetus gives oxygen to its mother.
• Percentage saturation is not a volume. If the question offers you a figure like 20 cm³ of oxygen per 100 cm³ of blood, it wants you to multiply.
• For carbon dioxide transport, name carbonic anhydrase, name hydrogencarbonate, and explain the chloride shift as a matter of charge balance rather than as a fact to be recited.

Questions

Written to the command words the boards use. Try them on paper before opening a scheme: the marks go to points made, not to length.

← Blood vessels and tissue fluid: what leaks out and what comes back · Digestion and absorption: one tube, four chemistries →