2.Iron Heme Hb

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    2.nd semester

    2.nd lecture Biochemistr of IronMetabolism

    2012/02/14

    Dr Rka Tth Rvszn

    Biochemistry and Molecular Biology Department

    Lectures for 2nd year Physiotherapyst

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    COMPULSORY READING:

    Lecture presentations with short explanations are available on the web page ofthe de artment: htt ://bmbi.med.unideb.hu .

    Username: student , password: student2011.Downloads/

    Educational materials in English/Physiotherapists/

    Biochemistry

    FURTHER READINGS:

    Biochemistry and Molecular Biology Syllabus III. (ed .by Prof Lszl Fss) chapter 5.1. th

    . -

    1075.p)

    Harvey, Ferrier: Biochemistry 6th ed. (Lippincott, 2011) chapter 21. Haem metabolism

    Supplementary

    Most important obligatory

    2

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    CONTENTS

    I. IRON METABOLISM

    1. Introduction

    .

    3. Transport of iron and Storage of iron

    4. Regulation of iron metabolism: hepcidin

    II. HEME METABOLISM

    1. Biosynthesis of heme, Porphyrias

    2. Degradation of heme, Jaundice

    III. HEMOGLOBIN, MYOGLOBIN

    1. Structure of hemoglobin

    2. Polymorphism of globins

    . , , , ,

    4. Abnormal hemoglobins: Sicle cell anemia, MetHb, HbA1c

    3

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    IRON METABOLISM

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    Iron is an essential metal for humans, involved in

    metabolism and transport of oxygene

    but free iron is dagerous both iron deficiency anaemia (affects over 30% of the

    wor s popu a on an emoc roma os s ron over oa

    are dangerous

    of iron absorption from the diet

    5

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    Iron is involved in the metabolism and

    ron con a n ng pro e ns:

    Hem containing proteins --myoglobin

    Electrons transporters- cytochromes in the electron transport chain

    2

    NADPH oxydaseTryptophan pyrrolase

    a a ases egra e 2 2NO synthetase

    FeS cluster proteins (electron transport,succinate DH, aconitase) Iron in the catalytic centre various oxidoreductases (RR,

    Homogentisate oxidase, Lys, Pro, Phe, Tyr hydroxilase)

    , ,

    lactoferrin) 6

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    free iron generates reactive oxygen species (H2O22OH

    -)

    forms complexes with anions, which are precipitated

    body

    7

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    Human iron metabolism

    Iron metabolism is the set of chemical reactions maintaining human homeostasis of iron. Iron

    is an absolute requirement for most forms of life, including humans and most bacterialspecies. Because plants and animals all use iron, iron can be found in a wide variety of food

    sources (meat, liver, dried leguminoses, dried fruits, fortified flour, cereals).

    ,

    acceptor. However, iron can also be potentially toxic. Its ability to donate and accept

    electrons means that if iron is free within the cell, it can catalyze the conversion of hydrogen

    peroxide into free radicals (Fenton reaction). Free radicals can cause damage to a wide

    variety of cellular structures, and ultimately kill the cell. In addition, free iron causes

    distorsion in the structure of macromolecules and forms complexes with anions, which are

    precipitated within the cells. To prevent that kind of damage, all life forms that use iron, bind

    . ,

    ability to do harm.Iron containing proteins

    Most well-nourished people in industrialized countries have 3-4 grams of iron in their bodies.

    Of this, about 2.5 g is contained in the hemoglobin needed to carry oxygen through the

    blood. Another 400 mg is devoted to cellular proteins that use iron for important cellular

    processes like storing oxygen in the muscle (myoglobin), performing energy-producing redox

    ,

    enzymes having Fe in the catalytic centre). 3-4 mg circulates through the plasma, bound to

    transferrin. Some iron in the body is stored. Physiologically, most stored iron is bound by

    ferritin molecules. The largest amount of ferritin-bound iron is found in cells of the liver

    8

    hepatocytes, the bone marrow and the spleen. The liver's stores of ferritin are the

    primary physiologic source of reserve iron in the body.

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    Iron distribution in the human bod

    g

    hemoglobin 2.5 68

    myoglobin 0.15 4transferrin 0.003 0.1

    ferritin, tissue 1.0 27

    ferritin, serum 0.0001 0.004

    enzymes 0.02 0.6

    Iron requirement (if the absorption efficiency is ~10%):,

    Iron sources: meat, liver, leguminoses, fruits

    9

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    Overview of iron metabolism

    dietary iron gut absorption plasma transferrin transport

    receptors on iron-requiring cells

    absorption

    internalization, acidification

    intracellularsynthesis of iron proteinsutilization

    mobile iron poolemog o n, myog o n,

    cytochromes, etc.)

    ferritin

    s orage(mainly in liver)

    hemosiderinNo physiologic excretion mechanism!But iron is highly recycled!10

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    11

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    How does the body get its iron?

    Most of the iron in the bod is hoarded and rec cled b the

    reticuloendothelial system (macrophages) which breaks down aged

    red blood cells. However, people lose a small but steady amount bysweating and by shedding cells of the skin and the mucosal lining of

    the gastrointestinal tract. The total amount of loss for healthy people

    day for men, and 1.52 mg a day for women with regular menstrual

    eriods. Peo le in develo in countries with astrointestinal

    parasitic infections often lose more. This steady loss means that

    people must continue to absorb iron. They do so via a tightlyregulated process that under normal circumstances protects against

    iron overload.

    12

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    Absorbing iron from the diet

    Like most mineral nutrients, iron from digested food or supplements is

    almost entirely absorbed in the duodenum by enterocytes of theduodenal lining. These cells have special molecules that allow them to

    move ron n o e o y. oo ng o oo ac a es e rea own o

    ligands attached to iron. To be absorbed, dietary iron must be in its2+ .

    of vitamin C. In addition, a ferric reductase enzyme on the

    enterocytes' brush border, Dcytb, reduces Fe3+ to Fe2+. A protein

    called divalent metal transporter 1 (DMT1), which transports all kindsof divalent metals into the body, then transports the iron across the

    en erocy e s ce mem rane an n o e ce .

    These intestinal lining cells can then either store the iron as ferritin (in

    sloughed off into feces) or the iron can move it into the body, using a

    transporter protein called ferroportin. Ferroportin transports Fe2+,

    13but tranferrin carries Fe

    3+

    , so iron has to be oxidized by hephaestin onthe capillary surface of enterocytes for further transport.

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    Absorption of iron

    Andrews (2005) N. Engl. J. Med., 353, 2508-2509.

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    Absorption of iron

    Stomach Small intestine

    +

    low pH,

    ferroxidasesCerulo-

    plasmin

    Fe3+

    vitamin Cand/or Steap homolog

    ferrireductases?

    HCP1

    (heme carrierprotein 1) 15

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    16

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    Structure of the transferrin (Tf)

    Tf is an 80 kDa serum glycoprotein synthesized mainly by the liver. Tf is bilobal in

    Structure of iron binding site of transferrin

    .

    high affinity.

    Iron is coordinated by Tyr, Asp and His residues. The binding of iron also needs an anion

    which is usually carbonate (CO 2-). The charge on the anion is balanced by arginine side

    chain. The iron-binding capacity of transferrin is strongly pH-dependent: high-affinity

    binding occurs at pH 7.4 (Ka ~ 1023 M1), but no binding occurs below pH 4.5. In a healthyindividuals only ~30% of Tf binding sites are saturated.

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    Structure of transferrin receptor 1 (TfR1)

    TfR1 is a homodimeric glycoprotein

    that consists of two 90 kDa subunits

    671 aa

    n e y su e on s.

    The Tf-TfR1 complex occurs

    .

    The TfR1-Tf interaction is reversible

    S-S

    Transmembrane

    28 aa

    iron content of transferrin.

    61 aa

    TfR2 Role: sensing iron stores. It isconstantly expressed on some iron sensing

    cells, such as hepatocytes and enterocytes

    18(no IRE in its mRNA).

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    How do cells get their own iron?

    Most of the iron in the bod is located on hemo lobin molecules of red blood cells.

    When red blood cells reach a certain age, they are degraded and engulfed by

    specialized scavenging macrophages. These cells internalize the iron-containing

    hemo lobin, de rade it, trans ort iron via ferro ortin molecules into the blood,

    which is then transported by the transferrin molecules to the cells expressing

    transferrin receptors. Most of the iron used for blood cell production comes from

    this cycle of hemoglobin recycling.

    All cells use some iron, and must get it from the circulating blood. Since iron is

    tightly bound to transferrin, cells throughout the body have receptors for

    transferrin-iron complexes on their surfaces and takes them up by receptor

    mediated endocytosis. Once inside, the cell transfers the iron to ferritin, the

    internal iron storage molecule, and recycles of complex of apotransferrin-TrfR

    back to cell surface where release apotransferrin to blood.

    19

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    Receptor mediated endocytosis

    - -

    binding of loaded transferrinapotransferrin release

    to its receptor

    cell membrane

    clathrin-coated pits

    internalization into

    coated vesicles

    2-15 minutes

    recycling of complex

    of apotransferrin-TfR1

    endosomal pH drop:

    iron release

    intracellular iron pool 20

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    21

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    Structure of ferritin

    24-mer of light chain 24-mer of heavy chain(L-chains catalyse the formation of iron core (H-chains have ferroxidase activity)

    err n s a wa er-so u e mo ecu e cons s ng o su un s a orms

    a hollow sphere that houses up to 4,500 atoms of iron. Each subunit is

    one of two isoforms, the heavy (21 kDa) and light (19 kDa) subunits.

    err n a es up an re eases ron rom s nner core roug

    hydrophilic channels found in the apoferritin shell. The core containscrystal-like Fe(III)-hydroxide-phosphate.

    22

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    The ratio of heavy-to-light subunits of ferritin

    pIMw (kDa)

    4.6

    HeLa

    H24L0 550

    Heart

    Kidney

    Liver

    5.7

    H0L24 460

    ironiron

    Nat. Apoover oaover oa

    23

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    Hemosiderin

    Hemosiderin is a water-insoluble iron-protein aggregates present in

    lysosomes and is a by-product of ferritin degradation through incomplete

    . ,

    marrow. Iron stored in hemosiderin is more inaccessible and less

    effective in producing free radicals than iron stored in ferritin. 24

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    metabolism

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    Iron is such an essential element of human life, in fact, that humans have nophysiologic regulatory mechanism for excreting iron. Human bodies tightly

    re ulate iron absor tion and rec clin and revent iron overload solel b

    regulating iron absorption.

    Those who cannot regulate absorption well enough get disorders of iron overload

    haemochromatosis . In these diseases, the toxicit of iron startsoverwhelming the body's ability to bind and store it. Haemochromatosis, is a

    hereditary disease characterized by excessive absorption of dietary iron resulting

    in a pathological increase in total body iron stores. Excess iron accumulates in

    tissues and organs disrupting their normal function. The most susceptible organs

    include the liver, adrenal glands, the heart and the pancreas; patients can present

    with cirrhosis, adrenal insufficiency, heart failure or diabetes mellitus. Iron

    overload may be also the consequence of repeated blood transfusions, or

    diseases that affect the gastrointestinal tract such as Crohns or celiac disease.

    Since so much iron is required for hemoglobin, iron deficiency anemia is the firstan pr mary c n ca man es a on o ron e c ency. xygen ranspor s so

    important to human life that severe anemia harms or kills people by depriving their

    organs of enough oxygen. Iron-deficient people will suffer or die from organ

    26electron transport.

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    Main lo ic of human iron

    metabolism regulation

    1. humans have no physiologic regulatory mechanism

    for excreting iron, but we continually loose iron,

    or bleeding (enteral infections)

    2. human bodies tightly regulate iron absorption andrecycling

    3. human bodies prevent iron overload solely by

    .

    (genetically or coupled to other diseases such as

    develops. 27

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    Summary of human iron metabolism

    dietary iron gut absorption plasma transferrin transport

    receptors on iron-requiring cellsExport of iron via

    ferroportin

    internalization, acidificationEngulfment of dead

    intracellularsynthesis of iron proteinsutilization

    by macrophages

    mobile iron poolemog o n, myog o n,

    cytochromes, etc.)

    ferritin

    s orage(mainly in liver)

    Loss of iron

    hemosiderinNo physiologic excretion mechanism!But iron is highly recycled!28

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    The body regulates iron levels by

    regulating absorption of iron inenteroc tes

    Factors affecting iron absorption

    .

    2. the extent to which the bone marrow is producing

    3. the concentration of hemoglobin in the blood

    4. the oxygen content of the blood

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    Re ulator roteins of iron

    metabolism

    -HFE

    -Hepcidin

    30

    The liver is the central regulator of iron homeostasis Research over the last decade

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    The liver is the central regulator of iron homeostasis. Research over the last decade

    has confirmed that the liver is the primary site of expression of many of the molecules

    res onsible for the re ulation of iron homeostasis. The hereditar hemochromatosis

    (abnormal accumulation of iron) associated molecules HFE (hefaestin), hepcidinexpressed at high levels in the liver. Mouse models of HH, where the genes have been

    disrupted or mutated all result in hepatic iron overload. Constitutive over-expression of

    hepcidin (negative regulator) in the liver results in iron deficiency anemia. Liver-specific

    deletion of HFE in mice recapitulates the phenotype of HH.These studies all suggest a

    major role for the liver in iron metabolism.Our bodies' rates of iron absorption appear to respond to a variety of interdependent

    factors, including total iron stores, the extent to which the bone marrow is producing new

    red blood cells, the concentration of hemoglobin in the blood, and the oxygen content of

    the blood. We also absorb less iron during times of inflammation. Recent discoveries

    demonstrate that hepcidin regulation of ferroportin is responsible for the syndrome ofanemia of chronic disease.

    e o y regu a es ron eve s y regu a ng eac s eps o a sorp on o ron n

    enterocytes. This is achieved within the crypt cells, which sense the availability of

    iron by taking up iron via both transferrin receptor 1 and 2.The affinity of transferrin-

    - , .

    with TfR1 in such a way that binding of HFE to the TfR enhances its affinity for iron-

    transferrin, resulting in an increase of cellular iron uptake. Depending on the amount of

    , , ,

    uptake of the iron, will express the appropriate amount of the Dcytb, DMT1 and

    ferroportin .31

    I i fl i t t t i d t i d

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    Iron influx into enterocyte is determined

    -Stomach

    Fe2+

    Small intestine

    villus

    cell

    (enterocyte) Fe3+

    low pH,

    vitamin Cand/or Steap homolog

    ferrireductases?

    ferroxidasesCerulo-

    plasmin

    ,

    responsible for the uptake

    of the iron from gut, will

    express the appropriate

    (heme carrier

    protein 1)

    Stomach Small intestine

    ,

    ferroportin proteins.

    crypt

    cell

    Fe2+

    low pH,vitamin C

    ferroxidasesCerulo-

    plasmin

    cell)Fe3+

    an or eap omo og

    ferrireductases?

    HCP1(heme carrier

    protein 1)

    32

    cryp ce s, sense e ava a y o ron

    by taking up iron via transferrin receptor 1,

    2 helped by the HFE protein.

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    Iron influx into enteroc te is determined

    by the set-point of precursor cells

    In response to iron deficiency anaemia:

    villus cells produce more Dcytb, DMT1 and ferroportin.

    Villus cell produce less Dcytb, DMT1 and ferroportin.

    33

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    Hepcidin, a circulating peptide hormone, is the master regulator

    of s stemic iron homeostasis, coordinatin the use and stora e

    of iron with iron acquisition. This hormone is primarily produced

    by hepatocytes in response to iron overload or inflammation. Its a negat ve regu ator o ron entry nto p asma. epc n

    functions to reduce serum iron levels by reducing intestinal iron

    types and achieves this by binding to the iron exporterferroportin on the surface of cells and inducing its

    internalisation and degradation. Ferroportin is distributed

    throughout the body on all cells which store iron. Thus,

    regu a on o erropor n s e o y s ma n way o regu a ng e

    amount of iron in circulation.

    34

    Regulatory pathways of hepcidin

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    Regulatory pathways of hepcidin

    Chua et al. (2007) Crit. Rev. Clin. Lab. Sci., 44, 413-459.

    35

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    Hepcidin has antimicrobial properties

    high iron levelin patients having hemochromatosismakes them more susceptible for microbial infection

    inflammation

    n ec onMacrophage Hepatocyte Hepcidin

    -

    Iron release from enterocytes

    and macro ha eslow iron level

    36

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    Iron and bacterial protection

    A proper iron metabolism protects against bacterial infection. If bacteria are to

    survive, then they must get iron from the environment. Disease-causing bacteriado this in many ways, including releasing iron-binding molecules called

    siderophores and then reabsorbing them to recover iron, or scavenging iron from

    hemoglobin and transferrin. The harder they have to work to get iron, the greater

    a metabolic price they must pay. That means that iron-deprived bacteria

    reproduce more slowly. So our control of iron levels appears to be an importante ense aga ns ac er a n ec on. eop e w ncrease amoun s o ron, e

    people with hemochromatosis are more susceptible to bacterial infection.

    To obtain a more perfect protection during bacterial infection, cytokines (such as

    - re ease rom e n amma on s es, w n uce e re ease o epc n.

    (Hepcidin alone is antifungal, and was discovered in urine during a screen forantimicrobial peptides.) Hepcidin functions to reduce serum iron levels, thus

    .

    this mechanism is an elegant response to short-term bacterial infection, it can

    cause problems when inflammation goes on for longer. Since the liver produces

    ,the result of non-bacterial sources of inflammation, like viral infection, cancer,

    auto-immune diseases or other chronic diseases. When this occurs, the

    37

    chronic disease, in which not enough iron is available to produce enough

    hemoglobin-containing red blood cells.

    Haemochromatosis: disorders of iron overload

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    Haemochromatosis: disorders of iron overload

    Hemosiderin is deposited allover the body. Haemochromatosis a hereditary

    disease characterized by excessive absorption of dietary iron resulting in a

    atholo ical increase in total bod iron stores. See models . Excess ironaccumulates in tissues and organs disrupting their normal function. The most

    susceptible organs include the liver, adrenal glands, the heart and the

    pancreas; patients can present with cirrhosis, adrenal insufficiency, heart failure or

    diabetes mellitus. (Iron overload may be also the consequence of repeated blood

    transfusions, or diseases that affect the gastrointestinal tract such as Crohns orceliac disease.) 38

    Crypt-programming model of hemochromatosis

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    Crypt-programming model of hemochromatosis

    39

    Li h idi d l f h h t i

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    Liver hepcidin model of hemochromatosis

    40

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    Regulation of ironme a o sm a e eve o

    What is regulated?

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    What is regulated?

    Ferritin concentration Number of tansferrin rece tor

    depends on iron amount inside of the cell

    .

    not necessary to uptake more iron, so less transferrin

    receptor is required to expressed on the cell surface,

    but more ferritine is required to store excess iron

    2, When iron level is low inside the cell:

    No need to express storage protein (ferritine), but more

    42

    paralely an a recyprocal way by the same regulatoryprotein!

    In human cells the best characterized iron sensing mechanism is the result of translational

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    In human cells, the best characterized iron-sensing mechanism is the result of translational

    regulation of mRNA of proteins involved in iron metabolism: transferrin receptors, and for

    ferritin.

    When iron level is low inside the cell an iron sensing protein (IRE-BP, a FeS cluster

    protein) binds to special mRNA sequences of ferritin and transferrin receptor mRNA and

    receptor synthesis (by stabilising its mRNA).

    When iron level is high iron binds to the iron sensing protein (IRE-BP) the protein changes

    shape with the result that the it can no longer bind the ferritin and transferrin receptormRNA, as a consequence the result is just the oposite seen above, so transferrin is readily

    translated , but no transferrin made. (Interestingly, in iron-bound state the IRE-BP functions

    as a cytosolic aconitase.)

    - -, .

    more transferrin receptors make it easier for the cell to bring in more iron from transferrin-

    iron complexes circulating outside the cell. But as iron binds to more and more IRE-BPs,

    they change shape and unbind the transferrin receptor mRNA. The transferrin receptor

    mRN is rapidly degraded without the IRE-BP attached to it. The cell stops producing

    transferrin receptors. When the cell has obtained more iron than it can bind up with ferritin or

    heme molecules, more and more iron will bind to the IRE-BPs. This will initiate ferritin

    .

    (Detailed mechanism: the special mRNA sequences (called iron response elements=IRE)

    located at different ends of the two mRNAs. If it is located at the 5 end, binding of IRE-BP

    43

    n ts trans at on o t e m . t s ocate at t e en , t protects m rom

    degradation leading to more protein synthesis. )

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    -

    IRE

    44

    SUMMARY

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    SUMMARY

    + e

    -Fe

    45

    Utilization of iron

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    Utilization of iron

    -

    hemoglobin, myoglobin, cytochromes, oxidases, peroxidases

    - ron-su ur c us er pro e ns:

    ferredoxin, succinate dehydrogenase, aconitase, etc.

    - Other iron containing proteins:

    amino acid hydroxylases (Phe, Tyr, Pro, Lys), acid phosphatase,

    homo entisinate diox enase, ribonucleotide reductase

    46

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    HEME / HAEM METABOLISM

    Structure of heme

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    Structure of heme

    COOHCOOH

    NN

    Fe

    Fe-protoporphyrin IX

    Porphyrins are cyclic compounds that readily bind metal ionsusually Fe2+or Fe3+,

    and formed by the linkage offour pyrrole rings through methenyl bridges.

    The most prevalent metalloporphyrin in humans is heme, which consists of one

    48

    errous e ron on coor na e n e cen er o e e rapyrro e r ng o pro o

    porphyrin IX.

    Heme is the prosthetic group for hemoglobin, myoglobin, the cytochromes, catalase,

    nitric oxide s nthase and eroxidase.

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    49

    Tetrapyrrole biosynthetic pathways

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    py y p y

    (5-aminolevulinate)(in most bacteria and plants)

    (in most eukaryotes)

    corin ringporphyrin ring

    50

    Porphyrins are cyclic compounds that readily bind metal ionsusually Fe2+or Fe3+.

    The most prevalent metalloporphyrin in humans is heme which consists of one ferrous (Fe2+) iron

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    The most prevalent metalloporphyrin in humans is heme, which consists of one ferrous (Fe2+) iron

    .

    Heme is the prosthetic group for hemoglobin, myoglobin, the cytochromes, catalase, nitric oxidesynthase, and peroxidase. These heme proteins are rapidly synthesized and degraded. (For

    example, 67 g of hemoglobin are synthesized each day to replace heme lost through the normal

    urnover o ery rocy es. oor na e w e urnover o eme-pro e ns s e s mu aneous

    synthesis and degradation of the associated porphyrins, and recycling of the bound iron ions.

    Structure of porphyrins

    Porphyrins are cyclic molecules formed by the linkage of four pyrrolerings through methenyl bridges(F

    Slide : ). Three structural features of these molecules are relevant to understanding their medical

    significance.

    1. Side chains: Different or h rins var in the nature of the side chains that are attached to each

    of the four pyrrole rings:

    Uroporphyrin contains acetate (CH2COO) and propionate(CH2CH2COO) side chains,Coproporphyrin contains methyl(CH3) and propionate groups,

    = , , .

    The methyl and vinyl groups are produced by decarboxylation of acetate and propionate side

    chains, respectively.

    2. Distribution of side chains: The side chains of porphyrins can be ordered around the

    e rapyrro e nuc eus n our eren ways, es gna e y oman numera s o . n y ypeporphyrins, which contain an asymmetric substitution on ring D (see Figure21.2), are

    physiologically important in humans. (Protoporphyrin IX is a member of the Type III series.)

    3. Porphyrinogens: These porphyrin precursors (for example, uro-porphyrinogen) exist in a

    51

    chemically reduced, colorless form, and serve as intermediates between porphobilinogen and

    the oxidized, colored protoporphyrins in heme biosynthesis

    Overview of heme synthesis

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    y

    Gly + Suc-CoA

    HEME

    8

    mitochondrionmitochondrion

    Fe2+

    pyridoxal

    phosphate

    protoporphyrin IX1

    7

    -aminolevulinic acid (ALA) protoporphyrinogen IX

    6

    Porphobilinogen (PBG)

    uroporphyrinogen IIIcoproporphyrinogen III3 4

    5

    -

    cytoplasmcytoplasm

    The organs mainly involved in heme synthesis are the liver and the bone marrow.52

    Biosynthesis of heme (1)

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    The major sites of heme biosynthesis are the liver, which synthesizes a number of heme

    proteins (particularly cytochrome P450 proteins), and the erythrocyte-producing cells of the bone

    marrow, which are active in hemoglobin synthesis. (Over 85% of all heme synthesis occurs in

    erythroid tissue.) In the liver, the rate of heme synthesis is highly variable, responding to

    .

    contrast, heme synthesis in erythroid cells is relatively constant, and is matched to the rate of

    globin synthesis.

    The initial reaction and the last three steps in the formation of porphyrins occur in mitochondria,whereas the intermediate steps of the biosynthetic pathway occur in the cytosol. (Slide. ).

    (Mature red blood cells lack mitochondria and are unable to synthesize heme.)

    .

    porphyrin molecule are provided by glycine (a nonessential amino acid) and succinyl

    coenzyme A (an inter-mediate in the citric acid cycle) that condense to form ALA in a

    reaction catalyzed by ALA synthase (ALAS) .This reaction requires pyridoxal phosphate

    (PLP) as a coenzyme, and is the committed and rate-limiting step in porphyrin biosynthesis.

    (There are two isoforms of ALAS, 1 and 2, each controlled by different mechanisms.

    Erythroid tissue produces only ALAS2,the gene for which is located on the X-chromosome.

    - .2. Formation of porphobilinogen: The condensation of two molecules of ALA to form

    porphobilinogen by Zn-containing ALA dehydratase (porphobilinogen synthase) This enzyme

    is extremely sensitive to inhibition by heavy metal ions, for example, lead that replace the

    53

    zinc. This inhibition is, in part, responsible for the elevation in ALA and the anemia seen in

    lead poisoning.

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    Overview of heme synthesis

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    Overview of heme synthesis

    Reaction catalyzed by ALA synthase is the rate-limiting reaction

    , .

    Aminomethyl -bilane

    e y ra ase

    55Side chains: A: acetyl; P: prppionyl; M. methyl; V: vinyl.

    Conversion of Uroporphyrins to Coproporphyrins

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    -

    | CH3

    2

    CH2 |

    Acetyl- Methyl-

    (A)4x

    56

    Conversion of Coproporphyrins to Protoporphyrins

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    COO-

    CH2

    2

    CH2

    | |

    | |

    Propionyl-

    (P)Vinyl-

    (V)

    2x

    57

    Steps of Heme Synthesis (7)

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    ro oporp yr nogen ox ase conver s e me y ene r ges e ween

    the pyrrole rings to methenyl bridges.58

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    Names of Porphyrins

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    , . ., .

    substituents found on the ring, and the number denotes how they are arranged.

    WORDS: uroporphyrin, coproporphyrin, protoporphyrin

    AP MP MPV

    , , ,

    In series I the substituents repeat in a regular manner: AP AP AP AP.

    Series II does not occur in natural systems.

    In series III the order of substituents in ring IV is reversed: AP AP AP PA.

    Series IV does not occur in natural systems.

    Porphyrin vs Porphyrinogen

    .

    The porphyrins contain a system of conjugated double bonds all around the tetrapyrrole ring,

    which makes the porphyrins more stable than the corresponding porphyrinogens. 60

    Regulation of Heme Synthesis

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    synthesis of new enzyme cytoplasmcytoplasm

    -ALA synthase

    Gly + Suc-CoA

    HEME

    8

    m oc on r onm oc on r on

    Fe2+

    -

    pyridoxal

    phosphateALA

    synthase

    1

    7

    -am no evu n c ac

    2

    6

    porphobilinogen

    uroporphyrinogen IIIcoproporphyrinogen III

    aminomethyl-bilane

    3 4

    5

    61

    Regulation of Heme and Globin Synthesis

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    - Substrate availability: iron (II) must be available for ferrochelatase.

    - Feedback regulation: heme is a feedback inhibitor of ALA synthase.

    - Subcellular localization: ALA synthase is in the mitochondria,

    w ere e su s ra e, succ ny o , s pro uce .

    ALA synthase is synthesized in the cytoplasm,

    its transport across the mitochondrial membrane may be regulated.

    - In erythropoietic cells, heme synthesis is coordinated with globin synthesis.If heme is available, globin synthesis proceeds. If heme is absent:

    - Effects of drugs:

    barbiturates and certain steroids can increase heme synthesis

    - , .

    62

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    Defects in heme s nthesis

    The porphyrias are classified depending on whether the enzyme

    deficienc occursIn the erythropoietic cells of the bone marrow: ErythropoieticIn the liver: Hepatic

    Either type may be hereditary or acquired.

    The symptoms are caused by accumulation of intermediates

    and deficiency of heme.

    Accumulated intermediares are converted by nonenzymatic (light,

    ox a ve e ec s s eps rom porp yr nogens o unuse u porp yr nswhich makes photosensitivity.

    orp yr a re ers o e purp e co or cause y p gmen - e por-

    phyrins in the urine.63

    Defects in heme synthesis

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    Pb poisoning3 1

    4

    25

    Pb

    6

    Porpyrias of erithropoietic origin1: erithropoietic porphyria

    2: hereditary protoporphyria

    Porphyrias of liver origin

    3: acute intermittent porphyria

    4: porphyria cutanea tarda

    5: hereditary coproporphyria6: variegate porphyria 64

    Porphyrias

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    1. individuals with an enzyme defect

    prior to the synthesis of the

    tetrapyrroles manifest abdominal

    - ,

    2. enzyme defects leading to the

    accumulation of tetrapyrrole

    intermediates show

    ,itches and burns (pruritus) when

    exposed to visible light.

    (Photosenstivity is a result of the

    porphyrinogens to colored

    porphyrins, which arephotosensitizing molecules that are

    formation of superoxide radicals

    from oxygen. These reactive oxygen

    species can oxidatively damage

    ,of destructive enzymes from

    lysosomes.)

    65red urine, injured skin

    Acute intermittent porphyria

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    Gl cine + Succin l-CoA ALA PBG // ... Heme Hemo roteins

    PBG

    deaminase

    ALAsynthase

    ALA PBG Heme

    no feedback inhibition!

    //

    .

    activity is sufficient to produce heme for erythropoiesis. In the liver, however, if heme is

    utilised or degraded by an elevated rate (e.g. certain drugs, hormones or ethanol are

    metabolised b c tochrome P450 containin enz mes, the induce the level of this heme

    containing enzyme) the decrease in the levels of heme induces ALA synthase. Under

    these conditions the elevated levels of PBG cannot be further converted by PBG

    deaminase. Both PBG (red urine) and ALA (neurotoxicity) are accumulated. Symtomps

    abdomen syndrom, neurological abnormalities. Can be treated by infusion of high

    concentration of heme. Barbiturates must be avoided beacuse they increase the level of

    ALA synthase.

    66

    Summary of heme synthesis

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    - It occurs in virtually all tissues but the highest rate is found in the

    ver an one marrow.

    - The first and the last three enzymes are located in the mitochondria.The middle 4 enzymes are located in the cytosol.

    - Heme is s nthetized from 8 l cine and 8 succin l-CoA molecules.

    - During synthesis the side chain modifications occur on the colorless.

    - The last step oxidizes it to porphyrin (methylen to methene bridges)

    .

    - Porphyrins are produced by nonenzymatic (light, oxidative effects)

    s eps rom porp yr nogens n porp yr as.

    67

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    68

    Degradation of heme

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    69

    A HEME

    Recycled!

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    degradation Hemeoxigenaseslpeen, macrophages

    Biliverdin

    Biliverdin reductase

    UDP

    glkuronil

    transzferz

    BLOOD

    LIVER

    (albumin)Bilirubin

    BILE Bilirubin

    Bacterial flora

    dconjugation, redcution

    Saturation of methenyl

    , INTESTINE

    KIDNEY

    70

    feces urineStercobilin Urobilin

    BilirubinThe high lipid solublity of bilirubin dictates

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    The high lipid solublity of bilirubin dictates

    - that it must be transported in the blood by a carrier(serum albumin)- that it is soluble in the lipid bilayers of cell membranes

    - that it must be conjugated to a water-soluble substance for excretion

    Bilirubin diglucuronide is excreted in the bile. It is subject to subsequent

    rans orma ons o o er spec es y e n es na ora.

    The clinical determination of plasma bilirubin distinguishes between conjugated

    .

    - Direct and indirect bilirubin values are used in the differential diagnosis

    ofhyperbilirubinemia.

    71

    Jaundice (also called icterus) refers to the yellow color of skin, nailbeds, and sclerae (whites of

    the eyes) caused by deposition of bilirubin, secondary to increased bilirubin levels in the blood

    h erbili rubinemia . Althou h not a disease, aundice is usuall a s m tom of an underl in

    disorder

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    disorder.

    Types of jaundice: Jaundice can be classified into three major forms described below. However, inclinical practice, jaundice is often more complex than indicated in this simple classification. For

    ,

    metabolism. a. Hemolytic jaundice: The liver has the capacity to conjugate and excrete over

    3,000 mg of bilirubin per day, whereas the normal production of bilirubin is only 300 mg/day. This

    excess capacity allows the liver to respond to increased heme degradation with a correspondingincrease in conjugation and secretion of bilirubin-diglucuronide. However, massive lysis of red blood

    cells (for example, in patients with sickle cell anemia, pyruvatekinase or glucose 6-phosphate

    dehydrogenase deficiency) may produce bilirubin faster than it can be conjugated. Unconjugated

    ,

    excreted into the bile, the amount of urobilinogen entering the enterohepatic circulation is

    increased, and urinary urobilinogen is increased.] b. Hepatocellular jaundice: Damage to livercells (for example,in patients with cirrhosis or hepatitis) can cause unconjugated bilirubin levels in

    the blood to increase as a result of decreased conjugation. Urobilinogen is increased in the urine

    because hepatic damage decreases the enterohepatic circulation of this compound, allowing more

    to enter the blood, from which it is filtered into the urine. The urine thus darkens, whereas stools

    , . .If conjugated bilirubin is not efficiently secreted from the liver into bile (intra-hepatic cholestasis), it

    can diffuse (leak) into the blood, causing a conjugated hyperbilirubinemia.] The similar thing

    hapens in case of neonatal jaundice.

    c. Obstructive jaundice: In this instance, jaundice is not caused by overproduction of bilirubin or

    decreased conjugation, but instead results from obstruction of the bile duct (extrahepatic

    cholestasis). 72

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    Normal Hemol tic aundice

    73

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    Normal Physiological (neonatal) jaundice

    In neonates, benign jaundice tends to develop because of two factors:

    - the breakdown of fetal hemoglobin as it is replaced with adult hemoglobin

    - immature hepatic metabolic pathways which are unable to conjugate and so excrete bilirubin as quickly as an adult.

    Infants with neonatal jaundice are treated with colored light called phototherapy.

    Phototherapy works through a process ofisomerization that changes the bilirubin into water-soluble isomers

    that can be passed without getting stuck in the liver.Wikipedia

    74

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    Normal Biliary obstruction

    75

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    76

    Functions of HemoglobinLung Circulation Tissues

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    Lung Circulation Tissues

    Hb.4O2O2O2inhaled

    respiratory

    chain

    TCAexhaled

    cycle

    2 2

    carbonic anhydrase

    2 + 2

    carbonic anhydrase

    +

    2 3H2CO3

    .

    Hb.carbamateH+ + HCO3

    -H+ + HCO3

    -

    77

    Quaternary structure of hemoglobin

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    Hemoglobin

    Quaternary structure: 4 subunits!

    Four heme, four Fe2+, four O2

    The 4 monomer are kept together by

    secondar bonds:

    Salt bridges

    Hydrogen bonds

    78

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    HgA1: 22

    79

    Structure of one subunit of hemoglobin

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    Tertiary structure of globin chain

    -helix A: Ser3- Gly18

    helix B: His20-Ser35

    helix C: Phe36-Tyr42

    helix D: His50-Gly51

    helix E: Ser52-Ala71

    helix F: Leu80-Ala88

    -

    helix H: Thr118-Ser138

    (Name of the loop between two helices is

    composed from names of the two helices: eg.

    AB, CD)

    The hem group is found in the apolar

    polar groups facing the surface.80

    Structure of heme

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    COOHCOOH

    N

    NN

    N

    Fe

    Heme is the prosthetic group for hemoglobin (myoglobin)

    Heme consists of one ferrous (Fe2+) iron ion coordinated in the center of he

    tetrapyrrole ring of proto porphyrin IX.

    Fe-protoporphyrin IX81

    Tertiary structure of hemoglobin

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    Haemoglobin is a globular verytightly stuffed compaque

    molecule. H dro hobe inside

    proximal His

    hydriphyl side chains outside.

    Heme is inside of the hydrophobe

    ocket. Isolated fee heme unableto keep Fe in 2+ state, only

    pocked inside the globin chain.

    If Fe is oxidized to Fe3+ ferri

    (methemoglobin) it cannot bind

    O2. Iron has 6 coordinative

    (covalent ) bindings:

    - .

    5.: His-F8 of globin (proximal His)

    This makes the bond between

    oxygen

    bindin site

    6.: O2

    82

    His-E7 helps to bound O2(distal His)

    (no O2 is show here)

    Polimorfism of globinsI. II. III.

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    I.Embrionyc haemoglobins

    2 2

    Hb Gower 2 22

    or an 22

    II.Foetal Haemoglobins

    Hb F 2 2

    III.Adult haemoglobins

    HbA1 (98%)

    HbA2 2% 22

    83

    Expression of hemoglobin genes

    during development is related tothe changing oxygen uptake

    Comparison of Mb and Hb

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    Myoglobin

    One ol e tide chain

    Hemoglobin

    Four ol e tide chains

    One heme, one Fe, one O2 Tertiary structure only

    Four heme, four Fe, four O2 Tertiary and quaternary structure

    O2 storage in muscle Regulated affinity to O2 binding

    O2 transport in RBC84

    Ox en-bindin to Hb

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    100Oxygen-binding curve for Hb

    - sigmoidal / cooperative

    - low affinity in the veins

    - hi h affinit in the arteries80

    ion

    - p50 25 mmHg

    40s

    atura

    20% venous

    pressure

    arterial

    pressure

    00 20 40 60 80 100

    pO2

    (mmHg)

    85

    O -bindin to Mb

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    100Oxygen-binding curve for Hb

    - hyperbolic / non-cooperative

    - high affinity for O2,

    higher than that of Hb

    80

    ion

    - p50 1 mmHgHbMb

    40s

    atura

    20%

    00 20 40 60 80 100

    pO2

    (mmHg)

    86

    O2-binding causes conformational changes

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    87

    The uaternar structure chan es

    I d Hb i i t f th l f th

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    In deoxyHb iron is out of the plane of theheme. Followin oxi en bindin iron moves

    into the plane. The movement of iron is

    followed by the movement of the protein

    .

    Upon oxygen binding:

    1

    1

    twists relative to 2

    2 heme-heme distance reduces

    centra cav ty constr cts

    In short, the deoxy state relaxes andswitches to the oxy state. These changes

    transmits the structural chan es to the other

    heme groups and INCREASES their O2

    binding. This is cooperativity.88

    v y

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    100Oxygen-binding curve for Hb

    60

    80

    turation

    0

    20%s

    pO2

    (mmHg)

    In other words the oxygen binding to the next subunits requires less energy

    89

    because part of the salt bridges are already broken. That is why the affinity

    becomes larger. This explains the sigmoid saturation curve.

    u y

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    O2 affinity is decreased by:

    1. 2,3BPG (2,3-bisphospho-glycerate)

    -produced by the shunt of the glycolysis in RBC

    -releases O2

    2. Low pH- Bohr effect,-metabolicall active tissues CO and H+

    -releases O2

    . -metabolycally active warm tissues

    90

    . m noac sequence

    -Foetal Hb binds O2 with more affinity than adult Hb

    Control: 1. 2,3-bisphospho-glycerate

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    2,3 BPG binds to the positively charged

    beta chains.

    -helps the relase of oxygen at tissues

    -reduces O2 affinity

    At high altitude the level of BPG increases

    facilitating the release of oxygen at tissues.

    91

    At low external oxygen more 2,3 BPG binds to the

    increased amount of deoxiHb, 2,3BPG will not

    inhibit its own production (BPG mutase). IncreasedBPG synthesis.

    Glucose S nthesis of 2 3-BPG

    Glucose 6 P

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    Glucose-6-P

    Biphosphoglycerate mutase

    , -

    2,3-biphosphoglycerate

    -

    ADP

    ATP

    -

    P ruvate

    ,

    At hi h altitude the level of BPG increases facilitatin the

    Lactate

    release of oxygen at tissues. At lowexternal oxygen more 2,3

    BPG binds to the increased amount of deoxiHgb, 2,3BPG will

    not inhibit BPG mutase. Increased BPG synthesis.

    92

    Control: 2. The Bohr effect

    Metabolically active tissues are rich on CO and H+

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    Metabolically active tissues are rich on CO2 and H+.

    + 2 2 .

    Why?

    NH3+

    R CO 2

    N

    R

    CO 2-

    N-terminus of

    + +-

    O

    His 146 of Asp 94 of

    These additional char es form additional salt brid es to further

    cross-link the Hb quaternary structure and stabilize the tense deoxy

    state. Hence, they lead to the release of O2.

    93

    Control: 2. The Bohr effect

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    low level of CO2

    high level of CO2

    o ncrease an 2 concen ra ons

    decrease the affinity of Hb for O2 94

    Control: 3. Temperature

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    Hb is a temperature controller. O2 binding to Hb is (usually) exothermic; oxygen release from Hb isendotermic, that is, heat is given out. This also means that when oxyHb arrives at muscle, heat is

    required to liberate O2. Whilst this isnt generally a problem to humans, it is for animals from colder

    .

    needed to free oxygen. At the other extreme, in the heavily worked flight muscles of some birds,

    efficient heat loss is essential to avoid overheating. Here O2

    release requires 3 times as much heat as it

    does in man.

    Control: 4. Amino acid se uence

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    HbA: 22

    HbF: 22

    One of the changes in the chain vs

    e c a n s s er, w c es n

    the central cavity.

    deoxyHbF for BPG relative to

    deoxyHbA

    This increases the affinity of HbFfor O2.

    96

    Abnormal hemoglobinsPoint mutations in the core region

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    Hemoglobin M

    Change E7 or F8 His to Tyr,

    therefore Fe2+ oxydizes to Fe3+,

    therefore it cannot bind O2.97

    Abnormal hemoglobinsMutations at subunit interfaces

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    Sickle cell anemia

    Hemoglobin S: Glu6Val in chains

    Wikipedia

    98

    Abnormal hemoglobinsMutations at subunit interfaces

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    mutation

    Altered surface of deoxyHbS causes polymerization

    Wikipedia

    99

    Abnormal hemoglobins

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    Thalassemias

    .the severity of the disease might vary.

    Glucose is spontaneously covalently bound to Hb.

    % of Hb glucosylated depends on blood sugar levels.

    Significance in the early diagnosis of diabetes mellitus.

    100

    CONTENTS

    I. IRON METABOLISM

    1. Introduction

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    .

    3. Transport of iron and Storage of iron

    4. Regulation of iron metabolism: hepcidin

    II. HEME METABOLISM

    1. Biosynthesis of heme, Porphyrias

    2. Degradation of heme, Jaundice

    III. HEMOGLOBIN, MYOGLOBIN

    1. Structure of hemoglobin

    2. Polymorphism of globins

    . , , , ,

    4. Abnormal hemoglobins: Sicle cell anemia, MetHb, HbA1c

    101

    Exam essa uestions

  • 7/31/2019 2.Iron Heme Hb

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    . , ,

    storage of iron. Regulation of iron uptake at body and cellular level.

    2. Heme synthesis, Porpyrias.

    3. Heme breakdown. Jaundice.

    4. Hemoglobin: structure and function, Regulation of O2 binding .Globin

    polymorphysm and abnormalities.

    102

    Example for Simple questions

    Give a short definition to

    1. Ferritin

    2. Transferrin3. Transferrin receptor

    1. List Heme containing proteins of human body!

    2. What is the mechanism of iron uptake to

    ll ?

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    3. Transferrin receptorcells?

    4. Hepcidin

    5. Ferroportin

    6. DMT1

    3. What is effects of hepcidine?

    4. List 3-5 intermediates of heme synthesis!

    -7. Hemocromatosis

    8. Porpyrins

    9. Heme

    .

    6. Classification of porphyrias?

    7. Types of jaundice and short explanation to

    them!10. ALA synthase

    11. Ferrochelatase12. Porhyrias

    8. List the factors which affect O2 binding of Hg!

    9. What is the composition of adult and foetalHg?

    13. Hemoxigenase

    14. UDP glucuronyl transferase

    15. Bilirubin

    16. Hemoglobin

    17. Myoglobin

    18. Coo erativit

    10319. Bohr effect

    20. Sicle cell anemia

    21. H A1c