Question 5

College Answer

Marks were awarded for an appropriate curve with values, an explanation of the nature of 
positive cooperatively and notes on those factors causing changes in the p50 or "shifts” in the 
curve. 
Most candidates were able to provide the required sigmoid shaped curve with appropriate key 
value points (p50, venous and arterial points). Better candidates were able to identify p50 as a 
measure of avidity or affinity for oxygen and commented on T- Tense and R- Relaxed states, 
the role and production of 2,3 DPG , binding to the beta chains (nature of the lack effect on 
foetal haemoglobin). Describing the mechanisms associated with factors shifting the curve and 
commenting on changes in oxygen content over the steep and flatter parts of the curves gained 
additional marks. Candidates are reminded to answer the question asked - no marks were 
awarded for a description of dissolved oxygen delivery. Some answers confused the Bohr and 
Haldane effects.

Discussion

The oxyhaemoglobin dissociation curve is a sigmoidal relationship between the partial pressure of oxygen and the oxygen saturation of haemoglobin:

• This curve describes the changing affinity of haemoglobin for oxygen which occurs with increasing PaO₂.
  • The flat upper plateau decreases variability in blood oxygen content even with large changes of PaO₂
  • The steep lower part allows the increased release of oxygen from haemoglobin with only a small change in PaO₂
• Positive cooperativity is the main reason behind the non-linearity of this relationship:
  • Haemoglobin is a heterotetramer
  • It is composed of four subunits
  • Each subunit binds oxygen independently.
  • Then, once an oxygen molecule is bound to it, the oxygenated subunit increases the oxygen affinity of the three remaining subunits
  • This is because of a conformational change produced by each subunit binding oxygen, which mediates the transition from the T state to the R state
• T and R state:
  • This refers to two distinct states of the haemoglobin tetramer molecule
  • The T ("Tense") state is the deoxygenated form (with 0 O₂ molecules)
  • The R ("Relaxed") state is the oxygenated state (with 4 O₂ molecules)
  • One pair of αβ subunits in the fully oxygenated R-state appears rotated by 15° with respect to the other pair of subunits
  • The binding of each oxygen molecule changes the state of the tetramer, changing the equilibrium constant for the next O₂ molecule to bind the next subunit more easily

The following physiological factors influence the affinity of hemoglobin for oxygen:

• The partial pressure of CO₂
  • Increasing CO₂ shifts the curve to the right
  • Hyperventilation and hypocapnia shifts the curve to the left
• pH, independent of CO₂
  • Decreasing pH (acidosis) shifts the curve to the right
  • Alkalosis shifts the curve to the left
• The concentration of 2,3-DPG inside the erythrocytes
  • Increased 2,3-DPG (eg. in response to hypoxia or erythropoietin) shifts the curve to the right
  • Decreased 2,3-DPG (eg. as a red cell storage lesion ) shifts the curve to the left
• The presence of unusual haemoglobin species
  • ​​​​​​​Methaemoglobin, carboxyhaemoglobin and foetal haemoglobin shift the curve to the left
  • Sulfhaemoglobin shifts the curve to the right
• Temperature
  • ​​​​​​​Hyperthermia shifts the curve right
  • Hypothermia shifts it left

Bohr, C., K. Hasselbalch, and A. Krogh. "Concerning a biologically important relationship–the influence of the carbon dioxide content of blood on its oxygen binding." Skand. Arch. Physiol 16 (1904): 402.

- this is an English translation of the original article, which was beautifully titled "Über einen in biologischer Beziehung wichtigen Einfluss, den die Kohlensäurespannung  des Blutes auf dessen Sauerstoffbindung übt".

Severinghaus, John W. "Simple, accurate equations for human blood O2 dissociation computations."Journal of Applied Physiology 46.3 (1979): 599-602.

Mills, Frederick C., and Gary K. Ackers. "Quaternary enhancement in binding of oxygen by human hemoglobin." Proceedings of the National Academy of Sciences 76.1 (1979): 273-277.

Eaton, William A., et al. "Evolution of allosteric models for hemoglobin."IUBMB life 59.8‐9 (2007): 586-599.

Mozzarelli, Andrea, and Stefano Bettati, eds. Chemistry and biochemistry of oxygen therapeutics: from transfusion to artificial blood. John Wiley & Sons, 2011.

Benesch, Reinhold, and Ruth E. Benesch. "The effect of organic phosphates from the human erythrocyte on the allosteric properties of hemoglobin."Biochemical and biophysical research communications 26.2 (1967): 162-167.

Bellingham, A. J., J. C. Detter, and C. Lenfant. "Regulatory mechanisms of hemoglobin oxygen affinity in acidosis and alkalosis." Journal of Clinical Investigation 50.3 (1971): 700.

Berg, Jeremy M., John L. Tymoczko, and Lubert Stryer. "Hemoglobin Transports Oxygen Efficiently by Binding Oxygen Cooperatively." in Biochemistry, 5th edition (Jeremy M Berg, John L Tymoczko, and Lubert Stryer (2002).

Benesch, Reinhold, and Ruth E. Benesch. "The Chemistry of the Bohr Effect."The Journal of Biological Chemistry 236.2 (1961).

Dash, Ranjan K., and James B. Bassingthwaighte. "Erratum to: Blood HbO2 and HbCO2 dissociation curves at varied O2, CO2, pH, 2, 3-DPG and temperature levels." Annals of biomedical engineering 38.4 (2010): 1683-1701.

Rossi-Bernardi, L., and F. J. W. Roughton. "The specific influence of carbon dioxide and carbamate compounds on the buffer power and Bohr effects in human haemoglobin solutions." The Journal of physiology 189.1 (1967): 1-29.

Arnone, A. "X-ray studies of the interaction of CO2 with human deoxyhaemoglobin."Nature 247, 143 - 145 (18 January 1974)

Perrella, M., D. Bresciani, and L. Rossi-Bernardi. "The binding of CO2 to human hemoglobin." Journal of Biological Chemistry 250.14 (1975): 5413-5418.

Weber, Roy E., and Kevin L. Campbell. "Temperature dependence of haemoglobin–oxygen affinity in heterothermic vertebrates: mechanisms and biological significance." Acta Physiologica 202.3 (2011): 549-562.

Barcroft, Joseph, and W. O. R. King. "The effect of temperature on the dissociation curve of blood." The Journal of physiology 39.5 (1909): 374-384.

Leach, R. M., and D. F. Treacher. "The pulmonary physician in critical care• 2: Oxygen delivery and consumption in the critically ill." Thorax 57.2 (2002): 170-177.