Haemoglobin

• human haemoglobin is tetrameric protein
• human haemoglobin has Mr of 64,400
• the four subunits of human haemoglobin each contain haem group with an Fe2+ ion
• tetrameric Hb can combine reversibly with 4 O2 molecules
• normal adult haemoglobin (HbA) has 2 identical alpha chains and 2 identical beta chains
• difference between Mb and Hb

  Mb carries charged amino acids at positions which are hydrophobic in Hb
• deoxyhaemoglobin structure

  • alpha chains have 141 amino acids
  • beta chains have 146 amino acids
  • haemoglobin in urea dissolves into 2 alpha-beta dimers (a1b1 and a2b2)
• little contact between the 2 alpha chains
• no contact between the 2 beta chains
• strong contacts between the chains in an a1b1 dimer (and an a2b2 dimer)
• Hb is tetrameric, Mb is monomeric
• Hb transports H+, CO2 and O2
• Hb-O2 binding is regulated by 3 molecules:

  • H+
  • CO2
  • 2,3-biphosphoglycerate
• regulators of haemoglobin binding to O2 bind to sites distant from the haem


  allosteric interactions
• haemoglobin-oxygen binding is cooperative
• sigmoidal shape of Hb-O2 binding means it is positively cooperative
• positive cooperative binding means binding of one O2 enhances the binding to the other haems
• cooperative binding makes Hb more efficient transporter
• p50 for haemoglobin is 26 Torr in red blood cells
• Mb binds O2 with an order of magnitude more tightly than Hb
• getting oxygen to cells

  labels

  A) haemoglobin

  B) myoglobin

  
• regulators weaken oxygen affinity to enable Hb to unload oxygen more easily
• lactic acid build up during exercise, leads to acidification of venous blood from dissolution of CO2, causing [H+] to rise
• when [H+] rises in the blood, oxygen dissociation curve shifts to the right so that


  more oxygen is released
• increased acidity enables the delivery of more oxygen without fall of oxygen tension
• 2,3-BPG is produced in red blood cells from glycolysis in concentrations of 4-5mM
• 2,3-BPG is generated by activity which uses oxygen so its presence is a signal that more oxygen is needed
• 2,3-BPG binds to deoxyhaemoglobin between the 2 beta subunits of Hb
• BPG is important because

  weakens oxygen affinity (shifts curve to the right), so more oxygen is released when passing through tissue capillaries
• increasing temperature shifts the curve to the right, increasing the release of O2 to active muscles
• deoxyhaemoglobin is the


  tense state
• oxyhaemoglobin is the


  relaxed state
• when Hb gets oxygenated, the distance between iron in beta chains decreases from 


  3.99 to 3.34nm
• T state is constrained and stabilised by hydrophobic bonds and 8 electrostatic bonds
• binding of 2,3-BPG between beta subunits stabilises the T state further
• influencing Hb levels in humans

  • iron levels for functioning Hb
  • altitude (high altitude, lower oxygen concentration, so higher RBC count)
  • exercise
  • erythropoietin drug stimulates Hb production and packing into RBCs
• binding one O2 molecule to iron pulls the Fe2+ ion 0.039nm closer into the porphyrin plane
• as iron moves when it is bond to O2, it brings His F8, so F helix, the EF corner and the FG corner follow
• when iron binds to oxygen it breaks the 8 electrostatic bonds
• shift in helix F causes change

  penultimate tyrosine residue moves out of H-bonded pocket with Valines breaking the electrostatic bonds that stabilise the T state