BICH 410: EXAM 2
79 Cards in this Set
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What is myoglobin?
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An intracellular protein found in muscle tissue.
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What is the function of myoglobin?
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To facilitate oxygen transport in rapidly respiring muscle tissue.
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How does myoglobin affect the solubility of oxygen in muscle tissue?
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- It increases the effective solubility of oxygen in muscle tissue.
- Helps facilitates oxygen diffusion.
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What is myoglobin's composition?
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Single polypeptide chain of 153 residues in 8 a-helices (a-helices are labeled A-H)
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How is oxygen bounded by myoglobin?
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By a heme.
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What is a heme?
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A porphyrin that consists of 4 pyrole rings linked by methylene bridges.
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How is metmyoglobin (MetMb)/ methemoglobin (MetHb) formed from regular myoglobin and hemoglobin?
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When the Fe(II) ferrous state central iron in the protophyrin is oxidized into Fe(III) ferric state.
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What happens to oxygen when it is bounded to metmyoglobin?
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The free heme in the solution will readily bind to oxygen, but the oxygen quickly oxides it into the ferric state.
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What is the binding arrangement of the iron atoms of the heme? Is the the same in both the ferrous or ferric state?
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In BOTH the ferric or ferrous state, the iron atoms prefer to bind six ligands in an octahedral geometry.
- 4 of the ligands are the nitrogen atoms of the 4 pyrrole rings of the heme
- 5th ligand comes from histidine F8
- 6th ligand occurs when oxygen binds to the ferrous heme/ water b…
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What is oxymyoglobin?
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It is the oxygenated heme of the myoglobin.
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How is the oxygen bounded to the heme structurally?
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60 degrees to the plane of the heme. Same side of the plane is His-E7 => makes the oxygen binding site a sterically hindered region.
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What is deoxymyoglobin?
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It is the unoxygenated heme of myglobin.
- in this form, the 6th coordination site is vacant
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What is the affinity of the free heme in solution for CO compared to O2?
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It has 25,000 times more affinity for CO than O2.
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What is the affinity of the heme in myoglobin/hemoglobin for CO compared to O2?
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CO only binds 250 times greater than O2
- because the His-E7 forces the CO molecule to tilt away from the preferred perpendicular alignment with the plane of the heme.
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How is oxygen bounded to the deoxymyoglobin structurally?
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- Ferrous iron atom has five ligands and lays 0.55 A above the plane of the heme towards His-F8 (gives the iron porphyrin the complex dome shape)
- When oxygen binds, the iron is pulled back into the plane of the heme such that it is only 0.26 A above the porphyrin
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What is the binding equilibria oxygen binding?
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Mb + O2 <=> MbO2
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Dissociation constant K =
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K = ([Mb][O2]) / [MbO2]
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What is fractional saturation (YO2)?
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The fraction of O2-binding sites occupied by O2.
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YO2 (fractional saturation) =
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YO2 = [MbO2]/ ([Mb] + [MbO2])
YO2 = [O2] / (K + [O2])
YO2 = pO2/ (K + pO2)
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What is p50?
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The value of pO2 when YO2 = 0.5
The partial pressure when half of the myoglobin binding sites are occupied with oxygen.
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YO2 (in terms of p50) =
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YO2 = p50/ (K + pO2)
YO2 = pO2/ (p50 + pO2)
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What is the fraction of unbound site in myoglobin?
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1 - YO2
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What is the ratio of fractional saturation to the free myoglobin?
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YO2 / (1 - YO2) = pO2 / K
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What is the Hill plot?
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A graph of log Y/(1-Y) versus log pO2
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What is the Hill Coefficient?
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- the midpoint of binding
- the slope of the hill plot at the point of log (Y/1-Y) = 0
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What is the Hill Coefficient for Myoglobin?
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1
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Hill coefficient = 1 means what?
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The oxygen atoms bind independently of each other.
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What is hemoglobin?
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It is a compact globular protein.
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What is the components of hemoglobin?
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- It is a tetramer
- four polypeptide chains
- each subunit contains a heme allowing hemoglobin to bind four oxygen atoms
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What type of tetramer is an adult hemoglobin?
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a2B2 - type tetramer
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What are the two different types of subunits in hemoglobin?
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alpha and beta
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How is the beta subunit of hemoglobin compared to the structure of myoglobin?
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- 7 amino acid residues shorter than Mb 153
- its last helix H is shorter than myoglobins
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How is the alpha subunit of hemoglobin compared to the structure of myoglobin?
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- 12 amino acid residues shorter
- has a shorter H helix
- lacks helix D
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Why is the tetrameric quarterinary structure of hemoglobin important to its function?
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when a molecule of oxygen binds to heme in Hb, the heme iron is drawn into the plane of the the porphyrin ring => sets off chain of conformation events
- dramatically enhancing the affinity of the heme for oxygen
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What happens to deoxyhemoglobin when exposed to O2?
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It shatters due to the change in the structure that occurs upon oxygen binding.
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What is the structure of hemoglobin like?
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- highly symmetrical and spherical
- hemes are located in the clefts between the E and F helices and are exposed to the aqueous solvent
- heme groups are far apart
- the closed two hemes are between a1 and B2 or a2 and B1 are separated by 25 A
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Describe the subunit interactions in hemoglobin.
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- occurs between a1 and B1(or a1 and B1) interface consisting of 35 residues ;contacts involve helices B, G and H and the GH corner; important for subunit packing
- occurs between a1 and B2 (or a2 and B1) interface consists of 19 residues; these contacts are called sliding contact ; inv…
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What is the binding curve for hemoglobin?
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It is sigmoidal shaped.
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What is the binding curve for myoglobin?
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It is hyperbolic shaped.
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Myoglobin binds oxygen under the condition of what?
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when Hemoglobin releases the oxygen.
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What is the Hill Equation?
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- it describes the degree of saturation of a multisubunit protein as a function of the ligand concentration
Ys = [S]^n / (K + [S]^n)
Ys: fractional saturation
S: ligand S
n: number of subunits (related to the degree of cooperativity among interacting ligand binding sites)
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Hill constant (n)
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- increases with degree of cooperativity of a reaction and provides a way of characterizing a ligand binding reaction
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Hill constant (n) =1
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- myoglobin - hyperbolic binding curve
- binding is NONCOOPERATIVE
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Hill constant (n) > 1
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- positively cooperative
- binding increases the affinity of the protein for further ligand binding
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Hill constant (n) < 1
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- negatively cooperative
- ligand binding decreases the affinity of the protein for subsequent ligand binding
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Ys / (1 - Ys) =
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- ratio of the fractional saturation to the free protein
- Ys / (1 - Ys) = [S]^n / K
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Why must the shift to the R-state must occur simultaneously at both the a1-B2 and a2-B1 interfaces?
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Due to the inflexibility of the a1B1 and the a2B2 interface.
- no one subunit can independently exist in the R-state
- no one aB dimer exist in the R state
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What interfaces can change upon oxygenation?
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a1-B2 and a2-B1
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Describe the transition from the T to the R-state?
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It requires a quaternary shift of the a1C-B2FG contacts one turn up the a1C-helix; the sliding contact shift one turn up the C-helix during the quaternary shift from the T to the R state.
- Result in a lot more sliding contacts
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How are the two states stabilized?
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- R-state is stabilized by oxygen binding
- when oxygen is not present, T-state is more stable => salt bridges electrostatically stabilize the T-state
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Where does the energy that cause the T-> R transition come from?
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The formation of the Fe-O2 bond.
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What is the equation for the bicarbonate buffer system of blood plasma?
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H2CO3 <=> H+ + HCO3-
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What is the pKa of carbonic acid at 37 degrees Celsius?
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3.57
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What is the physiological pH?
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7.4
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At the pH of 7.4, what is the concentration of H2CO3?
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It is miniscule
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Why does the bicarbonate system works well?
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- The critical level of the carbonic acid is maintained by equilibrium with dissolved CO2 gas produced in the tissues and is available as gaseous CO2 in the lung.
- The gaseous carbon dioxide from the lungs and tissues is dissolved in the blood plasma designated by CO2 and is hydrated t…
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What is the three equilibriums of the bicarbonate buffer system?
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1. H2CO3 <=> H+ + HCO3- (in blood of capillaries)
2. CO2(d) + H2O <=> H2CO3 (in blood of capillaries)
3. CO2 (g) <=> CO2 (d) (in alveoli of lungs)
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Net: CO2(g) + H2O <=> H+ + HCO3-
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What happens to the bicarbonate buffer system when you exercise?
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you generate H+ => drives equilibrium towards carbonic acid formation
- increase [CO2d]
- exhale more [CO2g]
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What happens to the bicarbonate buffer system when you hyperventilate?
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- increase [O2]
- decrease [CO2g]
- decrease [CO2d]
- decrease carbonic acid
- decrease H+ concentration
- decrease bicarbonate concentration
- increase in blood pH
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What happens to most of the Co2 produced int he muscles and tissues?
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They diffuse through the tissues to the plasma in the CO2(d) because of the slow rate of bicarbonate formation.
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What is carbonic anhydrase?
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- An enzyme found in erythrocytes.
- It catalyze the net reaction of the first two equilibria of the bicarbonate buffer system
- the enzyme catalyzes at the diffusion limit = the rate determining step is the diffusion of CO2 (d) to the enzyme
- this enzyme prevent high [CO2g], which wo…
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What is the Bohr Effect I?
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- On binding oxygen, the conformational change makes the hemoglobin a slightly stronger acid. It release protons upon binding oxygen.
- Conversely increasing the pH stimulates Hb to bind oxygen.
Hb(O2)nHx + O2 <=> Hb(O2)n+1 + xH+
n=1,2,3 x=0.6 under physiological conditions
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What is the Bohr Effect II?
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In the capillaries, pO2 is low, the H+ generated by bicarbonate formation is taken up by the Hb
- Binding of protons decreases the affinity for O2 => hemoglobin unloads its oxygen => more bicarbonate formation
- Conversely in the lungs, pO2 is high, oxygen binding by Hb releases the Bo…
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What is the major contributor to the Bohr effect?
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His-B146
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What is BPG (formerly DPG)?
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D-2,3-Bisphosphoglycerate
- it binds to hemoglobin and promotes oxygen release by stabilizing the deoxyHb (T) form
- 1 BPG binds per hemoglobin tetramer
- has a net charge of -5 at physiological pH
- binding site is located within the central cavity of Hb between the four subunits
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What does BPG do tho the saturation curve?
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- shifts the saturation curve to the right
- facilitates oxygen delivery
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Why do marathon runners train at high altitude?
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oxygen concentration is low => elevated BPG levels
- obtain high BPG blood concentration so you can use oxygen more efficiently and have a distinct competitive advantage at sea level
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How is fetal hemoglobin different from adult hemoglobin?
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- two gamma chain instead of two Beta chains (a2v2)
- gamma chain (histidine is substituted with a serine)
- cannot electrostatically interact with BPG => BPG binds much weaker to hemoglobin
- greater affinity for O2
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What is the T-state of hemoglobin?
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the quarterinary conformation of deoxyHb
- the ligand used to induce the state does not matter
- (i.e. H+, BPG, CO2)
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What is the R-state of hemoglobin?
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- the quarterinary conformation of oxyHb
- the ligand used to induce the state does not matter
- (i.e. O2, CO, CN-, NO)
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What are hemoglobinopathies?
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Genetic disorders caused by the synthesis of abnormal globin chains.
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Sickle Cell Anemia
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- most common and severe hemoglobinopathy
- caused by a single point mutation of the sixth amino acid of the B-subunit from a a glutamate to a valine; this residues is the third amino acid in the A helix
- HbS 6(A3)BGlu->Val
- sickle like shaped erythrocytes occurring at low oxygen co…
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Mutations that decrease the stability of hemoglobin
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decrease in stability result in increased rates of the degradation of hemoglobin => cause RBC to lyse open HEMOLYTIC ANEMIA
1/ Hb Savannah 24(B6)B Gly->Val
2/ Hb Bibba 136(H19)a Leu->Pro
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Mutations that effect heme binding
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1/ Hb Bristol 67(E11)B Val->Asp
2/ Hb Sydney 67(E11)B Val->Ala
3/ Hb Hammersmith 42(CD1)B Phe-Ser [heme loss->hemoglobin become unstable->severe anemia]
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Mutations that effect the oxidation state of the iron
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Only heterozygous individuals are found. Homozygous individuals never observed (fatal); ferric iron makes blood choclate brown in color and makes the skin bluish
1/ Hb (Iwate) 87(F8)a His-> Tyr
2/ Hb Hyde Park 92(F8)B HIs-> Tyr
3/ Hb Boston 58(E7)a His-> Tyr
4/ Hb Saskatoon 63(E7)B Hi…
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Mutations that favor the R state
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These mutations increase the affinity of hemoglobin for oxygen by eliminating a hydrogen bond or salt bridge or other stabilizing interaction of the T-state
1/ Hb Chesapeake 92(FG4)a Arg-> Leu
2/ Hb Philly 35(C1)B Tyr-> Phe
3/ Hb Yakima 99(G1)B Asp -> His
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Mutations that stabilize the T state
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T-state causes lower oxygen affinity
Hb Kansas 102(G4)B Asn-> Thr
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Mutations that affect BPG binding
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1/ Hb Syracuse 143(H21)B His->Pro [eliminate one of the electrostatic interactions and decreases the affinity of hemoglobin for BPG -> shifts equilibria to favor the R state-> high oxygen affinity)
2/ Hb Shepherd's Bush 74(E18)B Glu->Asp [add negatively charged aspartate -> repulses BPG …
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Mutations that affects the Bohr effect
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1/ Hb Cowtown 146(HC3)B His->Leu [T->R-state -> diminish Bohr effect -> increase oxygen affinity]
2/ Hb McKees Rocks 145(HC2)B Tyr-Stop [prematurely stops B-subunit before His146 -> diminish Bohr effect -> increase oxygen affinity]
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