1. 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 - 17000 daltons (monomeric) 153 amino acids Hemoglobin - 64500 daltons ( tetrameric)  -chain has 141 amino acids  -chain has 146 amino acids

2. Hemoglobin O 2 carrying capability Erythrocytes/ml blood: 5 billion ( 5 x 10 9 ) Hemoglobin/red cell: 280 million ( 2.8 x 10 8 ) O 2 molecules/hemoglobin: 4 O 2 ml blood: (5 x 10 9 )(2.8 x 10 8 )(4) = (5.6 x 10 18 ) or (5.6 x 10 20 ) molecules of O 2 /100 ml blood

3. A single subunit of Hemoglobin, an     tetramer

4. Myoglobin, monomeric

5. 3 o structure overlap: myoglobin,  -globin and  -globin  -Globin (blue)  -Globin (violet) Myoglobin (green)

6. Aromatic Heme

7. Iron in Hemoglobin binding O 2

8. Iron in Myoglobin binding O 2

9. Resonance in Iron binding O 2

10. Hemoglobin,     tetramer

11. O 2 binding: Hemoglobin & Myoglobin P 50 = 2 torr P 50 = 26 torr

12. O 2 transport capability, a comparison

13. Resting state vs exercise

14. O 2 Binding Changes 4 o Structure

15. 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.

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

17. R-state vs T-state Binding

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

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

20. 2,3-bisphosphoglycerate (2,3-BPG) The binding pocket for BPG contains 4 His and 2 Lys

21. Binding of bisphosphoglycerate

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

23. Loss of O 2 from Hemoglobin Carbamate: CO 2 combines with NH 2 at the N-terminus of globins

24. Chemical basis of Bohr effect

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

26. Combined Effects CO 2, BPG and pH are all allosteric effectors of hemoglobin.

27. CO 2 & Acid from Muscle

28. CO 2 & Hemoglobin Blood Buffering Metabolic oxidation in cells uses oxygen and produces CO 2. The pO 2 drops to ~20 torr and oxygen is released from incoming HbO 2 -. HbO 2 - Hb - + O 2 Release is facilitated by CO 2 reacting with the N- terminus of each hemoglobin subunit, by non-covalent binding of BPG and the Bohr effect.

29. Events at Cell sites The localized increase in CO 2 results in formation of carbonic acid which ionizes to give bicarbonate and H +. CO 2 + HOH H 2 CO 3 carbonic anhydrase H 2 CO 3 HCO 3 - + H + pKa = 6.3 The increase in [H + ] promotes protonation of Hb -. HHb Hb - + H + pKa = 8.2

30. Events at Cell sites The predominant species in this equilibrium at pH 7.2 is HHb. So, O 2 remains at the cell site, HHb carries a proton back to the lungs and bicarbonate carries CO 2. Charge stability of the erythrocyte is maintained via a chloride shift, Cl - HCO 3 -.

32. Events at Lung sites Breathing air into the lungs increases the partial pressure of O 2 to ~100 torr. This results in O 2 uptake by HHb to form HHbO 2. HHb + O 2 HHbO 2 Ionization of HHbO 2 then occurs and HbO 2 - carries O 2 away from the lungs. HHbO 2 HbO 2 - + H + pKa = 6.6 So, the predominant species at pH (7.4) is HbO 2 -.

33. Events at Lung sites The localized increase in [H + ] from hemoglobin ionization serves to protonate HCO 3 -. H 2 CO 3 HCO 3 - + H + pKa = 6.3 H 2 CO 3 CO 2 + HOH carbonic anhydrase The resulting H 2 CO 3 decomposes in presence of carbonic anhydrase and CO 2 is released in the lungs. Charge stability of the erythrocyte is maintained again via a chloride shift, HCO 3 - Cl -.

34. Sickle Cell due to Glu 6  Val 6