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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
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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