CW8: Myoglobin and haemoglobin

Question 1

How can you vary the concentration of oxygen in a buffer?

Answer 1

Have oxygen in the buffer originally

Saturate oxygen in a pressure above the buffer then apply the pressure so oxygen is transferred to the buffer medium

Question 2

What is haemoglobin’s function and strucutre?

Answer 2

• It is an oxygen-carrying protein in red blood cells
• It is a tetramer (has four sub-units) with polypeptide chains in an α₂β₂ complex
  
  • α type = 141 amino acids
  • β type = 146 amino acids
  • These values are similar to each other and to myoglobin; however, the structures of each type are not that similar (approx. 20% identity)
• Contains the essential prosthetic group (haem with Fe atom)
• Each polypeptide cahin is very similar to myoglobin

Question 3

What is the shape of the oxygen-binding curve of haemoglobin?

Answer 3

Sigmoidal

Question 4

Why is the shape of the oxygen-binding curve for haemoglobin sigmoidal whilst the shape for myoglobin’s is a rectangular hyperbola?

Answer 4

Mb only releases oxygen under stress conditions, i.e. when pressure is low

Hb releases oxygen at a low saturation in the tissues and is packed with oxygen at the lungs where there is a high partial pressure

Question 5

What is the pathway of communication for haemoglobin?

Answer 5

• Oxygen binds to Fe²⁺
• Fe²⁺ moves 0.7Å into the plane of the ring
• This pulls histidine side chain towards the ring (the small movement has a big effect in the tetramer)
• Histidine residue is in F-helix which moves by several Ångstroms (lever-arch effect)
• This breaks the electrostatic interactions at the subunit interface, allowing relative rotation of subunits

Question 6

What is the ‘postage stamp’ analogy of cooperativity?

Answer 6

The removal of the first stamp breaks two interfaces, the second and third breaks one interface each, but the last one comes for free

In terms of oxygen, one oxygen binding has an impact on two subunits, which healps the rest bind to the molecule

This helps to explain the shallow gradient at the start of the sigmoidal curve

Question 7

What affects oxygen binding to haemoglobin?

Answer 7

• H⁺ which favours the deoxy form
• CO₂ which favours the deoxy form
• Bisphosphoglycerate (BPG) binds one per tetramer which stabilises the deoxy form
• All of these effectors promote oxygen release in tissues

Question 8

Define ‘allosteric’.

Answer 8

Something that binds to the molecule somewhere other than the active site

Question 10

What is the effect of pH on oxygen binding to Hb?

Answer 10

Decreased pH by 0.4 means an additional 25% release of oxygen by haemoglobin (Bohr effet)

There is a similar loading at high pressures of oxygen, e.g. in the lungs

Question 11

What are two effects of CO₂ on Hb? What are the equations for these effects?

Answer 11

• Generates H⁺ (acid)
  
  • CO₂ + H₂ ⇋ H₂CO₃ ⇋ H⁺ + HCO₃^–
• Binds directly to Hb amine groups at N-terminal end of protein or on side chains, leading to negative charge and therefore change in interaction with other parts of the protein
  
  • –NH₂ + CO₂ → NHCOO^– (carbamate ion)

Question 12

What’s the difference between ‘di–’ and ‘bis–’?

Answer 12

di– = linked directly to each other, e.g. ADP

bis– = attached to different parts of a parent chain, e.g. BPG

Question 13

How does BPG affect the affinity of Hb for oxygen?

Answer 13

It reduces its affinity

Without BPG, the curve has no sigmoidal effect

Question 14

Where on the Hb tetramer does BPG bind? Why?

Answer 14

At the centre

In its deoxygenated form, there is a space large enough for a BPG molecule

(When oxygen is present, the hole where BPG binds closes up and this interaction doesn’t take place)

All surrounding amino acids are positive (histidine is positive due to the acidic conditions) so BPG is happy and Hb won’t want to bind with oxygen

Question 15

What is the subunit structure of foetal haemoglobin?

Answer 15

α₂γ₂

Question 16

Why does foetal haemoglobin bind to oxygen more tightly than adult haemoglobin?

Answer 16

It binds BPG less tightly than the adult haemoglobin, since the His143 is instead Ser143

This results in a less positive charge so oxygen is not released as readily so the foetus can take oxygen from maternal blood when required

Question 17

What is the cause of sickle cell anaemia?

Answer 17

Defective haemoglobin

There is a mutation in the β chain (Glu₆ to Val; negative to uncharged) leading to inappropriate interactions with subunit

This leads to loss of solubility and precipitation in red blood cells (the precipitate forms filaments which disrupt the overall RBC shape)

The function of this form of Hb (HbS) in homozygous individuals is very much impaired, but heterozygous have an increased resistance to malaria (selective advantage)

Question 18

How can you identify the sickle cell mutation?

Answer 18

Peptide fingerprinting:

• Chop the protein of interest with protease, e.g. trypsin (triptic digest)
• Perform 2D chromatography and electrophoresis in perpendicular directions, resulting in isolated protein spots on the gel
• The movement of the black dot to the green one above it is caused by a point mutation