08 Hemoglobin - Hemoglobin Course Goals Addressed...

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Unformatted text preview: Hemoglobin: Course Goals Addressed: • Understand the Relationship between Macromolecular structure and function: Learning Objective: • Describe the factors that allow Hemoglobin to transport oxygen • Explain the Bohr effect and its role in oxygen transport • Predict the possible effects of mutations on hemoglobin structure and function. Cellular Requirement for O2 Proteins Fats Carbohydrates (Nutrients) ADP Catabolism (Oxidation) O2 Waste (CO2/Urea/etc.) ATP NADP + NADPH Intermediates Anabolism (Biosynthesis) Oxygen Transport LUNGS pO2 = 100 torr deoxyHb deoxyHb MUSCLE CELL deoxyHb O2 + 4e– + 4H+ 2H2O RED BLOOD CELLS MbO2 O2 Hb(O2)n deoxyMb pO2 = ~20-30 torr Hb(O2)n O2 • Oxygen has limited solubility in Blood and Cytosol –Use Oxygen Carriers O2 Myoglobin and Hemoglobin • Myoglobin (Mb) – Binds O2 tightly in muscle cells – Facilitates O2 diffusion into muscle cells – Stores O2 in tissues (in marine mammals) myoglobin not a good oxygen transport molecule • Hemoglobin (Hb) – Transports O2 from lungs to peripheral tissues (in erythrocytes) The Globin Fold 8 helices (A-H) and loops in between all globins bind heme The Heme Prosthetic Group • permanent, non-proteinaceous • Incorporated during folding • responsible for reversible O2 binding iron binds the oxygen • Fe2+ has 6 coordination sites • 4 with N of pyrrole rings, • 2 perpendicular to ring • 6th coordination site: none deoxyhemoglobin 5th coordination site is occupied O2 oxyhemoglobin with proximal His CO carboxyhemoglobin Myoglobin • Small Intracellular Protein in Vertebrate Muscle • Single polypeptide (153 aa) with one bound heme eoxyHb • Facilitate deoxyHb O2 + 4e– + 4H+ – O2 Diffusion in Muscle RED– BLOOD O2 StorageCELLS in aquatic mammals Hb(O2)n Figure 7-1 MUSCLE CELL MbO2 deoxyMb pO2 = ~20-30 torr Hb(O2)n O2 2H2O O2 Myoglobin – Diffusion/Oxygen Storage! Fractional Saturation of Mb depends on: the binding constant of Mb for O2 the concentration of O2 (pO2) lower Kd = higher affinity for oxygen binding pO2 in tissue ~ 4 kPa KD = p50 = 0.4 kPa pO2 in lung ~ 13 kPa pO 2 p50 pO 2 Hemoglobin (Hb) a2 b1 • present in erythrocytes • makes blood look red • 34% of weight is Hb Different Hb subtypes: b2 • Hb A (adult): • two (141 aa) and two (146 aa) subunits • arranged as a pair of identical subunits (2 subunits) • Hb F (fetal): two and two chains a1 Each subunit has 1 heme, which binds 1 O2 O2 each hemoglobin molecule has 4 hemes that can bind 1 o2 each Lehninger, Figure 7-5, 7-6 Function of Hemoglobin: Oxygen Transport • O2 binding in lungs • O2 release in tissues LUNGS pO2 = 100 torr deoxyHb deoxyHb MUSCLE CELL deoxyHb O2 + 4e– + 4H+ 2H2O RED BLOOD CELLS MbO2 O2 Hb(O2)n deoxyMb pO2 = ~20-30 torr Hb(O2)n O2 O2 Hemoglobin and Myoglobin Bind Oxygen differently Myoglobin Fraction Saturated (θ) 1 0.8 p50 0.6 0.4 Hemoglobin myoglobin has a much smaller p50 than hemoglobin so they have different affinities for oxygen: helops with the function of hemoglobin - 0.2 0 0 20 40 60 pO2 (torr) 80 100 Hb has evolved to transport O2 Fraction Saturated (θ) pO2 In Tissues pO2 In Lungs 1 Θ = 0.98 0.8 Δ = 38% p50 0.6 Θ = 0.6 0.4 almost fully saturated in the lungs but much less saturated in the tissues (it was released into the tissues) 0.2 0 0 20 40 60 pO2 (torr) 80 100 Hb gains cooperativity by switching between 2 states T state (Low Affinity) R state (high affinity) subunits coordinate with each other to faciliatate from one state to another Lehninger Figure 7-10 Two Models of Cooperativity The Concerted Model The Sequential Model All or nothing mechanism Subunits switch one at a time T R Hb follows a little of both T Lehninger, Figure 7-14 R Movements of the Heme and the F Helix During the T —> R Transition blue in t state r in red state deoxy form: iron bent out of ring when oxygen binds to iron the heme is flattened out and the histidine is also pulled down along with the helix and the rest of the polypeptide etc etc Figure 7-8 Local structural changes around Heme are communicated to the rest of Hb By Janet Iwasa, Local changes around the Heme due to oxygen binding Structural changes within Hb when oxygen binds General structural rearrangements between T and R state T vs R State (1) Change at interface between 1 2 and 2 1 (2) R state is more compact, and relaxed (3) T state has additional salt bridges, which makes it more tense (4) In R state individual O2 sites have higher affinity for O2. - better Fe-O2 bond length - fewer steric repulsions associated with oxygen binding. Without cooperativity Hb could not efficiently transport oxygen cooperativity - ability to switch betwen the T and R state R state = higher affinity for oxygen Fractional Saturation (θ) 1 Tissues Lungs only r state = would bind oxygen in the lungs but wouldnt release enough oxygen in the tissues R state Hb 0.5 T state 0 pO2 only t state = would never bind enough oxygen in the lungs to deliver to the tissues When the partial pressure of O2 in venous blood is 30 torr, the saturation of myoglobin with O2 is ______ while the saturation of hemoglobin with O2 is ______. A) B) C) D) myoglobin has higher affinity for oxygen (so lower Kd) so will have a higher fractional saturation 0.55, 0.91 0.91, 0.55 2.8 torr, 26 torr 0.91, 0.97 Cooperativity is measured by the Hill coefficient (HC): HC greater than 1 is for positive cooperativity, less than 1 for negative cooperativity, and 1 for non-cooperative systems. What is the HC for Hemoglobin? A. B. C. D. 3 1 0 -1 MCAT Hemoglobin Variants Table 7-1 destabilize T state = will favor R state = increased affinity for oxygen = not as much oxygen released into tissues Ligand Binding can affect Protein Function • Allosteric regulation – 1 regulator binding affects binding of a ligand – Homotropic vs heterotropic – Positive vs Negative homotropic = actual ligand/substrate of the protein affects the binding of other molecules of that same ligand/substrate to other parts of the protein heterotropic - some other moelcule binds to protein and affects a different ligand binding to its site • Positive Cooperativity – 1 substrate bound = higher affinity forligand more substrates binds to binding site and increases the probability of a ligand – Concerted vs Sequential binding to another binding site – Positive homotropic allosteric regulation Allosteric regulation of protein function homotropic- once one is bound, the binding site is altered to increase the affinity for that molecule to increase binding homotropic, positive (= cooperative binding) -can also be negative heterotropic, negative -can aslo be positive hetereotropic - different molecule binding to a different site and this binding changes the shape of the orginal binding site The Bohr Effect chemicals that bind to Hb and decrease Hb’s ability to bind oxygen • H+ and CO2 are negative, heterotropic modulators of Hb so Hb is affected by pH • metabolizing tissue: H+ and CO2 accumulate bind to Hb and lower the affinity of Hb for O2 Hb releases O2 lower pH and higher Co2 • lungs: CO2 and H+ dissociate from Hb increases the affinity of Hb for O2 Hb binds O2 lower Co2 and higher pH • increase the efficiency of Hb as O2 transporter pH Dependence of O2 Binding to Hb Bohr effect Mechanism of Bohr Effect Protonation of side chains His-146+ forms salt bridge with nearby Asp-94 stabilizes low affinity T-state changes in pH change the protonation state of amino acids O2 is released as pH drops salt bridge stabilizes t state and it can only happen when one of the pairs to be protonated Roles of Hemoglobin and Myoglobin in O2 and CO2 Transport High pH (7.6) Low [CO2] Figure 7-12 Low pH (7.2) High [CO2] Oxygen triggers Hb to switch from its low affinity (T) state to its high affinity (R) state. What kind of allosteric effector is oxygen relative to Hb? activator - positive effector; inhibitor = negative effector A. Heteroallosteric; positive effector B. Homoallosteric; inhibitor C. Heteroallosteric; negative effector D. Homoallosteric; activator The major focus of oxygen transport in the blood compartment is the hemoglobin contained in red blood cells. In contrast, the carriage of carbon dioxide by the blood is predominantly in the form of: A. B. C. D. Dissolved gas Hemoglobin-bound gas Albumin-attached gas Bicarbonate ion MCAT 2,3-BPG is a negative regulator of Hb binds in central cavity in Hb; can only bind to T state and stabilizes it in the T state BPG binds to the positively charged central cavity of Hb By Janet Iwasa, T R pushes out BPG when it gets to the lungs and there is now enough oxygen, it is triggered to switch back to the R state and pushes BPG out because the central cavity is too small BPG allows for release of O2 No BPG pO2 In Tissues 5mM BPG pO2 In Lungs Oxygen transport at high altitude pO2 In Tissues hemoglobin is not fully saturated in the lungs pO2 In Lungs At 10,000 FT At Sea Level Fractional Saturation ( ) 1 0.8 39% 32% 0.6 0.4 0.2 0 0 20 40 60 pO2 (torr) 80 100 Increased 2,3-BPG on oxygen delivery pO2 In Lungs pO2 In Tissues At 10,000 FT 1 Fractional Saturation ( ) 0.92 0.8 0.89 32% 39% 0.6 0.6 increase BPG: bind a little oxygen less in the lungs but release more oxygen in the tissues 0.5 5mM BPG 0.4 8mM BPG 0.2 0 0 20 40 60 pO2 (torr) 80 100 FYI: Sickle Cell Anemia THIS SLIDE AND FURTHER WERE NOT COVERED IN LECTURE SO PROBABLY DONT NEED TO KNOW Glu ——> Val (aa 6 of -chain) -surface Val can bind to hydrophobic pocket on other Hb molecules Forms Long Fibers of Hb -Intermolecular hydrophobic interactions FYI: Erythrocytes: Elongated Fibers deform red blood cells giving the characteristic sickle shape. Normal Figure 7-17a Sickled FYI: Overlap between regions of Malaria and prevalence of the Sickle-Cell Gene: Relationship is unclear. Theory: malaria parasite completes life cycle in red blood cells. Sickled red blood cells are recycled faster, potentially clearing the infection before it can take hold. Figure 7-20 ...
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