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97 Cards in this Set
- Front
- Back
What are the functions of heme?
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- Transport of O2 (on hemoglobin and myoglobin)
- Electron transport (respiratory cytochromes) - Oxidation-reduction reactions (cytochrome P450 enzymes) |
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What are the sites of heme synthesis? What forms of heme are made in these locations?
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- Bone Marrow - hemoglobin
- Liver - cytochrome P450 enzymes - Virtually all cells (except mature RBCs) - other important cellular proteins |
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Where is hemoglobin synthesized? How much?
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- Bone marrow
- 6-7g synthesized per day to replace heme lost through normal turnover of RBCs |
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Where are cytochrome P450 enzymes synthesized? Function?
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- Liver
- Drug detoxification |
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Why can't hemoglobin be synthesized in mature erythrocytes?
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They lack mitochondria and other organelles
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What kind of molecule is Porphyrin? Use?
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- Cyclic, predominantly planar tetrapyrroles
- Capable of chelating to various metals - Form prosthetic groups for various biological molecules |
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What are the components of heme?
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- Porphyrin derivative: Protoporphyrin IX
- Single ferrous iron (Fe2+ = reduced form) |
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What porphyrin is found in heme? What is the name for this form when combined with ferrous (Fe2+) iron?
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Protoporphyrin IX:
- Specific isomer of porphyrin that contains specific substituent groups on the four pyrrole rings - Substituent groups provide important sites for binding to its apoproteins Forms Ferroprotoporphyrin IX |
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What happens if there is auto-oxidation of Ferroprotoporphyrin IX (heme)?
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Forms Ferriprotoporphyrin IX (with an "i")
- Called "Hemin" - Contains ferric Fe3+ iron |
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How many stages of heme biosynthesis are there? Where do these steps take place?
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7 major steps:
- 1st and last 3 (5, 6, and 7) take place in mitochondria - 2, 3, and 4 take place in cytosol |
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What are the molecules in the 7 steps to synthesize Heme?
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1. Succinyl CoA + Glycine
2. ALA: 5-Aminolevulinate 3a. PBG: Porphobilinogen 3b. Hydroxymethylbilane 4. Uroporphyrinogen III Isomer 5. Protoporphyrinogen IX 6. Protoporphyrin IX 7. Heme / Ferroprotoporphyrin IX |
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What are the enzymes in the 7 steps to synthesize Heme?
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1. ALAS: 5-Aminolevulinate Synthase
2. ALAD: ALA Dehydratase 3a. PBGD: Porphobilinogen Deaminase 3b. UROS: Uroporphyrinogen III Cosynthase 4. UROD: Uroporphyrinogen Decarboxylase 5. CPO: Coproporphyrinogen III Oxidase 6. PPO: Protoporphyrinogen IX Oxidase 7. Ferrocheletase |
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What happens in the first step of heme synthesis?
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- Substrate: Succinyl-CoA + Glycine
- Enzyme: ALAS - 5-aminolevulinate synthase - Cofactors: PLP - Product: ALA - 5-aminolevulinate (+ CO2 + CoA) |
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What happens in the second step of heme synthesis, after formation of ALA?
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- Substrate: 2 x ALA - 5-aminolevulinate
- Enzyme: ALAD - 5-aminolevulinate dehydratase - Cofactors: Zn2+ - Product: PBG - Porphobilinogen |
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What happens in part one of the third step of heme synthesis, after formation of PBG?
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- Substrate: 4 x PBG - Porphobilinogen
- Enzyme: PBGD - Porphobilinogen Deaminase - Cofactors: - Product: Hydroxymethylbilane (+ NH3 x4) |
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What happens in part two of the third step of heme synthesis, after formation of Hydroxymethylbilane?
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- Substrate: Hydroxymethylbilane
- Enzyme: UROS - Uroporphyrinogen III Cosynthase - Cofactors: - - Product: Uroporphyrinogen III Isomer |
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What happens in the fourth step of heme synthesis, after formation of Uroporphyrinogen III Isomer?
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- Substrate: Uroporphyrinogen III Isomer
- Enzyme: UROD - Uroporphyrinogen Decarboxylase - Cofactors: - - Product: Coprophorphyrinogen III (+ CO2 x4) |
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What happens in the fifth step of heme synthesis, after formation of Coprophorphyrinogen III?
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- Substrate: Coprophorphyrinogen III
- Enzyme: CPO - Coprophorphyrinogen III Oxidase - Cofactors: - - Product: Protoporphyrinogen IX |
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What happens in the sixth step of heme synthesis, after formation of Protoporphyrinogen IX?
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- Substrate: Protoporphyrinogen IX
- Enzyme: PPO - Protoporphyrinogen IX Oxidase - Cofactors: - - Product: Protoporphyrin IX |
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What happens in the seventh step of heme synthesis, after formation of Protoporphyrin IX?
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- Substrate: Protoporphyrin IX + Fe2+
- Enzyme: Ferrocheletase - Cofactors: - - Product: Heme |
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Where do the carbon and nitrogen atoms of the porphyrin ring in heme originate?
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All C and N are from Succinyl-CoA and Glycine
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What is the enzyme in the first step of heme synthesis? Location?
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ALAS: 5-Aminolevulinate synthase
- Found in the inner mitochondrial membrane, but encoded by a nuclear gene family - Therefore, nascent protein must be imported into mitochondrion |
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What does the enzyme 5-Aminolevulinate synthase (ALAS) require? What step? Mechanism?
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- Step 1
- Pyridoxal Phosphate (PLP) dependent enzyme - Condensation of glycine w/ succinyl-CoA takes place while amino group of glycine is in Schiff base linkage to PLP aldehyde; CoA and glycine carboxyl are lost during condensation |
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What are the two forms of 5-Aminolevulinate synthase (ALAS)?
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- ALAS1 - liver isoform
- ALAS2 - erythroid / reticulocyte isoform |
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How is feedback of the two ALAS isoforms different?
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- ALAS1 (liver) has feedback inhibitino by heme or hemin
- ALAS2 (RBCs) is not regulated by feedback repression |
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How is ALAS1 regulated in the liver?
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- Feedback inhibition by heme or hemin regulates heme biosynthesis in liver (ALAS1)
- Heme inhibits ALAS1 synthesis at both transcriptional and translational level and as its mitochondrial import - ~100 drugs or metabolites can stimulate ALAS1 - Many drugs are metabolized by cytochrome P450s in liver and thus increase synthesis of cytochrome P450 enzymes (increasing demand for heme) |
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How is ALAS2 regulated in the RBCs?
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- Heme biosynthesis is not regulated by feedback repression of ALAS2
- Heme stimulates synthesis of globin and ensures that heme and globin are synthesized in correct ratio for assembly of hemoglobin - Drugs that cause a marked elevation of ALAS1 (eg, phenobarbital) do not affect ALAS2 |
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What is the first pathway intermediate to include a pyrrole ring? When is it synthesized?
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- Porphobilinogen (PBG)
- Synthesized in step 2 in cytosol by ALA dehydratase (ALAD) |
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What is required for ALA dehydratase (ALAD) activity in step 2 of heme synthesis?
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Zn2+ complexed to an active site cysteine
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What step of heme synthesis is affected by lead poisoning? How does it affect this step?
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- Step 2: ALA Dehydratase (ALAD)
- ALAD requires a Zn2+ in the active site, lead and other heavy metals can displace Zn2+ and eliminate the catalytic activity |
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What are the implications of lead poisoning?
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- Increases ALA in urine (the substrate for step 2 of heme synthesis)
- Increases ALA in blood (causes neurological side effects) - Lead may also directly affect the nervous system - Clinical manifestations mimic acute porphyrias |
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What can cause a buildup of ALA levels in the blood? What are the implications?
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- Caused by lead poisoning (which eliminates catalytic activity of ALA dehydratase, ALAD)
- Toxic to brain perhaps because ALA has a similar structure as GABA; also ALA autoxidation generates reactive oxygen species (ROS) |
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What happens structurally in the third step of heme synthesis?
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Porphobilinogen Deaminase (PBGD)
- Head-to-tail condensation of four porphobilinogen molecules to form a linear tetrapyrrole (liberates 4 ammonium ions) Uroporphyrinogen III Cosynthase (UROS) - Directs the stereochemistry of the condensation reaction to yield Uroporphyrinogen III isomer |
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What happens structurally in the fourth step of heme synthesis?
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Decarboxylation of acetate side chains to methyl groups (by UROD)
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What happens structurally in the fifth step of heme synthesis?
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- Substrate (Coproporphyrinogen III) transported into intermembrane space
- Oxidase (CPO) converts specific propionic acid side chains to vinyl groups - Forms Protoporphyrinogen IX |
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What happens structurally in the sixth step of heme synthesis?
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Another mitochondrial oxidase (PPO) moves double bonds in the structure to form protoporphyrin IX
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What happens structurally in the seventh step of heme synthesis?
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Insertion of Fe2+ into Protoporphyrin IX to generate heme by Ferrochelatase
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What inhibits Ferrocheletase (the last step of heme synthesis)?
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- Lead poisoning
- Iron deficiency (anemia) - not enough Fe2+ to insert into Protoporphyrin IX |
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What happens if there is an absence of Fe2+?
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Ferrocheletase can insert Zn2+ into the protoporphyrin ring to yield a brilliantly fluorescent complex
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What are porphyrias?
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Defects in heme biosynthesis
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What causes Porphyrias?
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- Inherited / genetic disorders
- Acquired (rarely) disorders - Result from deficiency in specific enzymes of the porphyrin/heme biosynthetic pathway |
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How do you classify porphyrias?
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Based on the principal sites of heme biosynthesis and depending on the site of expression of the enzyme defect:
- Hepatic - Erythroid |
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How are Porphyrias inherited?
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* Autosomal dominant
- Exception: congenital erythropoietic porphyria which is autosomal recessive |
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What is the most common porphyria?
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Acute Intermittent Porphyria
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What is the most common erythropoietic porphyria?
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Erythropoietic Protoporphyria (EPP)
(also most common childhood porphyria) |
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What is the most common porphyria in childhood?
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Erythropoietic Protoporphyria (EPP)
(also most common erythropoietic porphyria) |
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What type of pophyria is extremely rare?
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Congenital Erythropoietic Porphyria (CEP)
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What causes the clinical symptoms in porphyrias?
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- Accumulation of intermediates upstream from the enzyme defect (measure in urine, blood, feces)
- Defects early in pathway (accumulation of ALA, prophobilinogen) result in neurologic dysfunction - Defects later in pathway (accumulation of cyclic tetrapyrroles, but not prophobilinogen) result in sunlight-induced cutaneous lesions; in presence of molecular O2, UV irradiation of cyclic tetrapyrroles generates ROS that can cause cellular damage |
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What happens if there are defects early in the biosynthetic pathway for Heme?
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Porphyria:
- Accumulation of ALA and prophobilinogen - Leads to neurological dysfunction |
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What happens if there are defects later in the biosynthetic pathway for Heme?
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Porphyria:
- Accumulation of cyclic tetrapyrroles - Sunlight-induced cutaneous lesions - In presence of molecular O2, UV irradiation of cyclic tetrapyrroles generates ROS that can produce cellular damage |
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What are the two ways that Porphyrias can present?
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Acute:
- Periodic acute attacks - Sx include abdominal pain, neurologic deficits, psychiatric symptoms, and reddish-colored urine Chronic: - Dermatologic diseases - May or may not include liver and nervous system |
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What are some triggers that can bring about acute attacks of Porphyria?
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- Nutritional changes (eg, hypoglycemia)
- Smoking - Certain drugs (barbiturates and sulfonamide antibiotics) - Steroid hormones, especially progesterone (some women develop attacks during second half of menstrual cycle when progesterone is high) |
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What is true about Ferrochelatase?
a) found in cytosol b) rate-limiting enzyme in heme synthesis c) requires glycine for activity d) activity stimulated in presence of lead e) catalyzes last step of heme synthesis |
Catalyzes last step in heme synthesis
- Found in mitochondria - First step is rate-limiting - Does not require glycine for activity - Activity inhibited by lead and iron deficiency |
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You are treating a patient with Porphyria Cutanea Tarda, most common porphyria. She came to your office w/ significant blistering on her hands following a day of gardening. Accumulation of what would cause photosensitivity?
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Accumulation of cyclic tetrapyrroles:
4. Uroporphyrinogen III Isomer 5. Protoporphyrinogen IX 6. Protoporphyrin IX |
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What is the function of Hemoglobin?
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- Transport O2 from lungs (high O2 concentration) to peripheral tissues where O2 tension is low
- Transports some CO2 and H+ that are generated in peripheral tissues back to lungs (14% of CO2 made is carried on Hb) |
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Why is it necessary for O2 to be carried on Hemoglobin? Consequences?
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- O2 has a very low solubility in plasma (non-cellular part of blood)
- As a consequence, >98% of O2 that reaches the tissues is carried bound to hemoglobin in RBCs |
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Why does CO2 not have to be carried on Hemoglobin?
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- RBCs carry Carbonic Anhydrase which catalyzes the rapid reversible hydration of CO2 to H2CO3 which then dissociates to HCO3- and H+
- CO2 and HCO3- are soluble in plasma and RBC cytosol - Most of the CO2 made in tissues returns to lung as those species, but 14% bound to Hb |
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What are the components of Hemoglobin?
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Heterotetrameric protein: α2β2
- Contains 4 heme prosthetic groups responsible for binding O2 |
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How does hemoglobin relate to myoglobin?
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- Subunits of hemoglobin are evolutionarily related to myoglobin (monomeric protein abundant in muscle that is designed to store O2)
- Both proteins contain a heme prosthetic group (1/myoglobin and 4/hemoglobin) - Fe2+ is ferrous form of iron responsible for binding O2 - Hemoglobin has a sigmoidal (cooperative) binding curve, whereas Myoglobin has a hyperoblic binding curve to O2 |
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How is hemoglobin affected by different forms of iron?
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- Fe2+ (Ferrous form) - capable of binding O2
- Fe3+ (Ferric form) - cannot bind O2, inactive form called Methemoglobin (metHb) |
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What kind of binding curve do Myoglobin and Hemoglobin have for O2? How do you explain the different binding curve for Hb?
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- Myoglobin: normal, hyperbolic binding curve
- Hemoglobin: sigmoidal, cooperative binding curve - D/t its more complex subunit structure, critical for its efficiency in loading O2 in lungs and unloading O2 in peripheral tissues |
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What are the benefits of the cooperativity of O2 binding to hemoglobin?
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Allows Hb to release a much larger fraction of its own O2 load at the pO2 levels found in the blood of working and even resting muscle
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How do the four subunits of hemoglobin lead to a cooperative/sigmoidal O2 binding relationship/
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- Binding of O2 to one subunit induces a conformational change that is partially transmitted to adjacent subunits
- Transmission of partial conformational change induces an increased affinity for O2 by these adjacent subunits |
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How does Carbon Monoxide compare to O2 binding to Hemoglobin?
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- CO has ~250-fold higher affinity for Hb than does O2
- When bound to the heme group of one subunit, it causes all four subunits to lock in the R conformation thereby limiting O2 release in peripheral tissues (needs to be in T state to release) |
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What is the T state? What does it prefer? Same for R state?
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- T state = tense, favors dissociation (lower affinity for O2)
- R state = relaxed, favors association (higher affinity for O2) |
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How does O2 binding change the conformation of a Hb subunit?
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- Without O2 bound, the heme Fe2+ is pulled away from the plane of the porphyrin ring by a His residue of the Hb polypeptide chain (a His ring N is bound to the Fe2+)
- When O2 binds, it pulls the Fe2+ back into the plane of the ring and that moves the His residue and its whole section of the polypeptide chain - That in turn causes the Hb subunits to shift relative to one another to an arrangement that favors the R-conformation |
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What is an allosteric regulator?
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Molecule that can bind to a protein and induce a conformational change that alters the affinity for substrate (or ligand such as O2) at some other site (allo means other)
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What are the allosteric regulators of O2 binding to Hb? Effects?
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All bind to Hb and reduce its affinity for O2:
- Protons (H+) - CO2 - 2,3-Diphosphoglycerate (DPG) |
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What are the effects of high amounts of H+, CO2, or 2,3-DPG?
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When these are present, the curve shifts to the right and O2 comes off of Hb
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What are the normal values for:
- pH? - pO2? - pCO2? - HCO3-? |
- pH: 7.35-7.45
- pO2: 80-100 mmHg - pCO2: 35-45 mmHg - HCO3-: 22-26 mM |
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What kind of effectors are H+ and CO2 on Hb binding of O2?
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Heterotropic Negative Allosteric Effectors that decrease the affinity of Hb for O2
- Heterotropic: they are not O2 - Negative: decrease affinity for O2 - Allosteric: bind to a site other than O2 site |
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What kind of effector is O2 on Hb binding of O2?
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Homotropic Positive Allosteric Effector
- Homotropic: it is O2 - Positive: increases affinity for more O2 - Allosteric: binds to a site other than the site the next O2 could bind |
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What kind of effector is O2 on Hb binding of H+ and CO2?
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Heterotropic Negative Allosteric Effector
- Heterotropic: not CO2 or H+ - Negative: decreases affinity for CO2 and H+ - Allosteric: binds to a site different than they would bind |
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What is the term for the reciprocal relationship between O2 and H+ binding to hemoglobin?
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Bohr effect or isohydric shift
- Changes in H+ binding result from a shift in the pKa of specific residues (mostly histidines) d/t microenvironment effects triggered by conformational changes in Hb structure |
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What is the mechanism of how increased H+ (↓pH) affects O2 binding?
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- In deoxyHb negative charge on Asp94 is near His146
- It is energetically favorable for N to be protonated (its pKa is higher) - Having His protonated in turn makes it favorable for Asp to stay near it, increasing the stability of the deoxyHb (T-state) - Conformation changes in going to oxyHb (R state) move His146 and Asp94 apart, pKa drops and H+ comes off |
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What kind of effector is DPG on Hb binding of O2? Mechanism?
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Heterotropic Negative Allosteric Effector
- Binds to a specific site in central cavity between β subunits by ionic interactions - This binding stabilizes the T state of deoxyHb |
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What effects does DPG have on the O2 binding curves?
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1. Without any DBG, Hb would be more like myoglobin (hyperbolic) and nearly useless for delivering O2 from lungs to tissues
2. DPG levels increase at high altitudes - There is less O2 at high altitudes, so tissues tend to become somewhat hypoxic - Increasing DPG lets RBC adapt to hypoxia by making it easier for O2 to dissociate from Hb |
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What situations/conditions stimulate DPG release?
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Causes of tissue hypoxia:
- High altitude - Smoking - Anemia |
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How long does it take to make changes in DPG? Implications?
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- A few days
- Therefore takes a few days to adapt to high altitude - Until then, strenuous aerobic exercise is difficult (more dyspnea) |
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How does temperature affect O2 association/dissociation?
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↑T → ↓O2 affinity
- During fever there are elevated metabolic rates so need increasing unloading of O2 |
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What are the developmental forms of hemoglobin?
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- ~2% HbF - Fetal: α2γ2 (until ~5-6 months)
- ~95% HbA1 - Adult 1: α2β2 (majority) - ~3% HbA2 - Adult 2: α2δ2 (very low) |
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Which chromosome contains the α-globin gene(s)?
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Chromosome 16 has two α-globin genes (total of 4 / person)
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Which chromosome contains the β-globin gene(s)?
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Chromosome 11 has a single β-globin gene (total of 2 / person)
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Where is hemoglobin synthesized during the lifetime?
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- Yolk Sac
- Liver - Spleen - Bone marrow |
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How do the different forms of hemoglobin (α2β2, α2δ2, and α2γ2) differ?
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Binding affinity for O2
- Higher affinity of fetal Hb means fetus' circulation can draw O2 from maternal blood at pO2 present in placenta |
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How is Sickle Cell Anemia inherited? Cause?
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Homozygous recessive disease
- Point mutation in adult β-globin gene that causes substitution of valine for glutamic acid at amino acid 6 - Patients mainly contain HbS |
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What is the most common hemoglobinopathy?
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Sickle Cell Anemia
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What are the effects of the mutation in Sickle Cell Anemia?
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- Valine substituted for glutamic acid at position 6 is on the surface of the β-chain and it should be hydrophilic
- Valine is hydrophobic and its presence creates a sticky patch on deoxyHb that leads to polymerization of Hb tetramers into long chains - Val6 makes critical contact with hydrophobic acceptor pocket of β-subunit of another molecule formed by Leu88 and Phe85 - Intracellular fibers cause sickle cell shape and reduced deformability of RBCs that leads to problems w/ their passage through microcirculation |
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What determines the amount/rate of polymerization of HbS?
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- Degree of deoxygenation (can be affected by pH, ionic strength, and temperature); deoxyHbS forms insoluble polymers
- Intracellular Hb concentration - Relative amount of HbF present (HbF inhibits polymerization d/t Glu87 on γ-chain |
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What occurs in a Thalassemia?
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Inherited mutations cause a decreased synthesis of adult hemoglobin (α2β2)
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What kind of mutations cause β-thalassemias?
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- β° mutations have absent β-globin chain synthesis
- β⁺ mutations have reduced (but detectable) β-globin chain synthesis |
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What are the implications of β-thalassemias?
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- Deficit in HbA
- Unpaired α-chains precipitate in RBC precursors, resulting in apoptosis |
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What are the types of β-thalassemias? Differences?
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- β-thalassemia major: two β-thalassemia alleles w/ severe, transfusion-dependent anemia
- β-thalassemia minor: heterozygotes have only one β-thalassemia allele and mild or asymptomatic microcytic anemias |
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What causes α-thalassemias?
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Mutations that result in reduced or absent synthesis of α-globin chains
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What are the implications of α-thalassemias?
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Unpaired β-chains are more soluble than unpaired α-chains, thus effects less severe than in β-thalassemias
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What is true of Oxygen:
a) binding converts Hb to T state b) dissociation from Hb is inhibited in patients w/ fever c) dissociation from Hb is enhanced at pH values below pH 7.4 d) binding to Hb causes conversion of a β strand to a random coil e) dissociation from Hb is inhibited in presence of CO2 |
Dissociation from Hb is enhanced at pH values below 7.4
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What is true about Methemoglobin?
a) similar O2 association/dissociation curve as myoglobin b) similar O2 association/dissociation curve as hemoglobin c) monomer d) cannot bind hemoglobin e) contains ferric iron |
Cannot bind hemoglobin (it contains ferric / Fe3+ form)
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