• Hemoglobin structure & synthesis

Hemoglobin
• is the iron-containing oxygen- transport 
metalloprotein in the red blood cells of 
vertebrates, and the tissues of some 
invertebrates.
Hemoglobin in the blood, transports oxygen from
the lungs or gills to the rest of the body (i.e. the
tissues) where it releases the oxygen for cell
use.
 Hb Variants

• HbA2
– 22
– Present in ~2% of adults

• Embryonic Hb
– 22
– Has  affinity for O2

• Fetal Hb
– 22
– Has  affinity for O2
http://oregonstate.edu/instruction/bb450/stryer/ch10/Slide27.jpg
 The subunit composition of hemoglobin tetramers
undergoes complex changes during development
• initially a ζ2ε2 tetramer.
• end of the first trimester, ζ and γ subunits
-----replaced by α and ε subunits, forming HbF
(α2γ2), the hemoglobin of late fetal life.
• synthesis of β subunits begins in third
trimester.
• β subunits do not completely replace γ
subunits to yield adult HbA (α2β2) until some
weeks postpartum
 Hb
• makes up about 97% of the red blood cell's dry
content, and around 35% of the total content
(including water)
• Hb has an oxygen binding capacity between 1.36
and 1.37 ml O2 per gram of Hb, which increases
the total blood oxygen capacity seventyfold.

• Other cells that contain hemoglobin =

– dopaminergic neurons in the substantia nigra, 
– macrophages, 
– alveolar cells,
– mesangial cells 
non-oxygen-carrying function as an antioxidant
 and a regulator of iron metabolism.
 Synthesis
• a complex series of steps.
• heme part, a series of steps in the mitochondria and
the cytosol of immature red blood cells,
•  globin protein parts, by ribosomes in the cytosol. 
• Production of Hb continues in the cell throughout its early
development from the proerythroblast to the reticulocyte in the bone
marrow.
• At this point, the nucleus is lost in mammalian red
blood cells, but not in birds and many other
species. Even after the loss of the nucleus in
mammals, residual ribosomal RNA allows further
synthesis of Hb until the reticulocyte loses its RNA
soon after entering the vasculature .
 HEME SYNTHESIS: Liver

•The liver is the main non-RBC source of heme synthesis

•Heme produced in the liver is used mainly for the

synthesis of the cytochrome P450 class of enzymes that
are involved in detoxification

Regulated at level of ALA synthase: Formation of 5-ALA

is the rate-limiting step in heme synthesis in the liver
Hemoglobin Structure

Beta-
Alpha-
type
type

Haem

Beta- Alpha-
type type
 Normal adult Hb
• HbA (alpha2/beta2)
– 97% +
• HbA2 (alpha2/theta2)
– 2-3%
• HbF (alpha2/gamma2)
– 0.5% or less

• NOTE ALL NEED ALPHA!

Iron Utilization
Heme synthesis
• PORPHYRINS and IRON
• are cyclic compounds
formed by the linkage of
four pyrrole rings through –
HC = methenyl bridges
• porphyrias = caused by abnormalities
in the pathway of biosynthesis, not
very prevalent
• Jaundice bilirubin in the, due to
overproduction or to failure of its
excretion.
• A characteristic property of the
porphyrins is the formation of
complexes with metal ions bound to
the nitrogen atom of the pyrrole rings
Natural Porphyrins having Substituent Side Chains
• Fischer shorthand formula
• methenyl bridges are
omitted and each pyrrole
ring is shown with the eight
substituent positions
• The arrangement of the
Uroporphyrin III. A (acetate) = -CH2COOH;
acetate (A) and propionate P (propionate) = -CH2CH2COOH
(P) substituents in the
uroporphyrin (in ring IV, the
expected order of the A and
P substituents is reversed).
• this type of asymmetric
substitution is classified as
a type III porphyrin.
Heme and protoporphyrin IX are both type III
porphyrins
• A completely symmetric
arrangement = as a type I
porphyrin.
• Only types I and III are
found in nature
• type III series is more
abundant and important
b/c includes heme .
• and its immediate
precursor,
•MITOCHONDRIA & CYTOSOL
MITOCHONDRIA
STARTING MATERIAL
B6
RATE LIMITING ENZYME
 O
 
OOC CH2 CH2 C S-C o A + OOC CH2 N H 3+
succinyl-CoA H+ glycine
-A m inolevulinic
A cid Synthase C o A-SH CO2
O

OOC CH2 CH2 C CH2 N H 3+
-am inolevulinate (A LA )

Heme synthesis begins with condensation of

glycine & succinyl-CoA, with decarboxylation, to
form -aminolevulinic acid (ALA).
 H O
Pyridoxal phosphate (PLP) O C
O
serves as coenzyme for - P
H2
C OH
Aminolevulinate Synthase O
(ALA Synthase), an enzyme O
evolutionarily related to 
N CH3
transaminases. H
Condensation with succinyl- P y rid ox al p h o sp h ate (P L P )
CoA takes place while the
amino group of glycine is in H2C COO
Schiff base linkage to the N+
PLP aldehyde. O HC H
O H2
CoA & the glycine carboxyl P C O
are lost following the O
O
condensation. 
N CH3
H
glycine-PLP Schiff base (aldimine)
• Cytosol
two molecules of ALA are condenses
ALA dehydratase is a zinc-containing enzyme
is sensitive to inhibition by lead ( lead poisoning.)
The Zn++ binding sites in the homo-octomeric mammalian
Porphobilinogen Synthase, which include cysteine S
ligands, can also bind Pb++ (lead).
Inhibition of Porphobilinogen Synthase by Pb++ results in
elevated blood ALA, as impaired heme synthesis leads to
de-repression of transcription of the ALA Synthase gene.
High ALA is thought to cause some of the neurological
effects of lead poisoning, although Pb++ also may directly
affect the nervous system. COO COO

CH2 CH2
ALA is toxic to the brain, perhaps due to:
CH2 CH2
• Similar ALA & neurotransmitter GABA
C O CH2
(-aminobutyric acid) structures.
CH2 NH3+
• ALA autoxidation generates reactive
NH3+
oxygen species (oxygen radicals).
ALA GABA
3) Acute intermittent porphyria
( phosphobilinogenin deaminase deficiency, the most common hepatic porphyria).
1. Symptoms - excrete PBG and ALA, high plasma PBG/ALA
- severe abdominal pain, nausea, vomiting, hypertension(cardiovascular)
then CNS - anxiety, insomnia, confusion, hallucination, paranoia then
peripheral neuropathy - fatal respiratory paralysis
2. a. PBG deaminase deficiency), autosomal dominant
b. Genetic and precipitated by phenytoin, phenobarbital drugs, alcohol, fasting,
hormones estrogen, stress, infection
c. lead poisoning
3. Biochemistry of neurotoxicity
a. heme deficiency   P450 drug metabolism
  tryptophan dioxygenase  brain tryptophan
 brain serotonin
 mitochondrial cytochromes i.e. a mitochondrial disease
b.  ALA  , mitochondrial damage, DNA damage
4. Therapy - heme arginate on admission (taken up by liver not b.m.)
5. Mouse model (PBG knockout) - called Vincent! - behavioural studies
Suspects - Van Gogh, King George III(?) (Absinthe for insomnia)
tryptophan kynuramine

heme
deficiency

serotonin 20
4) Congenital erythropoietic porphyria (Erythroid, Photosensitive)
-Fe overload of b.m., uroporphyrin overload of skin and erythrocytes
 hemolysis

1. Symptoms: skin lesions (light) , risk of infection in early infancy

Anemia (hemolysis)
Teeth reddish brown (fluorescence)
2. Uroporphyrinogen III synthase deficiency
• Uroporphyrinogen I and coproporphyrin I accumulation in the skin,
bone marrow, erythrocytes, urine, plasma
3. Treatment: activated charcoal (oral); b.m. transplant; gene therapy for bone
marrow; blood transfusion to stop erythropoiesis in b.m.
4. Only 200 diagnosed so far; werewolf, vampires (early childhood sensitive
to light skin blisters, infection  disfigures) is not porphyria

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5) Porphyria Cutanea Tarda - “heme deficiency and uroporphyrin
overload of liver” (Photosensitive; most common and readily treated).

1. Symptoms: lesions on backs of hands and face due to photosensitization by

uroporphyrin I, III or uroporphyrinogen. Diagnosis usually leads to diagnosis of
hemochromatosis.
2a. Uroporphyrinogen decarboxylase deficiency (liver but also erythrocyte in
type II): or hexachlorobenzene or dioxin induced CYP 1A2
(Turkey disaster).
Also Fe uroporphyrinogen III synthase - uroporphyrin I or
uroporphyrin.
b. Candidate hemochromatosis gene
c. provoked by alcohol, hepatitis C virus, estrogen (birth control, post-meno-
pause, prostate, pregnancy), Fe.
3. Biochemistry - inactivation of decarboxylase (-SH enzyme) by “ROS” from
reduced P450/P450 reductase/Fe
4. Therapy: - phlebotomy
- Fe chelators e.g. desferoxamine and ascorbic acid
- chloroquine  endocytosis and release uroporphyrin chloroquine
complex from hepatocytes

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6) Hereditary Coproprophyria - coproporphyrinogen oxidase deficient, chronic
fatigue syndrome. - hepatic porphyria
Symptoms: liver cancer

7) Porphyria variegata - protoprophyrinogen oxidase deficient. –hepatic and skin

porphyria
Gene locus 1q2320. 20,000 S. Africans descended from a Netherlands lady
in 1655 who went to S. African for marriage
Symptoms: skin and acute porphyria if exposed to sunlight.
No treatment

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8) Erythropoietic protoporphyria - association with liver cancer, red urine
“porphyrin and Fe overload disease” “Skin photosensitive”
a. Ferrochelatase partial deficiency or LEAD poisoning
b. Symptoms: photosensitive skin (infancy) and black liver
c. Biochemistry - protoporphyrin IX accumulates in erythrocyte membranes
and liver; intramitochondrial Fe accumulates in bone marrow - skin photo-
sensitivity results from protoporphyria IX effluxing erythrocytes into
plasma.
d. Therapy -  carotene tried but poor

I. Liver transplant; operation is difficult as operating room must be

kept dark (only yellow acetate filters)
II. Abdominal burns; transfused blood is destroyed as blood difficult
to store.

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