Hemoglobin and Oxygen Delivery Flashcards
Describe structure of Hemoglobin
-
4 proteins = globins, aka globin chains;
- 8 helices interrupted by turns
- 2 α type and 2 β type, arranged as 2 αβ dimers
- 4 heme prosthetic groups, one bound to each globin
- Globin chains within a dimer held together by hydrophobic bonds
- Dimers held together by ionic bonds
- Flexibility between the two dimers
Globin proteins = globin chains. Each has 8 helices, separated by turns. Heme binds in specific crevice between two helices, one for each globin.
HB is described as a tetramer made up of two dimers. The bonds between the two globins within a dimer are very stable, but the bonds between the dimers can be easily broken and reformed. This provides for flexibility, and is an important feature that allows HB binding affinity for oxygen to change in different conditions.
Structural importance of globin chain in Hb
Heme binds in crevice between two helices of each globin
2 α type = ζ and α; 4 β type = ε, γ, δ and β.
Developmental regulation is controlled by differentiation-specific transcription factors
Genes are transcribed only in a narrow stage of development in the 5-7 days from proerythroblast to enucleation
mRNAs are very stable. Translation continues after enucleation.
Notes:
- The globin genes are developmentally expressed, both in terms of cell differentiation in hematogenesis and in development of the person.
- Once the rbc become enucleated, regulation of globin synthesis is all at the translational level. Globin proteins are also quite stable.
Developmental regulation of globin chain genes
Hb gower = epsilon + zeta, expressed in the embryonic yolk sac. Alpha and gamma expressed in the fetal liver, then (by ~5 wks gestation) mostly in the bone marrow. After birth, synthesis of the gamma chain declines, while beta increases. By the end of the first year, most of the HbA = alpha+beta, andf HbF (alpha + gamma) is only a small percentage.
Notice that after 3 months gestation, alpha is the only alpha type hemoglobin. It’s a good thing that it’s expressed from 4 genes, but if there’s a problem with two or more, that causes alpha thalassemia, and the symptoms are apparent in utero. Defects in beta globin (Beta thalassemia or sickle cell) doesn’t present until after six months old, when gamma chains have decreased.
Delta chain not shown. Its expressed at low levels in both fetus and adult.
Hemoglobin F (HbF,α2γ2)
γ globin expressed only at low levels after ~6 months of age
- <1% total Hb in adults
- Found in small number of rbc called F cells
Higher levels of HbF can mitigate symptoms of β globin defects or deficiencies
HbF has higher affinity for O2 than HbA
Notes:
HbF has to pick up oxygen from mothers circulation, so it makes sense that it has to have a higher affinity than the adult form. The difference is due to the different amino acid sequence of the gamma chains which makes them less susceptible to a type of allosteric regulation (2,3-BPG).
In Sickle cell anemia, or beta thalassemia, when there’s a problem with the beta chain, the percentage of HbF increases, and people who have higher levels of HbF have fewer symptoms than those with lower levels. Hydroxyurea, a drug used to treat sickle cell, increases the amount of HbF.
What are the mechanisms used to coordinate heme and globin synthesis in erythroblasts?
- Heme synthesis coordinated with iron availability
- ALAS2 has iron response elements (IRE) in 5’ UTR
- Heme/hemin regulate Fe availability
- Heme synthesis coordinated with globin synthesis
- TS of ALAS2 is under control of same transcription factors that regulate globin synthesis
- Erythropoietin increases TS ALAS2 and αand β globin
- Heme increases TS of globins and stabilizes their mRNAs
- Low levels of heme activate a kinase that causes inhibition of translation
How can normal and abnormal forms of Hb found in blood samples be separated?
ELECTROPHORESIS
- The various forms of hemoglobin have similar size and structure, but can be separated by electrophoresis based on their different charges
- Top figure represents findings expected in a normal adult blood sample.
- Bottom shows standards for normal and some abnormal forms
Where does iron bind in Hb?
The iron binds:
- 4 nitrogens in the 4 pyrrole rings of protoporphyrin IX
- A histidine amino acid in the globin protein
- +/- oxygen
Under normal conditions, the ferrous (Fe2+) iron becomes oxygenated (binds O2 reversibly), but not oxidized (to Fe3+)
Oxidized Hb (Fe3+) = metheme, can’t bind O2
Describe the binding of heme to globin
The Fe2+ of heme binds to the globin chain via the proximal histidine on the F helix
The distal histidine on the E helix helps to stabilize the interaction of O2 with Fe2+
The distal histidine helps prevent oxidation of Fe2+ to Fe3+ and reduces Hb affinity for CO
Note:
- Remember that the proximal and distal histidines are just amino acids that are part of the globin protein.
Describe the conformational changes in Hb secondary to oxygen binding
In the deoxy state (T form) :
- Fe is puckered out from center of heme.
- The heme plane has a slight dome shape
- The F helix of the globin chain is not perpendicular, but at a slight angle
Transition:
- O2 binds Fe 2+ from opposite side of plane from globin
- Pulls Fe2+ down into center of plane, creating strain
Oxygenated state (R form):
- Movement of the globin chain relieves strain
- These changes are transmitted to the rest of the Hb molecule
Movement of the globin chain breaks some of the ioinic bonds between the alpha-beta dimers. This makes it easier for the next O2 to bind because there’s less strain to overcome.
In R form in Hb, two αβ dimers rotate __ degrees
~15 degrees
What causes transition of Hb from T-form to R-form?
Binding of O2
T form = tight, taut. Lots of ionic bonds between the dimers. Lots of strain to be overcome, so affinity for oxygen is low.
R form = relaxed. Fewer bonds between the dimers, more flexibility, less strain to overcome, so higher oxygen affinity.
Compare T and R form
T form = tight, taut
- Deoxygenated
- Low affinity
- Lots of ionic bonds between the dimers
R form = relaxed
- Oxygenated
- High affinity
- Fewer bonds between the dimers
The affinity of Hb for O2 increases > 300X in transition from T form to R form
Saturation of Hb changes with _______ of oxygen
Partial Pressure
The amount of oxygen bound to Hb (% saturation) changes as it circulates between lungs and tissues.
Hb is fully saturated in the lungs where partial pressure of oxygen is 100 mmHg (Hb in R form, affinity high). As it goes toward the peripheral tissues, partial pressure of O2 drops to 10 mmHg (Hb in T form, affinity low), unloads the O2.
Describe the O2 dissociation curve:
Sigmoidal curve suggests allosteric regulation
O2 is an allosteric activator
Allosteric inhibitors decrease Hb affinity for O2in the tissues
- 2,3 bisphosphoglycerate (2,3-BPG)
- H+
- CO2
None of the allosteric regulators has much effect in the lungs, where [O2] is high
Notes:
Myoglobin has only one globin chain with one heme, found in muscle cells, doesn’t move around the body. It binds O2 released from Hb and stores it until the muscle needs it. The saturation of Mb doesn’t change much with the partial pressure of O2. Mb is half saturated (p50) when the partial pressure of O2 is 1 mm Hg.
The p50 for Hb is ~26 mmHg, which is in the range found in the tissues. The curve is sigmoidal, suggesting allosteric regulation. We already know that binding of the first O2 affects the affinity for the second, so O2 is itself an allosteric activator of O2 binding to Hb. In addition, there are several allosteric inhibitors, and each of them is most effective at partial pressures found in the tissues, while having little effect in the lungs. This is very important because it helps Hb unload O2 in the tissues.
What’s the importance of sigmoidal curve?
A 10 mm Hg drop in oxygen tension
- 100 to 90 – little effect on hemoglobin saturation
- 60 to 50 – a much larger effect
Small decrease in lung function doesn’t have a major effect on tissue oxygenation
Larger effect in the middle of the curve allows more efficient unloading in tissues
Notes:
The O2 dissociation curve is very steep in the range of pO2 found in tissues. This means that minor changes in lung function don’t have much effect oh Hb loading of O2, but even small changes in pO2 in the tissues Hb unload O2.
2, 3-Bisphosphoglycerate (2,3-BPG)
- Made from a glycolytic intermediate
- Equimolar with Hb in rbc
- Binds deoxy Hb in positively charged pocket between β globins
- Stabilizes the T form
- Decreases O2 affinity in the tissues
Notes:
Allosteric inhibitors help Hb unload O2 by decreasing Hb affinity for O2 by stabilizing the T form.
2,3 BPG (sometimes called 2-3 diphosphoglycerate, of 2,3 DPG) only binds to the deoxy form, after O2 unloaded. Its binding stabilizes the T form and makes it harder for the O2 just unloaded to jump right back on.
Mutations that change the positively charged amino acids in the center pocket can make Hb variants with abnormally high affinity for O2 (less efficient at dropping of O2 in the tissues).
2,3 BPG and Adaptation to Altitude and Hypoxia
- The adaptation to high altitude is complex and takes several weeks to complete
- Increased number of rbc
- Increased hemoglobin concentration in rbc
- However, within a day there is increased synthesis of 2,3 BPG
- 2,3 BPG levels are also increased in conditions of hypoxia
- Chronic anemia
- Cardiopulmonary insufficiency (COPD)
Notes:
Acute mountain sickness develops at altitudes >2000m (25% incidence) 50% incidence at 4000m
At 4000m, barometric pressure = 460 mmHg (sea level = 160). Arterial pO2 45 mmHg (sea level 100). Hb saturation only 81%. O2 carrying capacity decreases to 160 mL/L (sea level 198).
Adaptation:
Hyperventilation decreases alveolar pCO2 and increases alveolar pO2 and arterial pH, which increases Hb O2 affinity.
Gradual increase in 2,3-BPG in resp to chronic hypoxia
Polycythemia = inc rbc concentration, due to erythropoietin
Within 1 week of acclimatization, Hb concentration can increase by 20% and arterial O2 content is nearly normal.
The Bohr Effect
Regulation of O2 binding by H+ and CO2
The Bohr effect is that more O2 is released from Hb when pH decreases or pCO2 increases.
- H+ is released as O2 binds and bonds between the dimers are broken.
- The reaction is reversible. In the tissues, where H+ is increased in metabolically active tissues, the reaction is pushed to the left, helping to unload the O2.
As Hb is converted from the T form to the R form, protons (H+) are released.
Increasing [H+] in tissues pushes the equilibrium to the left, helping to release O2
Effect of H+
As pH decreases, [H+] increases ==> O2 affinity decreases
H+ stabilizes the T form
Shifts dissociation curve to the right
Little effect at high [O2]
Notice that there is little effect in the lungs, where pO2 is high.
The Chloride Shift
- Happens in milliseconds
- Equalizes charges in RBC and plasma
- Helps regulate pH
- Increases the CO2-carrying capacity of blood
- Increases the unloading of oxygen in tissues
* Cl- acts as an allosteric inhibitor (a minor effect in humans) - Concentrations of Cl- and bicarb differ in venous vs arterial blood
CO2 transport on Hb
A smaller amount of CO2 is transported to the lungs on Hb as carbamino-hemoglobin.
CO2 binds N-terminal amino acid of globin chains.
This reaction also produces H+, so binding of CO2 promotes release of O2from Hb in the tissues.
Note:
Some of the CO2 does bind directly to Hb, on the N terminal amino acids of the globin chains. This has a more direct effect on Hb O2 affinity, but it also increases H+ and decreases affinity that way as well.
Summary of Hb function
- Cooperative binding of O2
- Allosteric inhibitors
- 2,3-BPG, H+, CO2
- Stabilize T form, shift curve to right
- Decrease Hb affinity for O2
- Help Hb drop off O2 in tissues
- Increasing temperature also decreases O2 affinity
Microenvironment of exercising muscle favors efficient release of O2
α Thalassemia
- Usually caused by deletion of 1 or more of the 4 α globin genes, severity depends on how many
- Hemoglobin Barts Syndrome = all 4 α genes deleted
- Usually fatal at or before birth; Hydrops fetalis = abnormal accumulation of interstitial fluid in a fetus or newborn
- Hb Barts = γ4
- Increased oxygen affinity makes it a poor transporter
- Hemoglobin H disease = 3 α genes deleted
- HbH= β4;
- Unstable tetramer found in patients with 3 α chains deleted
- Moderate to severe disease
β Thalassemia
- 170 different mutations
- Excess α chains precipitate, cause destruction of RBC (hemolysis), anemia
-
β Thalassemia Minor
- Heterozygous
- Usually asymptomatic except for mild anemia
-
β Thalassemia MAJOR (aka Cooley Anemia)
- Homozygous
- Transfusions for severe anemia
- Often suffer from iron overload
Notes:
Alpha chains can’t form stable tetramers, they precipitate and cause premature death of rbc precursor cells.
No symptoms until levels of gamma globin decline.
SCD
- Most common structural hemoglobinopathy
- Autosomal recessive
- Hemoglobin S = missense mutation (glu to val) in β globin
- Decreased solubility, but only the deoxy form is so
- insoluble that it precipitates in RBC and causes sickling
- Usually only homozygotes have symptoms –
- hemolytic anemia, vaso-occlussion leading to tissue infarction,
- painful crises, increased risk of stroke
- Precipitating factors include:
- Acidosis, Hypoxia, Infection, Stress
Notes:
Single nucleotide, missense mutation changes Glutamate (a charged amino acid) to valine (nonpolar). When Hb is in the deoxy form, the valine makes a protrusion that fits into a hydrophobic pocket. This forms a network of fibrous polymers in the rbc and causes them to sickle. Only in the deoxy state.
Anything that increases the amount of deoxy HB can increase the risk of a painful crisis. Infection increases expression of adhesion molecules and increases the time it takes for rbc to get through the capillaries.
Newborn screening and use of prophylactic antibiotics have helped to prevent crises and increase average lifespan.
5% of patients with HbS expect to experience 3-10 episodes extreme pain/yr. Pain crisis usually resolves within 5-7 days, but a severe crisis can last weeks.
Acquired Hemoglobinopathies
Carbon Monoxide Poisoning
- Most common fatal poisoning in US
- CO competes with O2 for binding to Hb, increases O2 affinity of remaining sites
- Levels of carboxyhemoglobin
- Normal <5%
- Smoker up to 9%
- Toxic >25%
- Fatal >95%
Notes:
- The toxicity of CO is due to tissue hypoxia and direct cell damage.
- CO also inhibits complex IV of the electron transport chain.
- The affinity of Hb for CO is 220X higher than affinity for O2
Methemoglobinemia (part 1)–general info
Occurs when:
Oxidation exceeds capacity of reduction
Mutation limits ability to reduce
Notes:
- Oxidation of the Fe2+ in heme to Fe3+ makes a form (called metHb) that can’t bind O2. This happens all the time in cells, so there is a system to deal with it and reduce Fe3+ back to Fe2+.
- If the rate of oxidation exceeds the rate of reduction, MetHb builds up, causing methemoglobinemia
- NADH cytochrome b5 reductase developmental regulation, nitrates in root vegetables – babies under 3 months old should eat pureed root vegetables.
