CBS Haemoglobin KEATS 23_24 PDF

Add Portrait Photo Faculty of Life Science and Medicine Stuart Knight Foundations of Medical Science Cell Biology and Signalling block Biochemistry Department Haemoglobin Teaching Objectives Describe...

Add Portrait Photo Faculty of Life Science and Medicine Stuart Knight Foundations of Medical Science Cell Biology and Signalling block Biochemistry Department Haemoglobin Teaching Objectives Describe the structure of adult haemoglobin HbA Explain the oxygen binding properties of haemoglobin Describe the Bohr effect Explain the significance of binding of 2, 3-bisphosphoglycerate (BPG) Outline the nature and biological significance of HbF Describe HbS and the cause of Sickle Cell Anaemia Dr Stuart Knight 2 Oxygen binding proteins Oxygen key component of ATP generation – final electron acceptor in oxidative phosphorylation (see NAM block) Oxygen transported from lungs to tissues bound to haemoglobin (Hb) in erythrocytes Oxygen stored in striated muscles bound to myoglobin (Mb) Mb has higher affinity for oxygen than Hb Mb is fully saturated at a lower oxygen level (partial pressure of oxygen: pO2) 50% Mb saturation : pO2 ~5 mm Hg 50% Hb saturation : pO2 ~27 mm Hg Fig 4.21 Biochemistry and Molecular Biology Sixth Edition Despo Papachristodoulou, Alison Snape, William H. Elliott, and Daphne C. Elliot Dr Stuart Knight 3 Structure of Hb and Mb Hb is a tetramer : 2 identical beta globin Fig 4.23 (b) subunits and 2 identical alpha globin subunits Biochemistry and Molecular Biology Sixth Hb is dimer of a dimer (α1β1)(α2β2) Edition Despo In adults this is HbA Papachristodoulou, Mb is a monomer Alison Snape, William H. Elliott, and Daphne C. Mb and Hb alpha and Hb beta globins have Elliott very similar structures Globins ~150 amino acids in length consisting of alpha helices Include a haem group buried within a hydrophobic pocket Bringas, M., Petruk, A.A., Estrin, D.A. et al. Tertiary Hydrophilic side of haem group faces the and quaternary structural basis of oxygen affinity in surface of the globin human hemoglobin as Each haem group can bind O2 revealed by multiscale simulations. Sci Rep 7, 10926 (2017). https://doi.org/10.1038/ s41598-017-11259-0 Dr Stuart Knight 4 Haem group Fe2+ (ferrous) ion can bond with six ligands: four by nitrogen atoms of haem group Fig 4.19 fifth by attachment to specific histidine of Biochemistry and Molecular Biology Sixth globin Edition Despo sixth is available for the reversible attachment Papachristodoulou, of oxygen Alison Snape, William H. Elliott, and Daphne C. Exists in two forms Elliott Oxyhaemoglobin (oxyHb) Deoxyhaemoglobin (deoxyHb) Hydrophobic residues found in interior of globin prevents oxygen from oxidising the Fe2+ Fig 4.20 to Fe3+ Biochemistry and Molecular Biology Sixth Haem with Fe3+ is unable to bind oxygen Edition This is met-haemaglobin (metHb) Despo Papachristodoulou, Alison Snape, William H. Elliott, and Daphne C. Elliott Dr Stuart Knight 5 Haemoglobin has cooperative oxygen binding Oxygen saturation curve for Hb has sigmoid shape. There is an increase in the affinity of Hb for oxygen as the occupancy of Hb increases. This is cooperative binding and an example allostery. Allostery occurs when the binding of oxygen to one globin changes the shape of the binding site of another globin. Affinity of Hb for oxygen increases as the site occupancy increases of Hb increases. When pO2 is high (lungs) cooperative binding Fig 4.21 Biochemistry and Molecular Biology Sixth Edition allows Hb to be fully saturated. Despo Papachristodoulou, Alison Snape, William H. Elliott, and Daphne C. Elliot Dr Stuart Knight 6 Tense and Relaxed forms (states) of haemoglobin Each globin subunit can exist in two forms (states): T (tense) conformation with low affinity for oxygen. R (relaxed) conformation with high affinity for oxygen. Binding of oxygen to first subunit causes a conformational change in the subunit and causes conformational change of a second subunit to R state. For example: Binding of oxygen to α1 changes the conformation of α1. Binding of oxygen to α1 changes β1 to R state. β1 has increased affinity for oxygen. Dr Stuart Knight 7 Transportation of carbon dioxide in blood mechanism percentage Small amount is dissolved in blood Carbon dioxide freely crosses erythrocyte plasma membrane Dissolved in blood plasma 10 Most carbon dioxide is converted to carbonic acid by carbonic anhydrase in erythrocytes Converted to bicarbonate 60 H2O + CO2 H2CO3 Carbonic acid dissociates to bicarbonate Bound to Hb 30 Bicarbonate exported via Cl-/HCO3- channel Some carbon dioxide reversibly binds to Hb to form carbaminohaemoglobin Dr Stuart Knight 8 Bohr Effect Ability of haemoglobin to bind oxygen decreases as the [H+] increases. Ability of haemoglobin to bind oxygen decreases as the pH decreases. pH 7.4 50% Hb saturation : pO2 ~27 mm Hg pH 7.0 50% Hb saturation : pO2 ~40 mm Hg Physiologically important biological adaptation. In peripheral tissues at distance from lungs haemoglobin released most oxygen. Important that haemoglobin becomes fully desaturated. Increase in [H+] due to production of carbon dioxide. https://en.wikipedia.org/wiki/File:Oxygen- Haemoglobin_dissociation_curves.svg Dr Stuart Knight 9 Impact of [H+] on oxygen binding of haemoglobin in periphery Tissues generate ATP using oxygen and produce carbon dioxide. Carbon dioxide must be transported to lungs to be expired. Carbonic anhydrase produces carbonic acid (H2CO3) from carbon dioxide. pKa carbonic acid is 3.5 and at physiological pH it is dissociated into bicarbonate (HCO3-) and protons (H+). Increase in H+ drives Hb to release oxygen. pKa deoxyHb is 7.8 and at physiological pH high proportion is undissociated (HA) – it is binding Fig 4.29 (a) protons. Biochemistry and Molecular Biology Sixth Edition Despo Papachristodoulou, Alison Snape, William H. Elliott, and Daphne C. Elliott Dr Stuart Knight 10 Impact of oxygen saturation on H+ binding of haemoglobin in lungs In lungs high pO2 results in increase in oxyHb. oxyHb is more acidic (pKa 6.8) and at physiological pH it is dissociated. Increase in [H+] within the erythrocytes shifts the dissociation of bicarbonate towards carbonic acid. HCO3- + H+ H2CO3 Increase in carbonic acid shift the equilibrium of carbonic anhydrase towards carbon dioxide. H2CO3 H2O + CO2 Carbon dioxide is expired. Fig 4.29 (b) Biochemistry and Molecular Biology Sixth Edition Despo Papachristodoulou, Alison Snape, William H. Elliott, and Daphne C. Elliott Dr Stuart Knight 11 Fetal haemoglobin HbF and other globins in development During development different haemoglobins are expressed. Embryonic haemoglobin. HbE (Gower1) ζ2ε2 HbE (Gower2) α2ε2 HbE (Portland) ζ2γ2 Fetal haemoglobin. HbF α2γ2 Adult haemoglobin. HbA α2β2 Dr Stuart Knight 12 Fetal haemoglobin has a higher affinity for oxygen At birth: 20% HbA - 80% HbF. HbF has a higher affinity for oxygen than HbA. In placenta HbF is able to be saturated in preference to HbA. 50% HbA saturation : pO2 ~27 mm Hg. 50% HbF saturation : pO2 ~19 mm Hg. Fig 5.4 Norma Frizzell and George M. Helmkamp Jr Medical Biochemistry https://doctorlib.info/medical/biochemistry/7.html Dr Stuart Knight 13 BPG and oxygen binding of haemoglobin 2, 3 - bisphosphoglycerate (BPG) is allosteric regulator of oxygen binding of haemoglobin. BPG is generated by metabolising tissues. BPG binds to internal position of Hb but only in the T-state (deoxyHb). BPG is negatively charged and binds to a positively charged pocket. Binding of BPG lowers the affinity of Hb for oxygen. Oxygen binding curve of HbA in absence of BPG is similar to Mb. HbF has variation in sequence resulting in reduced BPG binding – increases the difference in affinities between HbF and HbA. Fig 4.28 Biochemistry and Molecular Biology Sixth Edition Despo Papachristodoulou, Alison Snape, William H. Elliott, and Daphne C. Elliott Dr Stuart Knight 14 Sickle cell anaemia Recessively inherited anaemia characterised by formation of sickle shaped cells. Inherited mutation of β-globin: glu 6 val. Acidic to polar substitution. Located on surface of haemoglobin. HbS. Sickle cell disease – two copies of mutant gene. Sickle cell trait / carrier – one copy of mutant gene. Substitution creates hydrophobic “sticky” patches on normally charged surface of the β- https://www.sciencemag.org/sites/default/files/styles/ article_main_image_-_1280w__no_aspect_/public/ globin. Sickle_Cell_Blood_Smear.JPG?itok=Xgfz21-X Dr Stuart Knight 15 HbS aggregation Conformation change in deoxyHb results in the alpha helix containing position 6 shifting to position more on the surface In HbA glutamic acid is stabilised by hydrogen bonds with aqueous environment In HbS to prevent valine being in an aqueous environment two haemoglobins aggregate together This aggregation continues to form polymers Dr Stuart Knight 16 Formation of sickle shaped cells Polymers of HbS disrupt the erythrocyte membrane and prevents the deformability resulting in sickle shaped cells Sickle shaped cells can be trapped in capillaries and lyse Dr Stuart Knight 17 Haemoglobin and other diseases low Fe2+ intake in diet may lead to anaemia. Hereditary haemolytic anaemia - Imbalance of globin chain production. α –thalassaemia : deficient α-globin production (reduced or absent). Excess β chains. In adult : HbH (β4) and fetus: Hb Barts (γ4). β -thalassaemia : deficient β -globin production (reduced or absent). Hereditary Persistence of Fetal Haemoglobin (HPFH). Dr Stuart Knight 18 Thank you Dr Stuart Knight [email protected] Henriette-Raphael House 1.17