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MCP I > Unit III- Hemoglobin > Flashcards

Unit III- Hemoglobin Flashcards

1
Q

Life Needs Oxygen

A
  • most living organisms need oxygen to carry out basic metabolic functions
  • the rate of oxygen transport in tissues is inversely proportional to the square of the distance it must diffusion
  • this makes the diffusion rate of Oxygen through tissue thicker than ~1mm too slow to support life
  • the evolution of larger organisms required the aquisition of oxygen carriers and circulatory systems
  • hemoglobin is the predominant oxygen carrier in the circulatory system
  • myoglobin in an intracellular oxygen transport and storage protein
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2
Q

Oxygen transport the big picture

A
  • Atomosphere: 760 Torr
  • Blood in alveolar capillaries: 100 torr
  • Blood in muscle capillaries: 20 torr
  • cytoplasm of myocyte: 5 torr
  • Km of Cyt oxidase for O2- <1 torr
  • oxygen transport is downhill, we just need to speed up diffusion
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3
Q

Myoglobin folding

A
  • myoglobin is a small intracellular protein of 153 amino acids that is highly abundant in vertebrate muscle
  • Mb has a globular shape with 44 X 44 X 25 A.
  • the heme group is tightly wedged in a hydrophobic pocket formed between helices E and F
  • myoglobin functions in intracellular transport and temporary storage of oxygen needed for aerobic metabolism of muscle
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4
Q

Quaternary Structure

A
  • hemoglobin is a tetrameric protein with the quaternary structure alpha2beta2 (dimer of alphabeta protomers)
  • Hb has a globular shape with approximate dimensions 64 x 55 x 50 A
  • each alpha-globin and beta-globin subunit is bound non-covalently to a prosthetic group (heme). So the tetrameric Hb molecule contains four hemes

ApoHb- empty Hb tetramer consisting of alpha1beta1, alpha2beta2
HoloHb- is ApoHb + 4 Hemes

-4 hemes = 4 O2 binding sites

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5
Q

Assembly of hemoglobin

A
  • hemoglobin monomers first assemble into rather stable, rigid alphabeta heterodimers. Two of these dimers come together to form a tetramer. The association between two alphabeta dimers in the tetramer is loose and flexible
  • hemoglobin an switch between T (low affinity for O2) and R (high affinity)
  • this is the basis for the cooperativity resulting in the sigmoid binding curve
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6
Q

Heme: the prosthetic group

A
  • heme is a big heteroxycle with Fe coordinated in the center
  • Heme is Fe-protoprophyrin IX, which in turn is a porphyrin with the specific substituent side chains depicted
  • prophyrin is a heteroxyclic ring system- with four pyrrole rings linked by briding carbons
  • the nitrogens of each pyrrole faces inwards to the center, creating a metal chelating site where the Fe binds
  • four methyl, two vinyl, two proprionate substituent groups
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7
Q

deoxyHb

A
  • hemoglobin with no oxygen bound
  • the iron is 5-coordinate, coordinated by 5 nitogen atoms, four from pyrroles, one from the Proximal Histidine side chain
  • this iron has electronic state Fe (II) which is known as ferrous
  • the distal hisidine is too far away and there is an empty space on the distal side of Fe
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8
Q

O2 binds the heme iron from the side opposite the proximal histidine

A
  • O2 binds with one of its atoms providing the 6th ligand of Fe
  • the other atom of O2 forms a H-bond with the distal histidine, which thus increases the affinity for O2
  • in turn, O2 is bound between the Fe (II) and Histidine E7 also known as distal histidine
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9
Q

Binding of Oxygen to Heme

A

-oxygen binding to the heme group has dramatic consequences for the physciochemical properties of Hb. First of all O2 changes the absorption properties of Hb and consquently its color
DeoxyHb- dark red
OxyHb- brilliant scarlet

-small molecules like CO, NO, and H2S bind the heme with higher affinity than oxygen, acting as poisons

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10
Q

Hemoglobin nomenclature

A
  • apo/holo refers to whether or not the cofactor is present
  • ferro/ferri indicates reduced/oxidized iron
  • oxidation state refers to whether the Fe is Fe2+ (ferrous) or Fe3+ (ferric)
  • oxygenation state- whether oxygen is bound or not and is close to the coordination state
  • deoxyhemoglobin is ferrous. Ferric hemoglobin is called met-hemoglobin and does not function as oxygen carrier
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11
Q

Oxygen dissociation curves of Hb (and Mb)

A
  • O2 concentration in our circulatory system is:
  • ~20-30 torr in venous blood (depends on level of exercise)
  • ~100 torr in arterial blood
  • P50 is the pressure at which binding is half-maximal, or the pressure value at which 50% of the maximal O2 load has been release- 26 torr in hemoglobin, 2.6 torr in myoglobin
  • Mb hyperbolic curve, Hb sigmoidal curve (cooperative interaction)
  • Mb binds O2 under conditions in which Hb releases it
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12
Q

Cooperativity

A
  • Hb displays a characteristic sigmoidal binding curve. What does it mean?
  • The sigmoidal shape of this curve can be thought as the convulution of two molecular states: the high affinity of the R state, the low affinity of the T state
  • the two Hb states are in equilibrium and O2 binding drives the T R conversion
  • at high O2 concentration the dissociation curve is close to the R curve; as O2 concentration decreases, it approaches the T curve
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13
Q

Regulation of Hemoglobin in vivo

A
  • the cooperativity of O2 binding to Hb is a classical model for allosteric interaction
  • in the case of Hb, binding of O2 to one site increases the affinity of other binding sites on the same protein. Other small molecules that bind to Hb decrease the affinity for O2
  • the allosteric nature of O2 binding results from the peculiar quaternary structure of the tetramer which is eqilibrium between two forms (T and R as oxy)
  • positive effectors- O2
  • negative effectors- BPG, CO2, H+, and Cl-
  • myoglobin, which is a monomer, has no T/R transition and shows no allosteric effects
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14
Q

Allostery and Cooperativity

A

Allostery: refers to an effect of something happening at another site on the active site. The other site can be another active site in a multimeric protein

Cooperativity: a type of allostery in which what is happening at one site promotes the same thing happening at another identical site, as oxygen binding at one site increases the affinity of the other sites

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15
Q

Allosteric Regulation of Hemoglobin

A
  • positive effectors shift the O2 dissociation curve to the left
  • negative effectors shift the O2 dissociation curve to the right
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16
Q

BPG regulates O2 affinity of Hb

A
  • purified deoxygHb (stripped) has much greater O2 affinity than does hemoglobin in whole blood
  • this is due to the presence of a physiological inhibitor, known as BPG
  • deoxy Hb bound to BPG displays reduced affinity for O2, therefore BPG shifts to O2 dissociation curve to the right
17
Q

Physiological Role of BPG

A
  • BPG binds deoxyHb in 1:1 molar ratiowith Kd ~15 uM but binds only weakly to oxyHb. Because the concentration of BPG in erythrocytes is 4-8 mM, BPG is basically always bound to deoxyHb
  • the deoxyHb:BPG complex displays reduced affinity for O2, so at the pO2 present in capillaries of respiring tissues more O2 can be released
  • in the presence of BPG, Hb has a P50 ~26 torr
  • in the absence of BPG, Hb has much higher affinity (P50~3 torr)
  • in arterial blood the pO2 is 100 torr: Hb is 95% sat
  • in venous blood the pO2 is 20 torr: Hb is 43% sat
  • this means that in vivo (in the presence of BPG) Hb is a very efficient O2 carrier, which unloads ~52% of its O2 content passing through capillaries
18
Q

Structural Basis for BPG binding to deoxyHb

A
  • the crystal structure of deoxyHb bound to BPG reveals that one molecule of BPG binds per tetramer of Hb
  • one equivalent of BPG is bound at the tetramer interface, where it interacts with several lysins, histidines and N-termini protruding from the surface of B subunits
  • the presence of BPG stabilizes the deoxyHb state and decreases Hbs oxygen affinity by keeping it in the deoxy conformation
19
Q

Varying concentration of BPG in blood

A
  • individuals acclimated to high altitude were found to have altered BPG levels in their blood
  • increase BPG to squeeze more O2 out of the hemoglobin
20
Q

Bohr effect

A
  • ph Modulates the affinity of Hb (but not Mb) for O2
  • the conformational changes in Hb associated with O2 binding alter the pKa of several Hb side chains
  • oxygenation of Hb makes it a stronger acid
  • in the lungs: high pO2- higher pH (favors R state)- Hb has higher affinity for O2, more O2 is loaded
  • muscles: low pO2- lower pH (stablizes T state)- Hb has lower affinity for O2, more O2 is released
21
Q

CO2 regulates O2 affinity of Hb (but not Mb)

A
  • CO2 combines reversibly with the N-terminal amino groups of blood proteins to form carbamates
  • DeoxyHb binds more CO2 in this way than does OxyHb
  • in capillaries where theres low O2 and high CO2, CO2 stabilizes the T state of Hb stimulating the interconversion R and T the subsequent release of O2
  • H+ released from carbomates further reduces the affinity of OxyHb for O2, facilitating its release

-H+ and CO2 synergize to unload O2 in the capillary where O2 is needed

22
Q

Fetal hemoglobin

A
  • HbF has a composition alpha2gamma2 in which HbA’s Beta subunits are replaced by a subunit variant known as gamma
  • HbF has a higher affinity for O2 than maternal Hb
  • this is partially explained by the fact that deoxy-HbF has lower affinity for BPG
23
Q

Normal hemoglobins

A

-Hemoglobin A: alpha2beta2
predominant form of HB in erythrocytes

-Hemoglobin A2: alpha2delta2
this is a minor component of the Hb found in red cells after birth

  • Hemoglobin F: alpha2gamma2
  • fetal development
24
Q

Hemoglobin variants

A
  • 900 naturally occuring variants
  • 90% from single amino acid substitutions in one of the globin polypeptide chains
  • 300,000 individuals with serious hemoglobin disorders are born every year
  • 5% of the worlds population have an inherited variant hemoglobin
25
Q

Molecular Pathology of Hemoglobinopathies

A
  • changes in surface residues
  • changes in internally located residues
  • changes stabilizing methemoglobin
26
Q

HbS and molecular basis for Sickle Cell Anemia

A
  • single substitution Glu to Val at position 6 of chain beta
  • up to 10% of the African American population and as many as ~25% of the African population are heterozygotes for the gene for sickle-cell hemoglobin
  • homozygotes have sickle cell anemia in hemolytic anemia
  • individuals who carry one copy of the gene for HbS, usually have normal life, evolutionary selected protection against the disease
27
Q

Molecular Disease

A
  • Glu to Val mutation at position B6 causes HbS to aggregate and polymerize into rigid extended fibers that span the length of the cell
  • in the deoxyHbS fiber only one of the two Val6B per Hb molecule contacts a neighboring molecule, in this contact the Val side chain occupies a hydrophobic surface pocket on the B subunit of the adjacent molecule whose Val-6B does not make an intermolecular contact
28
Q

Other common varients of Hemoglobin

A
  • HbC- subunit composition alpha2 beta2. same residue mutated in HbS Glu is mutated to Lys
  • HbH- subunit composition B4. It binds oxygen very tightly and non-cooperatively, hence providing very inefficient oxygen delivery to tissues
  • HbBarts- subunits composition gamma4. Like HbH, it displays high affinity, non-cooperative binding to O2, with poor oxygen delivery properties
29
Q

Detection of abnormal Hb by native gel electrophoresis

A
  • Hb variants can be easily detected by non-denaturing gel electrophoresis
  • For instance HbS is less negatively charged than HbA, resulting in slower migration on native cellulose acetate gel

MCP I (24 decks)

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