Haemoglobin as an allosteric tetrameric protein to transport oxygen

Question 1

Haemoglobin

Answer 1

• iron-containing O2 transport metalloprotein in the RBCs of vertebrates
• transports O2 from the lungs or gills to tissues where it is released in proportion to consumption
• pressure of half-saturation P50 = 26 Torr
• sigmoidal binding curve

Question 2

tetrapyrolle

Answer 2

4 pyrolle rings e.g. in the porhphyrin of Haeme

Question 3

haem

Answer 3

functional prosthetic group of Hb

• porphyrin (tetrapyrolle) + Fe2+
• hydrophobic (2x vinyl groups) and hydrophilic (2x propriotic acid groups) ends
  
  • vinyl groups bind to N of 2 histidines (E7, F8) in the globin to form Hb
    
    • E7 blocks water and tf generation of metHb (Fe3+)
• involved in:
  
  • O2 transport in Hb and Mb
  • electron transport (cty-C, Fe2+ <–> Fe3+)
  • redox enzymes (e.g. catalase reduces H2O2 to water)

Question 4

subunit cooperativity

Answer 4

at the lungs:

• binding of first O2 @ lungs to T-Hb is slow
• elicits conformational change that increases affinity at the other binding sites for O2
• rapidly converts to R-Hb (oxyHb)

at the tissues:

• @ low pO2 tissues, one O2 dissociates
• induces other O2s to dissociate rapidly to form T-Hb

Question 5

allosteric effectors

Answer 5

• Hb alone is not a good transporter of O2, requires allosteric effectors:
  
  • CO2 (Bohr effect)
    
    • +[CO2]/-pH shifts dissociation curve R, such that deoxyHb is more stable in acidic environment of the tissues
    • this promotes oxyHb to release O2
  • 2,3-biphosphoglycerate
    
    • binds between beta subunits of deoxyHb, stabilizing it and preventing re-binding of O2 @ tissues

Question 6

adaptation

Answer 6

• shift of Bohr curve to the R after several days on low O2 environment
• increases O2 dissociation at the acidic tissues by stabilizing deoxyHb
• compensates for less O2 picked up at the lungs by unloading more at tissues
• elevated BPG raises P50 from 26 to 31 Torr
  
  • +P50 means little change in delivery at rest after adaptation (37%) from at sea level (38%)
  • higher P50 of Hb means it will more readily transfer O2 when pO2 is low (altitude, and at tissues)
• increased EPO production from kidney leads to +RBCs

Question 7

haemoglobinopathies

Answer 7

• over 800 variants of Hb known
• links to ethnic groups (inherited)
• most have no physiological consequences, others are deleterious mutations
• e.g. homozygous sickle cell disease (HbSS)
  
  • hydrophobic Val where Glu should be binds to hydrophobic pocket in deoxyHb resulting in crystalline structure
  • discovered in 1950s
  • causes blockage in kidneys but also kills malaria parasite (common in equatorial Africa)
• e.g. Hb Hiroshima - increases O2 affinity and decreases Bohr effect
  
  • disrupts stable salt bridge formation in deoxyHb
  • removes His that binds a Bohr effect proton
• e.g. Hb Hammersmith - unstable Hb, loses haeme
  
  • replaces a ‘gatekeeper’ Phe with hydrophilic Ser
  • water gets in and RBC lifespan decreases to ~14-20 days
  • +metHb

Question 8

What percentage of the dry cell RBC content is Hb?

Answer 8

97%

Question 9

How does Hb carry CO2?

Answer 9

~15% of total CO2:

• as carbaminohaemoglobin
• on beta terminal amino groups of deoxyHb
  
  • Hb-NH-COO-

other ~85% CO2:

• carbonic anhydrase sticks OH from H2O onto CO2, producing HCO3- (bicarbonate) –> transported in plasma
• H+ produced is buffered by HPO42- and proteins including Hb

Question 10

What is the function of NO carried by Hb?

Answer 10

• released with O2 to relax vascular walls and enhance gas diffusion

Question 11

Who won the Nobel prize for solving the structure of Hb?

Answer 11

John Kendrew and Maz Perutz, 1962

Question 12

What was the first protein structure to be solved?

Answer 12

Myoglobin, then haemoglobin (NP: Kendrew and Perutz)

Question 13

What is the character of oxygen association in Hb, and how is it achieved?

Answer 13

• first investigated by Christian Bohr
• variable sigmoid binding character
• achieved by subunit cooperativity and allosteric effectors
• acid stabilizes deoxyHb (shifts curve R) tf more O2 can be unloaded at tissues by oxyHb

Question 14

Haemoglobin adapts to

Answer 14

altitude

Question 15

How does foetal haemoglobin differ?

Answer 15

• foetal/HbF has different subunits
  
  • adult/HbA has alpha 2 beta 2
  • foetal/HbF has alpha 2 gamma 2
• has a higher affinity for O2
• has a lower affinity for BPG
  
  • tf deoxyHb is more unstable, readily picks up O2

Question 16

What causes haemoglobinopathies?

Answer 16

mutations in AA sequence

Question 17

Myoglobin

Answer 17

• monomeric, 8 alpha helices, 7 kinks
• binds O2 in muscle, higher affinity
• similar structure to Hb
• precursor of globin, adapted to become Hb
• hydrophillic outside, phobic centre containing haeme subunit w/Fe2+
• hyperbolic binding curve
  
  • binds O2 efficiently @ low pO2 i.e. capillaries of active muscle