1. Dr Gihan Gawish Hemoglobin

2. Dr Gihan Gawish Hemoglobin   Synthesized in RBC precursor cells: reticulocytes and erythroblasts   Synthesis is tightly controlled and dictated by the concentration of heme  Tetramer of 2  -globin and 2  -globin chains  Best described as a dimer of the heterodimer (  )

3. Dr Gihan Gawish Hemoglobin Structure In most humans, the hemoglobin molecule is an assembly of four globular protein subunits. Each subunit is composed of a protein chain tightly associated with a non-protein heme group. Each protein chain arranges into a set of alpha- helix structural segments connected together in a globin fold arrangementalpha- helixglobin fold

4. Dr Gihan Gawish Hemoglobin Structure A heme group consists of an iron (Fe) ion (charged atom) held in a heterocyclic ring, known as a porphyrin. The iron ion, which is the site of oxygen binding, coordinates with the four nitrogens in the center of the ring, which all lie in one plane. The iron is also bound strongly to the globular protein via the imidazole ring of the F8 histidine residue below the porphyrin ring. A sixth position can reversibly bind oxygen, completing the octahedral group of six ligands.

5. Dr Gihan Gawish HEMOGLOBIN VARIANTS TypeStructureComments HbA (95%) 2222 HbA 2 (4%) 2222 Functionally, this variant is indistinguishable from HbA Mutations in  -globin are without effect HbF (1% in adults – predominate form in the fetus during the 2 nd and 3 rd trimesters of pregnancy)  2  2 His 143 (  )  Ser (  ) Interaction with 2,3-BPG is weaker resulting in an increased affinity for O 2 and a greater stabilization of the R state. This allows for a more efficient transfer of O 2 from maternal to fetal hemoglobin

6. Dr Gihan Gawish INTERACTIONS WITH O 2 * Can bind up to 4 O 2 molecules * Binding of O 2 is cooperative: the binding of 1 O 2 influences the binding of another

7. Dr Gihan Gawish DEOXYGENATED VS. OXYGENATED HEMOGLOBIN  As deoxygenated hemoglobin becomes oxygenated, significant structural changes take place  the proximal hisitidine and its helix shift  one heterodimer rotates and slides relative to the other  existing noncovalent bonds are broken and replaced by new ones  Approximately 30 amino acids participate in the noncovalent (hydrogen and/or electrostatic) interactions between the 2 heterodimers  Interactions between the two heterodimers are stronger in the T (tense)-state = deoxygenated hemoglobin  These interactions are weaker in the R (relaxed)-state = oxygenated hemoglobin  The R-state has a higher affinity for O 2 than the T-state

8. Dr Gihan Gawish

9. DEOXYGENATED VS. OXYGENATED HEMOGLOBIN (CONT.)  The transition of hemoglobin from the T- to the R-state is not well-defined  Best explained as a combination of a sequential and a concerted model  It is unknown whether the  and  subunits differ in O 2 affinity and which subunit binds to (or releases) O 2 first.

10. Dr Gihan Gawish INTERACTIONS WITH ALLLOSTERIC EFFECTORS  Allosteric proteins are typically multisubunit proteins  Small molecules know as allosteric effectors bind to the protein at sites that are spatially distinct from the ligand binding site and exert either a positive or negative effect on ligand binding  These effects are accompanied by changes in tertiary and/or quaternary structure

11. Dr Gihan Gawish INTERACTIONS WITH ALLLOSTERIC EFFECTORS Hemoglobin is modified negatively (i.e. decreased affinity for O2) by a number of allosteric effectors including H+, CO2 and 2,3-bisphosphoglycerate (2,3-BPG) It is unknown whether the  and  subunits differ in O2 affinity and which subunit binds to (or releases) O2 first.

12. Dr Gihan Gawish INTERACTIONS WITH ALLLOSTERIC EFFECTORS (CONT.)  As the curve shifts from A to B the affinity for O 2 decreases  The effects of these molecules appears to be additive  Increasing temperature will also shift the curve to the right

13. Dr Gihan Gawish Bohr effect CO and H bind to hemoglobin (allosteric site), which changes conformation of molecule and changes binding site for O at tissues: CO binds to hemoglobin, decrease in affinity for O, allowing better delivery of O

14. Dr Gihan Gawish The Bohr Effect  Term that is used to describe the rightwards shift in the O 2 saturation curve with increasing H + concentration (decreasing pH)  N-terminal amino group of the  -chain and side chains of His 122  and His 146  are the residues most involved  These residues are more extensively protonated in the T-state. When hemoglobin binds O 2, protons dissociate. In acidic media, protonation inhibits O 2 binding. Lungs (high pO 2 )  Favors O 2 saturation  Forces protons from the molecule to stabilize the R-state Capillary Bed (Peripheral tissues) (lower pH) O 2 -saturated hemoglobin will acquire some protons, shift towards the T-state and release O 2 for tissue uptake

15. Dr Gihan Gawish The Bohr Effect

16. Dr Gihan Gawish Effect of CO 2 : increased pCO 2 in venous capillaries decreases the affinity for O 2 1. CO 2 reacts reversibly with the unprotonated N- terminal amino groups of the globin polypeptides to form carnation-hemoglobin 2. In peripheral tissues, carbamination (H 2 CO 3 ) followed by hydration/dissociation (H + + HCO 3 - ) generates additional protons available to participate in the Bohr Effect and facilitate CO 2 -O 2 exchange (more O 2 can be released)  Shifts the equilibrium towards the T-state thereby promoting the dissociation of O 2

17. Dr Gihan Gawish Transport and Removal of CO 2  Blood transports two forms of CO 2 to the lungs: carbamino-hemoglobin and H 2 CO 3 /HCO 3 - (carbonic acid- conjugate base pair) 1. Carbamino-hemoglobin: exposure to low pCO 2 results in the reversal of the carbamination reaction by mass action and O 2 binding is again favored. CO 2 is expelled by the lungs. 2. H 2 CO 3 /HCO 3 - : in the pulmonary capillaries RBC carbonic anhydrase converts H 2 CO 3 into CO 2 and H 2 0, which are expelled in their gaseous forms into the atmosphere

18. Dr Gihan Gawish Effect of 2,3-Bisphosphoglycerate  Byproduct of anaerobic glycolysis in the RBC  It is found at high concentrations (~4-5 mM) in RBCs nearly equal to the concentration of hemoglobin  Reacts with only deoxygenated hemoglobin in a positively charged cavity where the two  -subunits juxtapose - stabilizes the T-state  Its concentration is responsive to various physiological and pathological conditions. For example, when pO 2 is decreased, as in chronic tissue deprivation of O 2, the level of 2,3-BPG increases. This results in a stabilization of the T-state and further rightward shift of the curve facilitating O 2 release to the deprived tissues.  Usually the rightward shift of the O 2 saturation curve has an insignificant effect on the O 2 saturation in the lungs

19. Dr Gihan Gawish Genetics Mutations in the genes for the hemoglobin protein in a species result in hemoglobin variants.genes some of which cause a group of hereditary diseases termed the hemoglobinopathies in humans.

20. Dr Gihan Gawish Genetics The best known is sickle-cell disease, which was the first human disease whose mechanism was understood at the molecular level.sickle-cell disease A (mostly) separate set of diseases called thalassemias involves underproduction of normal and sometimes abnormal hemoglobins, through problems and mutations in globin gene regulation. thalassemiasgene regulation These diseases also often produce anemia