Respiratory: Function of Haemoglobin in Oxygen Carriage

Question 1

Draw oxygen dissociation curve

Answer 1

Question 2

Describe structure of haemoglobin

Answer 2

• four polypeptide globin chains
• each containing a haem molecule made up of protoporphyrin ring with a central iron atom in the ferrous state (Fe2+)
• Quaterney structure
• Molecular weight of approximately 65,000 Da
• two alpha (α) chains consist of 141 aa residues
• two beta (β) chains consist of 146 aa residues
• Genes for α chains chromosome 16
• Genes for β chains chromosome 11

Question 3

Forms of Hb

Adult Hb

1. HbA
2. HbA2

Fetal Hb

1. HbF

Answer 3

• Adult HbA 2 α, 2 β 95%
• HbA2 2α, 2δ chains 2.2-3.5%
• HbF 2α, 2γ chains, higher affinity for O2 than HbA
  
  • at birth 50-95%
  • decline after 6 months -> HbA
  • <1% adult Hb

Question 4

Structure of haem

Answer 4

• each globulin chain attached to a iron containing haem
• Haem = orgainic part + iron atom in ferrous state (Fe ²⁺)
• organic part is protoporphyrin made up of four pyrrole rings
• Haem = red
• The Fe2+ forms six bonds
  
  • 4 to nitrogen atoms in the centre of the protoporphyrin ring
  • one to a histidine residue (the ‘proximal histidine’) on globin chain -> one subunit
  • one with O2 molecule
• ‘Distal’ histidine residue near O2 binding site
  
  • prevents haem groups on other haemoglobin molecules oxidizing the Fe2+ to Fe3+
  • prevents carbon monoxide binding irreversibly to the Fe2+

Question 5

Haem synthesis

Answer 5

• Mitochondria
• Protoporphyrin synthesized from condensation of glycine and succinyl coenzyme A
• Protoporphyrin combines with iron in the ferrous state (Fe2+) to form haem
• Globin chains are formed in the ribosomes
• Hb molecule = tetramer 4 globin chains + haem bound to each

Question 6

Haem destruction

Answer 6

• red cells removed by retiulo-endothelial system
• globin chains broken down to aa
• aa’s re-entre aa pool
• iron reused by bone marrow to synthesis Hb
• protoporphyrin ring opened to form biliverdin
• biliverdin metabolised to bilirubin, bound to albumin, carried to liver
• in liver bilirubin conjugated with glucuronic acid and excreted in the bile then small bowel
• In GIT bilirubin converted to stercobilin, reabsorbed into plasma and excreted by kidneys as urobilinogen
• Small amounts of free haemoglobin may be released into the plasma

Question 7

Carriage of Oxygen and O2 Binding Mechanism

Answer 7

• one haem group bind reversibly to one O2
• each Hb bind to 4 O2
• allosteric protein
• i.e. the binding of O2 to one haemoglobin subunit increases the affinity of the other subunits for O2
• deoxyhaemoglobin a different quaternery structure from oxyhaemoglobin
• Haemoglobin is described as ‘tense’ when deoxygenated and ‘relaxed’ when oxygen has bound

Question 8

Hüfner Constant

Answer 8

• The amount of oxygen (in ml) carried by each gram of haemoglobin
• 1.39 ml/g

Question 9

Explain the shape of the oxygen dissociation curve

Answer 9

• sigmoid shape due to cooperative binding between the component globin chains
• lowest affinity for O2 when first molecule binding
• each successive O2 molecule binding has higher affinity
• when venous sats 75% only last O2 molecule binding/unbinding - efficient

Question 10

Haemoglobin-Oxygen Binding

What factors affect the P50

Answer 10

The factors that affect the P50 and therefore reflect O2 affinity are:

• Temperature
• Hydrogen ion concentration or pH
• Carbon dioxide (CO2) concentration
• 2,3-diphosphoglycerate (DPG) concentration

H+, CO2 and 2,3-DPG are all allosteric effectors as they favour the conformation of deoxyhaemoglobin. They act at different sites so their effects are additive.

The P50 of HbA is 3.5 kPa, whereas the P50 of HbF is 2.5 kPa. This reflects the higher affinity that HbF has for O2

Question 11

What is the Bohr effect?

What is the double Bohr effect?

Answer 11

Bohr effect is the reduced affinity oxygen has for haemoglobin when pH is low and the higher affinity it has when pH is higher

The ‘double Bohr’ effect helps to increase fetal oxygenation. Uptake of fetal CO2 by maternal blood shifts the maternal oxy-haemoglobin curve to the right and the fetal curve to the left.

Question 12

2,3-Diphosphoglycerate

Answer 12

• highly anionic organic phosphate
• promotes the release of oxygen from haemoglobin
• produced by a side-shunt reaction in glycolysis
• present in large quantities in the erythrocyte
• binds with the beta globin chains of deoxyhaemoglobin, altering the protein conformation and reducing oxygen affinity
• causes right shift of O2 dissoociation curve
• promoted production in anaemia
• produced in aclimatization to altitude (offset by left shift caused by hyperventilation alkalosis)
• decreased in stored blood (low levels after 14 days in SAG-M: saline, adenine, glucose, mannitol)

Question 13

Factors affecting P50

What factors casue a left shift (increased affinity) of O2 dissociation curve?

What factors cause a right sift (decreased affinity) of O2 disccosiation curve?

Answer 13

Left shift

• low temperature
• alkalosis
• hypocapnia
• CO
• low 2,3-DPG

Right shift

• high temperature
• acidodid
• hypercapnia
• high 2,3-DPG

Question 14

Abnormal Hb: globin chains

Answer 14

• Genetic defects
• Inpaired production
  
  • Thalassaemias: excess either α or β chain precipitate -> haemolysis + anaemia
  • gene for α chain on Ch16 (4 genes) so deletion between 1-4. 4 deletion incompatable with life.
  • β due to gene mutation, become apparent between 3-6 mnths (swithc to HbA). Excess α chains comine with any β,γ,δ chains making HbA2 and HbF
• abnormal structure of the globin chain
  
  • haemoglobinopathies
  • sickle cell disease
  • valine is substituted for glutamic acid at position 6 of the β globin chains
  • single base mutation in the β globin gene
  • homozygous state = HbSS = both β globin genes are abnormal -> sickle cell anaemia.
  • lower P50 => oxy-haemoglobin dissociation curve shifted left

Question 15

Abnormal Haemoglobins: Abnormal Ligands and Fe2+ Oxidation

Answer 15

• Other ligands can combine with the iron in haemoglobin
• Carbon monoxide
  
  • affinity for haemoglobin is 300 times that of oxygen
  • shifts the oxy-haemoglobin dissociation curve to the left
  • reduces the availability of binding sites for O2
  • most common source of CO is tobacco smoke
  • smokers COHb levels of 5-10%.
• Iron oxidised from ferrous (Fe²⁺) to ferric (Fe³⁺)
  
  • Fe³⁺ unbale to bind with O2
  • Haemoglobin naturally scavenges nitric oxide
  • If a patient is treated with nitric oxide
  • If a patient is treated with prilocaine or nitrates

Question 16

Artificial Oxygen Carrying Solutions

Answer 16

Perfluorocarbons:

• synthetic compounds
• can dissolve O2
• potential alternative means of oxygen delivery
• Initial results promising
• research abandoned because of adverse effects caused by the emulsified perfluorocarbon preparation required for clinical use.

Modified Haemoglobin Solutions

• Stroma-free haemoglobin extracted from erythrocytes
• Hb dissociates into α/β dimers after extraction (non-functional, cause nephrotoxiciy)
• Chemicals have been used to stabilize hb and reduce the O2 affinity to promote O2 release.
• Genetic engineering techniques used to insert a modified Hb gene into E.coli plasmids to produce stabilised Hb in large quantities
• use of bovine Hb suggested (concerns re prion contamination)
• free Hb has a much shorter intravascular half life than an erythrocyte and can lead to raised bilirubin levels
• excellent scavenger of nitric oxide and the resulting vasoconstriction may cause hypertension
• Liposome- encapsulated haemoglobin is currently undergoing investigation