Hemoglobin

Question 1

Overview of heme synthesis

Answer 1

• 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

Question 2

First reaction in heme synthesis

Answer 2

• 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)

Question 3

Second reaction in heme synthesis

Answer 3

• Formation of porphobilinogen (PBG) via condensation of 2 d-ALA molecules
• Catalyzed by ALA dehydratase
• Occurs in cytoplasm

Question 4

Third reaction in heme synthesis

Answer 4

• 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)

Question 5

Reactions 4-7 of heme synthesis

Answer 5

• 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

Question 6

Final reaction (8) of heme synthesis

Answer 6

• 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

Question 7

Regulation of heme synthesis

Answer 7

• 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)

Question 8

ALA-S2 gene

Answer 8

• 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

Question 9

Transferrin

Answer 9

• 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

Question 10

Inherited disease states

Answer 10

• 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

Question 11

Congenital erythropoietic porphyria

Answer 11

• 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

Question 12

Acute intermittent porphyria

Answer 12

• 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

Question 13

Properties of hemoglobin (Hb)

Answer 13

• 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)

Question 14

Myoglobin (Mb) vs Hb

Answer 14

• 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)

Question 15

Hb evolution and mutation

Answer 15

• 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

Question 16

Expression of Hb genes during life

Answer 16

• 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)

Question 17

Hb structure

Answer 17

• 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

Question 18

Hb saturation curves and modifications

Answer 18

• 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

Question 19

Alkaline Bohr effect

Answer 19

• 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

Question 20

2,3-Diphosphoglyceric acid effect

Answer 20

• 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

Question 21

CO2 effect on Hb

Answer 21

• 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)

Question 22

Formation of HbA1c

Answer 22

• Non-enzymatic glycosylation of Hb
• Normal range: 4-6%
• Diabetics: 7-16%

Question 23

Hemoglobinopathies

Answer 23

• 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)

Question 24

Sickle cell Hb

Answer 24

• 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

Question 25

HbC

Answer 25

- 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

Question 26

HbE

Answer 26

- 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

Question 27

Unstable Hb

Answer 27

- 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

Question 28

MetHb and HbM

Answer 28

- 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)

Question 29

Hb w/ altered O2 affinity

Answer 29

- 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