CBS - Haemoglobin A/F/S

Question 1

Describe the haemoglobin protein.

Answer 1

Haemoglobin (Hb) is a tetrameric heme protein found in erythrocytes (red blood cells, RBC) where it is responsible for binding oxygen in the lung and transporting the bound oxygen throughout the body where it is used in aerobic metabolic pathways.

It also facilitates the return of carbon dioxide from the tissues to the lungs

Question 2

Describe the myoglobin protein.

Answer 2

Myoglobin (Mb) is a monomeric heme protein found mainly in muscle tissue.

Its major physiological role is to facilitate oxygen transport in rapidly respiring muscle.

Mb receives O2 from Hb

Question 3

Describe the cooperativity of Hb and Mb.

Answer 3

In terms of cooperativity, Mb follows a typical hyperbolic
curve, and Hb follow a sigmoidal curve.

A sigmoidal curve indicates cooperativity.

A macromolecule exhibits cooperative binding if its affinity for its ligand
changes with the amount of ligand already bound.

Question 4

What is the Bohr effect?

Answer 4

An increase in pH (and decrease in CO2 concentration) increases H’s affinity for O2.

Question 5

What are the implications of the Bohr effect in the peripherial tissues and in the lungs?

Answer 5

In peripheral tissues (where there is a lower pH due to binding of CO2 and H+) there is a decreased affinity for O2 (this is good as we want O2 released)

In lungs (where there is a higher pH due to the release of CO2 and H+) there is an increased affinity for O2 (which is good because we want O2 binding).

Question 6

What lowers the blood’s affinity for oxygen?

Answer 6

The substance D-2,3-biphosphoglycerate lowers the blood’s affinity for oxygen.

Question 7

Describe the change of heamoglobin globin chains during development.

Answer 7

During development in the womb, the foetus has high amounts of α and γ chains (HbF).

Once born, the levels of γ chains rapidly drop, and are replaced with β chains, making HbA.

Throughout, we have a constant high amount of α globin chains.

Question 8

Describe heme as a prosthetic group.

Answer 8

Heme is incorporated into proteins during synthesis.

It is stabilized by hydrophobic residues found in interior of the protein: a protective environment that prevents oxidation of Fe2+ (ferrous) to Fe3+ (ferric) or “rusting”.

In this state it can not react with O2.

Heme is essential for oxygen to bind to the RBC.

It can only bind with O2 in the ferrous 2+ state, and so that needs to be maintained.

Question 9

Describe the globin fold and how it holds the heme group.

Answer 9

The two histidine residues of the alpha and beta chains are on opposing sides of the heme ring.

The 4 hydrophobic residues constitute the heme prosthetic group. This provides four planar coordination sides, and then we find that the proximal histidine (F8) directly coordinates the iron centre, whereas the distal histidine (E7) does NOT coordinate the 6th side.

Thus, this is where the oxygen will bind reversibly.

The distal histidine assists with stabilizing the O2- bound form and also destabilizing the CO-bound form. CO binding is only 200x stronger in Hb (compared to 20,000x to free heme).

Question 10

How does the haemoglobin molecule move when oxygen binds?

Why is this movement functionally relevant?

Answer 10

The iron centre is slightly shifted from the plane of the heme by 0.4 Armstrong (10^-10 metres).

When oxygen binds, the iron centre now moved into the plane of the heme, and the oxygen binds onto the iron centre.

When the iron moves into the haem plane, there is a partial electron redistribution from the iron centre to the oxygen. This, the iron goes into a partial Fe3+ character, whilst the oxygen becomes a partially superoxide anion.

Remember, the iron is connected to the proximal histidine. When it moves into the centre, it caused a rearrangement of the entire alpha1beta1 alpha2beta2 interface.

As we add more and more oxygen molecules bound to Hb, the binding of subsequent oxygen molecules becomes easier. That is why we see a sigmoidal oxygen binding curve.

Question 11

How is Hb involved in the transport of CO2 and H+?

Answer 11

About 15-20% of the CO2 produced is transported by Hb.

The production of bicarbonate yields H+ (causing a pH decrease). Hb also transports about 40% of the H+ produced.

Protons bind to various protein sites. The CO2 reacts with Hb N- termini to produce carbamino terminal residues.

Question 12

What are the structural determinants of the Bohr effect?

Answer 12

The protonated His HC3 (β subunit) is hydrogen bonded to Asp FG1 (β subunit) , thus stabilising the T-state (deoxy form) and conferring a high pKa value.

Back at the lungs, the Hb shift to the R-state changes HC3 back to its normal pKa, releasing the H+

As the pH decreases (going to peripheral tissues) protonation of HC3 promotes release of O2 favouring transition to the T-state.

The formation of carbaminoHb (N-termini) also contributes to the Bohr effect due to the presence of salt bridges that stabilise the T-state.

In short, this is a positive feedback mechanism; as CO2 is produced, we have a decrease in pH which stabilises the T state, therefor favouring a lower oxygen affinity state, favouring the release of oxygen.

The opposite happens in the lungs.

Question 13

What is the advantage of the change in BPG concentration in higher altitudes?

Answer 13

At normal altitudes, there is the normal curve for picking oxygen at the arterial pO2 at sea level, and releasing it at the venous pO2. This means that about 38% of the oxygen content gets picked up between these two partial pressures.

Between the two partial pressures, if the curve were to stay the same and we moved to higher altitudes (thus changing the partial pressures), we would only pick up 30% of the uploaded oxygen for physiological needs – which is not enough.

By increasing the BPG concentration, we shift the curve to the right, we have re-established 37% oxygen uptake.

Question 14

We know that increasing BPG effectively decreased oxygen affinity for Hb, but how does it do so?

Answer 14

The T shape has a larger pocket, and so the BPG is the right charge and can only fit in the T state, thus stabilising it and increasing it, thus decreasing oxygen affinity.

Question 15

How do we overcome the BPG effect in foetuses?

Answer 15

In HbF, the β chain features the H143S (histidine to serine) mutation.

Thus BPG- induced stabilisation of the T-state is less efficient in HbF compared to HbA resulting in a higher O2-binding affinity of the former.

Question 16

Describe sickle-cell anaemia.

Answer 16

A genetic disease resulting from a mutation that converts Glu6 (acidic) in the β-chains to Val (nonpolar).
This substitution creates hydrophobic
“sticky” patches on the normally
charged surface of the β-chains.

The oxygenated molecules are soluble, but upon de-oxygenation, the conformation of HbS differs considerably from HbA, and it aggregates into insoluble fibers.

Where we would normally have glutamate 6 interacts with a hydrophobic pocket that also has a number of hydrophobic residues. When valine is present, the hydrophobic patch becomes favoured, and because of the structure, the Hb start to polymerise.

These fibers deform the RBCs into spiny or sickle-
shaped cells.

Question 17

What can be used to combat the blockage of capillaries in SCA due to the cell shapes?

Answer 17

oxygen therapy is used to reduce this, as oxygenated form has less chance of forming polymers than deoxygenated.

Question 18

(from workshop)

Most proteins are colourless. Explain why haemoglobin is red.

Answer 18

The pure globin, chains are indeed colourless. In haemoglobin, each globin subunit is associated with a tightly bound haem group with a central Fe 2+ ion, which gives the protein its bright red colour.

The environment of the haem group affects the actual absorption spectrum, so that oxyhaemoglobin is bright red (present in arterial blood) and deoxyhaemoglobin (present in venous blood) is more purple-red in colour.

Question 19

(from workshop)

Briefly explain how the technique of electrophoresis can separate different proteins from each other.

Answer 19

The sample of protein (in buffer of pH 8) is loaded close to one end of a gel (or supporting medium). A voltage difference is applied across the gel.

The proteins will be attracted to the anode, and move according to their net charge at pH 8. Proteins with different net charges (due to their different amino acid sequences) will then migrate to different positions.

Question 20

(from workshop)

What is the amino acid substitution in the most common haemoglobin variant (Hb S) compared to Hb A?

At what position and in which subunit is the substitution located?

Answer 20

Glutamate (in HbA) is replaced by valine (in HbS) at position 6 in the β-chain (at position 3 in helix A; this is located on the surface of the β-subunit)

Question 21

(from workshop)

Explain (in terms of the ionising properties of the amino acid side chains) why a haemoglobin variant such as Hb S migrates more slowly than Hb A during electrophoresis at pH 8.0.

Answer 21

HbS has valine (which has a neutral, uncharged side group) in each β-subunit, instead of glutamate (which is negatively charged at pH 8) in HbA.

Overall, HbS will have 2 less negative charges than HbA and will move more slowly to the anode.

Question 22

(from workshop)

Patients with the sickle cell condition usually have good health for most of their lives, unless some external stress or change of circumstances triggers a ‘sickling crisis’.

In what way could Jason’s respiratory tract infection have been responsible for the sudden ‘sickling’ episode that resulted in the leg pains?

Answer 22

Some patients with the full sickle cell condition have relatively good health, with minor episodes which can be treated at home with pain killers. Others may have repeated sickling crises which cause severe pain and need hospital treatment.

In Jason’s case, a respiratory tract infection can result in sticky mucus in the airways, and a reduction in the effective transfer of oxygen to the blood supply (hypoxia). Lower oxygen levels will lead to a greater proportion of the HbS in the red cells being in the deoxy-form and a few cells may start to sickle. Once a few cells have sickled in the peripheral circulation, this blocks or slows down blood flow through the capillaries (infarction).

This in turn leads to local areas of lower oxygen concentration, and more cells will sickle. The ‘sickling crisis’ results. Blockage of blood flow will lead to local pain and tissue damage. If the damage is not too great, the situation can be reversed when oxygen becomes available again.

Question 23

Why would someone who suffers from sickle cell anaemia have yellow eyes?

Answer 23

There is a rapid rate of destruction of red cells containing HbS.

When red cells are destroyed, the haem group of haemoglobin is broken down in the spleen and liver to bilirubin, which is an orange brown pigment.

The liver cannot cope with the excess bilirubin above its normal rate of excretion in the bile and some of this bilirubin diffuses in the blood to other tissues and gives a yellow-orange colouration to the skin, eyes, nail bed (jaundice).

Question 24

15. Can the Sickle Cell Anaemia condition be diagnosed prenatally, from a blood sample from the fetus (at 2 or 3 months gestation)?

Answer 24

Even if an uncontaminated blood sample could be obtained from the fetus, the predominant form of haemoglobin before birth is HbF which has γ-chains instead of β-chains.

So the condition could not be detected by electrophoresis of the haemoglobin,, since the change is in the β-chain.

However, we know that the HbS gene has a single base difference in codon 6 (T replaces A) and therefore DNA testing (from a chorionic sampling) could allow the condition to be detected before birth.

Question 25

List four other countries / regions in which the gene for Hb S is widely found in the population.

What explaination(s) have been put forward for such common frequency of the gene?

Answer 25

The distribution of the HbS gene in the world overlaps many of the regions where malaria is endemic. e.g. West Africa, Central Africa, Northern coastal regions of Africa, Greece, Southern Italy, Turkey, parts of India, the West Indies.
The HbS gene is also found in populations which have migrated from these areas to other countries such as the US and the UK.

There is good evidence that children in Africa who are heterozygous for HbS have less severe infections of the local malaria parasite (P. falciparum) than their normal siblings. The hypothesis is that the gene is widespread because the population as a whole is protected against malaria, even though the homozygous individuals die young (under local conditions).

A simple explanation may be that the parasite cannot complete its life cycle in the short lived sickle cell.