Hemoglobin Flashcards
1
Q
Overview of heme synthesis
A
- 85% occurs in BM for RBCs
- 15% occurs in liver (mostly for CYP450)
- 8 rxns in forming heme
- First one and last 3 are in mito, intermediate 4 are in cytoplasm
- There is only feedback inhibition (heme inhibits ALA synthetase) in the liver (no inhibition in BM)
- We are responsible for steps 1, 2, 3, and 8
2
Q
First reaction in heme synthesis
A
- Formation of d-ALA (aminolevulinic acid), by condensation of succinyl CoA and glycine
- Catalyzed by ALA synthetase, requires pyridoxal phosphate (vit B6)
- Is the rate limiting step of heme synthesis in LIVER
- Will be inhibited by heme (negative feedback) in LIVER
- Occurs in the mito (where succinyl CoA is)
3
Q
Second reaction in heme synthesis
A
- Formation of porphobilinogen (PBG) via condensation of 2 d-ALA molecules
- Catalyzed by ALA dehydratase
- Occurs in cytoplasm
4
Q
Third reaction in heme synthesis
A
- Four porphobilinogen are linked head-tail to form a linear tetrapyrrole, hydroxymethylbilane
- One NH4+ is released for each methylene bridge formed
- Occurs in cytoplasm via nz porphobilinogen deaminase
- The product hydroxymethylbilane undergoes ring inversion during cyclization in the fourth reaction (side chains switch from acetyl/proprionyl to proprionyl/acetyl in the lower left quadrant only)
- The rest of the molecule retains the A/P pattern of side chains (clockwise)
5
Q
Reactions 4-7 of heme synthesis
A
- Hydroxymethylbilane is cyclized by uroporphyrinogen III cosynthetase and ring inversion occurs (see 3rd rxn)
- Subsequent reactions alter side chains and degree of saturation of the porphyrin ring
- The ring re-enters the mito btwn rxns 5 and 6
6
Q
Final reaction (8) of heme synthesis
A
- An atom of ferrous (2+) iron is incorporated into protoporphyrin IX to form heme
- Occurs in mito and is catalyzed by ferrochelatase (heme synthetase)
- Heme is prosthetic group for Hb, myoglobin (Mb), catalase, and Cyt C
- Heme will negatively feedback ALA synthesis, only in liver
7
Q
Regulation of heme synthesis
A
- In liver (non-erythroid tissue), heme directly feeds back to inhibit synthesis of ALA (step 1)
- In BM (erythroid tissue), depressed levels of Fe lead to decreased heme synthesis, and high levels of Fe enhance heme synthesis
- This is due to the ALA-S2 gene (found only in erythroid tissue)
8
Q
ALA-S2 gene
A
- The gene that encodes BM ALA synthetase nz contains an IRE (iron response element), which isn’t present in the ALA-S1 gene (liver isoform)
- When there is excess iron, Fe-S clusters (ISCs) bind to IRP (Iron regulatory proteins), preventing the IRPs to bind to IRE
- When the IRE is not bound, the synthesis of ALA-S2 gene is initiated, leading to higher amounts of ALA synthetase and increasing heme synthesis
- When Fe levels are low, ISCs are not created and IRP are free to bind to IRE, preventing synthesis of ALA synthetase and inhibiting heme generation
- Overview: high Fe-> ISCs+IRP-> ALA-S2 synthesized-> more heme production
- Overview: low Fe-> IRP+IRE-> no ALA-S2-> less heme production
9
Q
Transferrin
A
- Plasma transport protein for Fe (transported in ferric, or 3+ state, while in plasma)
- Upon binding to the receptor, transferrin is internalized
- Within the endosome the pH is lowered (to 5.5) and Fe dissociates from transferrin
- Transferrin is recycled to the surface in an empty state, ready to be used again
- Iron is converted to ferrous form (2+, the form used within cells)
- Fe2+ is used for heme synthesis or is stored in ferritin proteins, which condense to form hemosiderin structures
- Empty form of ferritin is apoferritin
10
Q
Inherited disease states
A
- Porphyrias: disease characterized by deficiencies of an nz in the heme synthesis pathway
- Manifested by cutaneous photosensitivity (from excess tissue porphyrins), and defects of the nervous system
- Subdivided into erythropoietc or hepatic based on where the excess porphyrins occurs
11
Q
Congenital erythropoietic porphyria
A
- Deficiency of uroporphyrinogen III cosynthetase (cyclizes hydroxymethylbilane, step 4)
- There is a buildup of uroporphyrinogen (which is normally not made, but in this case is made non-enzymatically) and other porphyrins in RBC, marrow, plasma, urine, and feces
- RBCs are prematurely destroyed and urine of patients is red due to excretion of porphyrins
- Skin is photosensitive and teeth fluoresce (buildup of porphyrins) and patients are anemic
- Rx is mostly IV hematin (form of heme), can also blood transfusion and BM transplant
12
Q
Acute intermittent porphyria
A
- Porphobilinogen deaminase is depressed and there is a compensatory increase in ALA synthetase
- In the liver and urine there are large amounts of ALA and porphobilinogen
- Manifests as intermittent abdominal pain and neurologic disturbances
- Rx by IV hematin
13
Q
Properties of hemoglobin (Hb)
A
- Normally heme contains ferrous (Fe2+) iron
- When Fe is oxidized to ferric (Fe3+), Hb is called methemoglobin (MetHb), which does not bind O2
- In deoxyHb, Fe2+ is bound to 4 nitrogens from the heme and a nitrogen from the histidine residue of the globin protein
- The 6th Fe coordination site is unoccupied (where O2 binds)
- Upon O2 binding to the 6th position of Fe, Hb becomes oxyHb and the structure of hemoglobin changes (from taught to relaxed)
- The relaxed state of Hb (oxyHb) gives the other subunits of Hb tetramer a higher affinity for O2 (cooperativity)
14
Q
Myoglobin (Mb) vs Hb
A
- Mb is a single chain (unlike Hb which is 4 chains)
- Mb does not demonstrate cooperativity, b/c of its single chain
- Mb has a rectangular hyperbolic curve of O2 binding (due to lack of cooperativity
- Hb has sigmoidal curve of O2 binding, enabling Hb to release more O2 at tissues (result of cooperativity)
15
Q
Hb evolution and mutation
A
- Normal adult Hb composition: 4 chains consisting of 2 alpha and 2 beta chains (A2B2)
- Adults also have small amounts of A2D2 (A2 Hb) and A2G2 (Fetal, F, Hb)
- Abnormal Hb chains: sickle cell Hb (HbS) is A2B(s)2, HbBarts (G4) and a-thalassemia Hb (HbH) is B4
- S Hb (sickle cell) has Glu changed to Val @ position 6 on the B chain
- Thalassemia: decreased synthesis of A or B chain
- A, B, D, G genes on 2 different chroms
16
Q
Expression of Hb genes during life
A
- A chain is always expressed at all stages
- G chain is expressed w/ A (A2G2) for development until birth (HbF), at which time B chain begins to be expressed
- By 3 moths HbF is almost completely absent and B is almost entirely expressed (A2B2)
17
Q
Hb structure
A
- Heme sits in the hydrophobic pocket btwn the F and the E helices of the globin chain
- This prevents water and substances from getting to heme and oxidizing it to ferric form
- 75% of globin is helix
- Hb and Mb chains fold similarly
- Interchain contacts are electrostatic, when in deoxyHb form these contacts are in place and keep the molecule taught
- Upon O2 binding and conversion to oxyHb, the electrostatic contacts are broken and the molecule is relaxed
18
Q
Hb saturation curves and modifications
A
- P50: pO2 required to half saturate Hb
- Lower P50 means higher affinity for O2 (curve shifted to the left)
- Higher P50 means lower affinity for O2 (curve shifted to the right)
- Modifications of O2 affinity: Alkaline Bohr effect (pH changes), CO2, 2,3-diphosphoglyceric acid (DPG), higher temp
19
Q
Alkaline Bohr effect
A
- O2 affinity is pH dependent
- The more protons (lower pH), the lower the affinity for O2
- This means an increase in the P50 and a shift to the right in the saturation curve
- Due to H+ binding directly to Hb, creating salt bridges, stabilizing deoxyHb and thus reducing its binding capacity for O2
- Most important at tissues, where pH is lower (affects cooperatively by changing affinity- easier for O2 to be released)
- higher temp has the same effect: shifts the saturation curve to the right
20
Q
2,3-Diphosphoglyceric acid effect
A
- DPG binds to positively charged AA residues of deoxyHb (most important: Val1- the amino terminal AA)
- Stabilizes the deoxyHb by binding btwn the 2 B subunits, decreasing the affinity for O2
- Thus DPG shifts the Hb saturation curve to the right, helping to remove all the O2 from Hb in tissues
- In fetal Hb, there is a change in residue 143 (his-ser), which reduces HbF’s affinity for DPG
- In turn this increases HbF’s affinity for O2, allowing the fetus to more efficiently take oxygen from maternal blood in placenta
21
Q
CO2 effect on Hb
A
- CO2 binds to the same AA as DPG, Val1 (the amino terminal AA)
- Binds more to the B chains than A chains
- Carbamino groups bind to + charged side chains and form salt bridges to stabilizes deoxy form
- 10-15% of CO2 is carried by Hb (rest as carbonate)
22
Q
Formation of HbA1c
A
- Non-enzymatic glycosylation of Hb
- Normal range: 4-6%
- Diabetics: 7-16%
23
Q
Hemoglobinopathies
A
- Most are single AA substitutions, heterozygous, and not associated w/ any disease
- Normal (HbA) AAs at position 6 and 73: Glu, Asp
- HbC: Glu6->Lys (point)
- HbS: Glu6-> Val (point)
- HbC(harlem): Glu6-> Val and Asp73-> Asn (double point)
- Hb gun hill: 5 AA deletion near heme binding site results in unstable Hb (del)
- Hb constant spring: A chain extended, chain termination mutant leading to unstable mRNA (point mutation, leads to A-thalassemia)
- Hb wayne: frameshift
- Lepore: due to unequal crossing-over of D and B genes, creating a DB gene product (lepore) and anti-lepore (BD) on other chrom (leads to B-thalassemia)
24
Q
Sickle cell Hb
A
- HbS defect due to position 6 AA substitution: Glu to Val (polar AA-> hydrophobic AA)
- This change leads to aggregation of HbS and rigidity of the RBC
- RBCs assume abnormal shapes (sickled) and lysis occurs in capillaries
- Only the DEOXY form of Hb sickles, there is no effect on oxyHb
- HbS has lower affinity for O2 and this aids in O2 deposition in tissues (helping the anemia)
- But this also makes it easier for RBCs to sickle (happens mostly in tissues where O2 is low)
- In the capillaries acidosis results from stasis, lowering pH and shifting saturation curve tot he right, further exacerbating the release of O2 and inducing more sickling
- Can have severe crises of anemia
25
Q
HbC
A
- Autosomal recessive, a person must be homozygous recessive for symptoms
- Glu6->Lys6 AA substitution in the B chain, resulting in mild hemolytic anemia
- This is b/c HbC precipitates to form crystals w/in the RBCs in the OXY form (HbC less soluble than HbA)
- RBCs w/ HbC are less deformable and have shorter survival
- Can lead to abdominal pain, joint pain, enlarged spleen, and mild jaundice
- But do not develop severe crises like SCD
26
Q
HbE
A
- Most common Hb variant (SE asians), due to change in Glu26->lys26 in B chain
- Results in splice site alteration in mRNA and low levels of HbE
- Heterozygous state is asymptomatic but causes micricystosis w/o anemia
- Homozygous state has more severe microsystosis and hypochromia but little anemia
- Synthesized inefficiently compared to HbA, and thus has the same appearance clinically to mild B-thalassemia
- Should always be considered in differential form microcystosis
27
Q
Unstable Hb
A
- Due to altered residue involved in contact w/ heme
- Leads to denaturation of affected globin chain in RBCs w/ formation of Heinz bodies (oxidized Hb)
- Ex: Hb Koln, Hb Hammersmith
28
Q
MetHb and HbM
A
- MetHb: Fe2+ oxidized to Fe3+
- Occurs in normal HbA, cannot bind O2
- MetHb reduced by cytochrome B5 reductase)
- Can be drug-induced, hereditary (Cyt B5 reductase deficiency)
- HbM: mutation of proximal His or distal His residue in globin chain generally to Tyr
- Abnormal residue around heme allows Fe to be oxidized to Fe3+
- Results in cyanosis (no O2 binding), lowers Hill’s N (measure of cooperativity)
29
Q
Hb w/ altered O2 affinity
A
- Increased O2 affinity: picks up O2 from lungs well but doesn’t drop it off at tissues adequately (may need Rx, may see microcystosis and high EPO)
- Decreased O2 affinity: doesn’t pick O2 up from lungs well but drops it off at tissues adequately (occasional anemia/cyanosis, no Rx)
- These can be due to mutations in: A1B2 contacts, residues that are part of salt bridges at COOH terminus, changes in DPG binding site
- Ex of increased O2 affinity: Hb rainier has COOH salt bridge mutation, resulting in stabilization of oxy form (shifts curve to the left)
- Ex of decreased O2 affinity: Hb kansas has A1B2 contact mutation resulting in stabilization of deoxy form (shifts curve to the right
