1. Biochemistry Sixth Edition
Berg • Tymoczko • Stryer
Chapter 7:
Hemoglobin:
Portrait of a Protein in Action
Copyright © 2007 by W. H. Freeman and Company

2. Erythrocytes (Red cells)

3. Hemoglobin and Myoglobin
These are conjugated proteins. A simple protein has only a polypeptide chain. A conjugated protein has a non-protein part in addition to a polypeptide component. Both myoglobin and hemoglobin contain heme.
Myoglobin daltons (monomeric) 153 amino acids
Hemoglobin daltons ( tetrameric) a-chain has 141 amino acids b-chain has 146 amino acids

4. Hemoglobin O2 carrying capability
Erythrocytes/ml blood: 5 billion ( 5 x 109 )
Hemoglobin/red cell: million ( 2.8 x 108 )
O2 molecules/hemoglobin: 4
O2 ml blood: (5 x 109)(2.8 x 108)(4) = (5.6 x 1018)
or (5.6 x 1020) molecules of O2/100 ml blood

5. A single subunit of Hemoglobin, an a2b2 tetramer

6. Myoglobin, monomeric

7. 3o structure overlap: myoglobin, a-globin and b-globin
a-Globin (blue)
b-Globin (violet)
Myoglobin (green)

8. Aromatic Heme

9. Iron in Hemoglobin binding O2

10. Iron in Myoglobin binding O2

11. Resonance in Iron binding O2

12. Hemoglobin, a2b2 tetramer

13. O2 binding: Hemoglobin & Myoglobin
P50 = 2 torr
P50 = 26 torr

14. O2 transport capability, a comparison

15. Resting state vs exercise

16. O2 Binding Changes 4o Structure

17. Allosteric Proteins There are two limiting models of allosterism:
Monod, Wyman & Changeux: Two State, concerted
Koshland, Nemethy & Filmer: One State, sequential
Allosteric effectors (modulators) bind to a protein at a site separate from the functional binding site (modulators may be activators or inhibitors)
Oxygen binding and release from Hb are regulated by allosteric interactions
Hemoglobin cooperativity behaves as a mix of the above two models.

18. Concerted, two state model
Monod, Wyman & Changeux

19. R-state vs T-state Binding

20. Sequential, one state model
Koshland, Nemethy & Filmer

21. Decreasing O2 affinity 2,3-bisphospho-glycerate (2,3-BPG)
Lowers the affinity of oxygen for Hemoglobin

22. 2,3-bisphosphoglycerate (2,3-BPG)
The binding pocket for BPG contains 4 His and 2 Lys

23. Binding of bisphosphoglycerate

24. The Bohr Effect Bohr Effect:
Lowering the pH decreases the affinity of oxygen for Hb

25. Loss of O2 from Hemoglobin
Carbamate:
CO2 combines with NH2 at the N-terminus of globins

26. Carbamate formation
Covalent binding at the N-terminus of each subunit

27. Combined Effects CO2 , BPG and pH are all allosteric effectors of
hemoglobin.

28. CO2 & Acid from Muscle

29. CO2 & Hemoglobin Blood Buffering
Metabolic oxidation in cells uses oxygen and produces CO2 .
The pO2 drops to ~20 torr and oxygen is released from incoming HbO2-.
HbO2- <===> Hb- + O2
Release is facilitated by CO2 reacting with the N- terminus of each hemoglobin subunit, by non-covalent binding of BPG and the Bohr effect.

30. Events at Cell sites
The localized increase in CO2 results in formation of carbonic acid which ionizes to give bicarbonate and H+.
CO2 + HOH <===> H2CO carbonic anhydrase
H2CO3 <===> HCO3- + H+ pKa = 6.3
The increase in [H+] promotes protonation of Hb-.
HHb <===> Hb- + H+ pKa = 8.2

31. Events at Cell sites
The predominant species in this equilibrium at pH 7.2 is HHb.
So, O2 remains at the cell site, HHb carries a proton back to the lungs and bicarbonate carries CO2 .
Charge stability of the erythrocyte is maintained via a chloride shift, Cl- <==> HCO3- .

33. Events at Lung sites
Breathing air into the lungs increases the partial pressure of O2 to ~100 torr.
This results in O2 uptake by HHb to form HHbO2.
HHb + O2 <===> HHbO2
Ionization of HHbO2 then occurs and HbO2- carries O2 away from the lungs.
HHbO2 <===> HbO2- + H+ pKa = 6.6
So, the predominant species at pH (7.4) is HbO2-.

34. Events at Lung sites
The localized increase in [H+] from hemoglobin ionization serves to protonate HCO3- .
H2CO3 <===> HCO3- + H+ pKa = 6.3
H2CO3 <===> CO2 + HOH carbonic anhydrase
The resulting H2CO3 decomposes in presence of carbonic anhydrase and CO2 is released in the lungs.
Charge stability of the erythrocyte is maintained again via a chloride shift, HCO3- <==> Cl-.

35. Sickle Cell due to Glu 6  Val 6

37. Binding relationships
The binding of O2 to myoglobin can be shown by the equilibriuim: Mb + O2 <===> MbO (1)
The dissociation constant for the loss of O2 is: [Mb][O2] Keq = KD = (2) [MbO2]
Define the fraction of sites, Y, occupied by O2 as: [MbO2] sites bound Y = = (3) [Mb] + [MbO2] total sites

38. Binding relationships
Substituting from equation (2) into (3): [MbO2] Y = = K [MbO2] K [MbO2] O O2
or: [O2] pO2 pO Y = = = K + O2 K + pO2 p pO2
Evaluating K at Y = 0.5 gives K = p50 for O2

39. Biochemistry Sixth Edition
Berg • Tymoczko • Stryer
End of Chapter 7
Copyright © 2007 by W. H. Freeman and Company