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Flashcards in Hemoglobin Deck (29):
1

Overview of heme synthesis

-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

First reaction in heme synthesis

-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

Second reaction in heme synthesis

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

4

Third reaction in heme synthesis

-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

Reactions 4-7 of heme synthesis

-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

Final reaction (8) of heme synthesis

-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

Regulation of heme synthesis

-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

ALA-S2 gene

-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

Transferrin

-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

Inherited disease states

-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

Congenital erythropoietic porphyria

-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

Acute intermittent porphyria

-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

Properties of hemoglobin (Hb)

-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

Myoglobin (Mb) vs Hb

-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

Hb evolution and mutation

-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

Expression of Hb genes during life

-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

Hb structure

-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

Hb saturation curves and modifications

-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

Alkaline Bohr effect

-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

2,3-Diphosphoglyceric acid effect

-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

CO2 effect on Hb

-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

Formation of HbA1c

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

23

Hemoglobinopathies

-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

Sickle cell Hb

-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

HbC

-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

HbE

-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

Unstable Hb

-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

MetHb and HbM

-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

Hb w/ altered O2 affinity

-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