Protein Function

Question 1

What is haem?

Answer 1

• The oxygen binding site in haemoglobin and myoglobin.
• Consists of a central Fe atom bound to the 4 N atoms of a protoporphyrin ring.
• Fe2+ can make 2 additional bonds (one on either side of the plane):
  
  • to a histidine residue (proximal histidine) on haemoglobin/myoglobin
  • to 1 oxygen molecule

Question 2

What are the functions of myoglobin and haemoglobin?

Answer 2

• Both are oxygen binding proteins.
• Myoglobin carries oxygen to muscle tissues.
• Haemoglobin binds oxygen on RBCs and delivers it to tissues for cellular metabolism. Also removes the resulting waste product, carbon dioxide.

Question 3

Describe and compare the structure of myoglobin and haemoglobin.

Answer 3

Myoglobin

• Compact monomeric protein.
• Consists of 8 alpha helices (75%) and contains 1 haem group - His93 in 8th helice is covalently linked to Fe.

Haemoglobin

• Tetrameric protein.
• 2 alpha chains and 2 beta chains (each has conformation very similar to that of myoglobin) - each contains 1 haem prosthetic group.

Question 4

Describe the binding of oxygen by myoglobin.

Answer 4

• Each myoglobin molecule binds to 1 oxygen: binds tightly and easily.

1. Fe in deoxyhaemoglobin is slightly below the plane of the ring.
2. Oxygen binding causes Fe movement into plane of the ring.
3. Movement of His F8 and small change in overall protein conformation.

• Oxygen binding to myoglobin shows a hyperbolic dependence on oxygen concentration.

Question 5

Describe the binding of oxygen to haemoglobin.

Answer 5

• Each haemoglobin binds 4 oxygen molecules: binds oxygen loosely and with difficulty.
• Binding of oxygen causes change in haemoglobin conformation from low affinity T state (haem groups not exposed) to high affinity R state.
• Binding affinity thus increases as more oxygen molecules bind to Hb subunits (allosteric effect) - cooperative binding of oxygen.

Question 6

What is the effect of the sigmoidal oxygen binding curve of haemoglobin?

Answer 6

• Oxygen can be delivered efficiently to the tissues: high affinity in lungs (high pO2) so easier uptake, low affinity in tissues (low pO2) so easier release.
• More sensitive to small differences in oxygen concentration.

Question 7

How is Hb oxygen binding regulated and how does this affect the binding curve?

Answer 7

Via allosteric effectors:

• activators enhance high affinity R state (shift curve to left)
• inhibitors enhance low affinity T state (shift curve to right)

Question 8

Give an example of and Hb oxygen binding inhibitor.

Answer 8

2,3-Bisphosphoglycerate (BPG)

• Decrease the affinity for oxygen (shifts curve to right) - more efficient transporter.
• BPG concentration increases at high altitudes, promoting oxygen release at tissues.
• 1 BPG binds per Hb tetramer - sits in the middle and interacts with positive lysine and histidine residues of beta 1 and 2 subunits.
• Present in RBCs at approx. 5 mM.

Question 9

What is the Bohr Effect?

Answer 9

• H+ and CO2 can both bind to Hb molecules - lower oxygen affinity.
• Ensures the delivery of oxygen is coupled to demand - metabolically active tissues produce large amounts of H+ and CO2.

Question 10

Why is carbon monoxide (CO) toxic?

Answer 10

• CO combines with ferromyoglobin and ferrohaemoglobin and blocks oxygen transport.
• Binds to Hb 250x more readily than oxygen.
• CO never releases Hb so CO saturation = cell death. Fatal when COHb is > 50%.
• CO binding also acts to increase the oxygen affinity of unaffected subunits.

Question 11

Which types of haemoglobin are found in adults?

Answer 11

HbA (alpha2 beta2) = 90%
HbA2 (alpha2 delta2) = 2-5%
HbF (alpha2 gamma2) = <2%

Question 12

Which chromosomes are Hb genes located on?

Answer 12

• Chromosome 16: 2 copies of alpha-globin gene (alpha 1 and alpha 2)
• Chromosome 11: beta-globin-like genes (beta, gamma and delta)

Question 13

What type of Hb is dominant in foetal blood and why?

Answer 13

HbF as higher binding affinity for O2 allows transfer of oxygen from maternal oxyhemoglobin to foetal deoxyhaemoglobin.

Question 14

Describe how the sickle cell anaemia mutation causes RBCs to sickle.

Answer 14

• Single base pair mutation of glutamate to valine in beta globin, forming HbS.
• Glu is a hydrophilic molecule on surface of protein but valine is hydrophobic - forms a hydrophobic pocket and polymerisation of deoxygenated HbS.
• Abnormally shaped sickled cells are more prone to lysis (causing anaemia) and are more rigid (block microvasculature).

Question 15

What are thalassaemias?

Answer 15

• Group of genetic disorders where there is an imbalance between the number of alpha and beta globin chains.

Beta-thalassaemias

• Decreased (beta-thalassemia minor) or absent (beta-thalassemia major) beta-globin chain production.
• Alpha chains are unable to form stable tetramers, instead form: HbF, Hb Bart’s (gamma chain tetramer) or alpha chain precipitate.
• Symptoms appear after birth

Alpha-thalassaemias

• Decreased or absent alpha-globin chain production.
• Several different levels of severity due to multiple copies of the alpha-chains present.
• Beta chains can form stable tetramers (Hb H) with increased affinity for oxygen.
• Onset before birth.