Haemoglobin and Oxygen Carriage

Question 1

Describe the structure of haemoglobin?

Answer 1

Composed of four polypeptide globin chains, each containing a haem molecule.

Each haem molecule is made up of protoporphyrin ring with a
central iron atom in the ferrous state (Fe2+).

Hb has a quaternary structure and a molecular weight of 65,000 Da.

Question 2

What is meant by a Quaternary structure?

Answer 2

Definition of Quaternary protein:
1. Primary – sequence of aa’s

2. Secondary – angles created in aa chain to form differing angles
3. Tertiary – Changes in the shape that form the detailed shape of the protein i.e. crevasse for haem binding region created
4. Quaternary – interaction between the protein chains (i.e. 4 globin chains)

Question 3

How many amino acids are on an alpha and a beta globin chain?

Answer 3

Alpha globin chain contains 141 amino acids.

Beta globin chain contains 146 amino acids.

Question 4

Which chromosomes contain the genes for alpha and beta globin chains?

Answer 4

Alpha globin: Chromosome 16

Beta globin: Chromosome 11

Question 5

Describe the structure of a Haem molecule?

Answer 5

Each Haem is made up of a protoporphyrin

Protoporphyrin is made of 4 pyrrole rings which give the haem its red colour. Iron its ferrous state (Fe2+) lies in the centre of the protoporphyrin.

Fe2+ forms 6 bonds:
* 4 with nitrogen atoms
1 to a histidine “proximal” residue located on the
* globin chain to form 1 subunit of haemoglobin
* 1 with an oxygen molecule (O2)

Near to the oxygen binding site, there is another distal histidine which acts to perform 2 important functions:
1. Prevents oxidation of Fe2+ to Fe3+ by other haem groups on Hb molecules
2. Prevents irreversible binding of CO to ferrous iron

Question 6

What are the different non pathological forms of haemaglobin?

Answer 6

1. HbA (2 alpha 2 beta) 95% of adult Hb
2. HbA2 (2 alpha 2 delta) 2.2-3.5% of adult Hb. Has a poor efficiency for O2 carriage.
3. HbF (2 alpha 2 gamma) <1% of adult Hb

Question 7

What is the amount of HbF present at birth and why is it advantageous in utero?

Answer 7

50-95% of haemoglobin at birth.

HbF has a very high affinity to allow greater oxygen transfer in a more general hypoxic environment.

Levels decline 6
months post-natally to ~2%

Question 8

Describe how haemoglobin is synthesised?

Answer 8

Synthesis occurs in mitochondria of RBC where:
1. Protoporphyrin is synthesized from the condensation of glycine and succinyl coenzyme A.

2. Protoporphyrin combines with iron in the ferrous state (Fe2+) to form haem.
3. Globin chains are formed in the ribosomes.

Four of the above will produce 1 haemoglobin molecule

Question 9

Describe the removal of Hb from the body?

Answer 9

1.Globin chains are broken down to amino acids and re-enter the amino acid pool
2.Iron is reused by bone marrow to synthesise haemoglobin
3.Protoporphyrin ring is opened to form biliverdin which is then metabolised to bilirubin.

Bilirubin excretion:
* Binds to albumin and carried to the liver to be conjugated with glucuronic acid and is excreted in the bile and then into the small bowel.
* GI tract: Bilirubin converts to either stercobilin – some of which may be reabsorbed and excreted in the urine as urobilinogen.

Question 10

Describe the allosteric binding of O2 to Hb?

Answer 10

Binding of O2 to one haemoglobin subunit increases the affinity of other subunits for O2.

As one molecule of oxygen binds to haem, it pulls the ferrous
group into the plane of the porphyrin ring to flatten it. This causes change in ionic interactions holding the 4 subunits together and they reform in a different position altering the
quaternary structure to help facilitation of oxygen binding.

Deoxygentaed Hb can be referred as having a tense structure and oxygenated Hb as being relaxed

Question 11

What is the Huffner Constant?

Answer 11

This is the amount of oxygen in ml, carried by each gram of haemoglobin. Based on Hb’s molecular weight.

Theoretical value 1.39g/ml.
Actual value 1.34g/ml due to other forms of haemoglobin which have a reduced affinity for O2.

Question 12

Describe the shape of the oxygen dissociation curve?

Answer 12

Sigmoid shaped.

Reflects the allosteric binding of O2. The affinity of haemoglobin for oxygen is lowest for the 1st oxygen molecule to bind.

As the first O2 molecule binds conformational change making it easier for further O2 to bind.

The curve becomes less steep for for the 4th molecule as although O2 still binds easily to Hb there is only one site remaining.

Question 13

What are average venous oxygen saturations and what is the significance of this with regards Hb Oxygen binding?

Answer 13

Average venous oxygen saturations are 75%

The significance of this is that it is only ever the final oxygen molecule that binds and unbinds increasing the efficiency of the system.

Question 14

What is meant by the P50 and what is the P50 for HbA and HbF?

Answer 14

P50 refers to the partial pressure of O2 at which there is 50% oxygen haemoglobin saturation. It is used as a reference point to help describe left and right shift on the oxygen dissociation curve.

HbA 3.5 Kpa
HbF 2.5Kpa

Question 15

Which factors affect the Haemaglobin oxygen binding affinity?

Answer 15

• pH (acidaemia causes a right shift)
• Increased CO2 (results in a reduced pH which causes a right shift)
• Temperature (increased temp causes a right shift)
• 2,3-Diphosphoglycerate (causes a right shift)

Question 16

What is the double Bohr effect?

Answer 16

The Bohr effect describes the right shift of the HbA oxygen dissociation curve in the presence of increased CO2 levels.

The Double Bohr effect describes the above as well as the leftward shift of the HbF oxygen dissociation curve in the presence of increased CO2 levels.

The double Bohr effect helps increase fetal oxygenation: as it reduces the HbA O2 binding in the relatively hypoxic envrionment, meaning more O2 is available for fetal Hb. The leftward shift on HbF oxygen dissociation curve increases the HbF uptake.

Question 17

What is 2,3-Diphosphoglycerate?

Answer 17

Highly anionic organic phosphate which promotes the allosteric deoxyhaemoglobin state.

It is produced by a side-shunt reaction in glycolysis and is present in large quantities in the erythrocyte. It binds to beta globin chains of deoxyhaemoglobin altering its protein conformation.

Its production is promoted in anaemic states, as it promotes oxygen delivery in anaemic states.

Question 18

What happens to 2,3 diphosphoglycerate levels at altitude?

Answer 18

At altitude there are increased concentrations of 2,3 diphosphoglycerate. This causes a right shift in the curve.

This right shift is counteracted by the respiratory alkalaemia caused by hyperventilation.

Question 19

Think beyond standard haemaglobinopathies.

What are the different types of abnormal Haemaglobin?

Answer 19

Abnormal haemaglobin can be categorised into:
* Absence or abnormality of a globin chain
* Abnormal ligand bound to the Haem group
* Oxidised Fe2+

Globin chain abnormalaties:
* Thalassaemia: Abnormal ratios of α and β chains are produced, which result in excess haemolysis.
* Sickle Cell: single base mutation in β gene resulting in abnormal Hb, again more prone to haemolysis.

Abnormal Ligands:
Other ligands may combine with iron – most commonly carbon monoxide which has an affinity to haemoglobin 300x greater than oxygen. This reduces the available binding sites as well as shifting the Oxygen dissociation curve to the left.

Oxidised Fe2+
Iron can be oxidised from the ferrous (Fe2+) to the ferric (Fe3+) form which is unable to bind with oxygen.

There are other haemaglobinopathies such HbC which you do not need to know in depth.

Question 20

What are the different types of thalassaemia?

Answer 20

Alpha thalassaemia: Usually as a result of DELETIONS of 1 or all 4 of the alpha genes on chromosome 16. Varies in severity depending on number of deletions. (4 deletions is incompatible with life)

Beta thalassaemia: gene mutation and results in reduced production of β chains. The excess α chains combine with whatever β, δ or γ chains are present, forming excess HbA2 and HbF.

It usually becomes apparent between 3-6 months of age when the switch to HbA occurs.

Question 21

Where is the gene mutation in Sickle Cell disease, and what are the different forms of the disease?

Answer 21

Sickle cell is caused by a single base mutation in the β globin gene where valine is substituted for glutamic acid at position 6 of the β globin chains.

**Homozygote disease HbSS: **both β globin genes are abnormal which results in sickle cell anaemia The P50 is lower than normal and the oxy-haemoglobin dissociation curve is shifted to the left. (reducing tissue oxygen delivery)

Heterozygote (trait) HbAS: clinically much less severe, known as carrier status.

Question 22

In what scenarios might Iron oxidise and what is the significance of this?

Answer 22

Iron can be oxidised from the ferrous (Fe2+) to the ferric (Fe3+) form which is unable to bind with oxygen. This may occur in the following scenarios:

1. Haemoglobin naturally scavenges nitric oxide, therefore If a patient is treated with nitric oxide
2. If a patient is treated with prilocaine or nitrates