Hema lec 6

Hemoglobin synthesis and function ➢ Changes during fetal life and early infant life
Prepared by: Ms. Arbee Castro ➢ Change also after birth

HEMOGLOBIN
➢ Synthesized within maturing erythrocytes
➢ Metalloprotein (combination of metal +
protein)
➢ Composed of four polypeptide chain with its
own heme
-4heme+4globin chains
➢ O2 delivery to tissues
➢ Transport waste products like CO2
Protoporphyrin IX and iron (makes up heme
➢ Maintaining blood ph (act as buffer)
molecule)
➢ Protoporphyrin IX – synthesized in
Components of hemoglobin
mitochondria and partly cytoplasm of nucleated
4 constituents
red cells
1. globin- 2 sets of dimers
➢ Fe2+ is added forming ferroprotoporphyrin
2. 4 protoporphyrin IX
IX – forms heme molecule
3. 4 iron atoms (Fe2+)
➢ Form chemical bonds with globin - forms
4. one 2-3diphosphoglycerate (2-3DPG)
hemoglobin molecule
Note: Transient component of hemoglobin
2-3diphosphoglycerate
Depending on form Hb molecule, it can either
➢ 2-3DPG – produced in the Embden Meyerhof
release or bind 2-3DPG
pathway specifically in Rapaport Luebering
shunt
➢ Binds with hemoglobin – causing decrease
O2 affinity
Note:
Decrease affinity = oxygen will be delivered to
the tissues
➢ Plasma 2-3DPG decreases – Hb 2-3DPG
released causing increase O2 affinity to
hemoglobin
Note:
Inverse relationship between 2-3DPG and
affinity of hemoglobin for oxygen

Globin chains
SYNTHESIS OF HEMOGLOBIN:
➢ Varied sequence of amino acids-
➢ Synthesis of hemoglobin begins in the
polytpeptide chains
proerythroblasts and continues even into the
➢ α, β ,𝜸, , ε, 𝜻 (alpha, beta, gamma,
reticulocyte stage of the red blood cells
epsilon, zeta)
➢ Differ is sequence of amino acids>

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➢ The process begins in the mitochondrion
with the condensation of succinyl-CoA and
glycine to form 5-aminolevulinic acid.
➢ succinyl-CoA, then, binds with glycine to
form a pyrrole molecule.
➢ In turn, four pyrroles combine to form
protoporphyrin IX, which then combines with
iron to form the heme molecule.
➢ Finally, each heme molecule combines with
a long polypeptide chain, a globin synthesized
by ribosomes, forming a subunit of hemoglobin
called a hemoglobin chain .
➢ Each chain has a molecular weight of about
16,000; four of these in turn bind together
loosely to form the whole hemoglobin
molecule.
STRUCTURE OF HEME:
➢ Heme is a cyclic tetrapyrrole i.e. consists of
four molecules of pyrrole.
➢ This imparts a red color to erythrocytes
GLOBIN CHAINS:
➢ There are four globin chains in each
molecule of adult hemoglobin (Hb-A)
➢ These are designated as α and β ➢ There
are two α (α1 α2) and two β (β1 β2)
chains
➢ These four chains form two dimers
➢ i.e. α1 β1 and α2 β2
➢ Hb-A (adult hemoglobin) consists of
β1, β2 (two beta chains)
α1, α2 (two alpha chains)
NON PATHOLOGICAL VARIANTS OF
HEMOGLOBIN
>IN THE EMBRYO;
➢ Gower 1 (ζ2ε2)
➢ Gower 2 (α2ε2)
➢ Hemoglobin Portland (ζ2γ2)
>IN THE FETUS:
➢ Hemoglobin F (α2γ2)
>IN ADULTS:
➢ ​Hemoglobin A (α2β2) the most common
with a normal about 97% in adult (newborn
20%)
➢ Hemoglobin A2 (α2δ2) - δ chain synthesis
begins late in the third trimester and in adults,
it has a normal range up to 2.5% (<0.5%NB)
➢ ​Hemoglobin F (α2γ2) ​- In adults
Hemoglobin F is restricted to a limited
population of red cells called F-cells. However,
the level of Hb F can be elevated in persons
with sickle-cell disease and ​beta-thalassemia.
Less than 1% in adult and upto80% in newborn
QUATERNARY STRUCTURE OF HB:
➢ Hemoglobin tetramer is composed of two
identical dimers, α1β1 and α2β2
➢ The two globin chains within each dimer are
held tightly together by inter chain hydrophobic
interactions between α and β subunits.
➢ The two dimers are held together primarily
by polar bonds and able to move with respect
to each other.
➢ The weaker interactions between these
mobile dimers result in two different relative
positions in Deoxyhemoglobin compared to
Oxyhemoglobin
➢ Bonds between a and b units “a” – very
strong bonds
➢ Bonds between two ab are of lesser
strength – “b”
➢ They move relative to each other –
approaching, separating as well as twisting
➢ Change into tense form or relaxed forms
➢ T form – tense form (T form) , beta chains
are farther apart and the molecule binds
2-3DPG and with the formation of anionic salt
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bridges between b chains. Presence of 2-3DPG
encourages O2 delivery to tissues
Note: When plotted with oxyhemoglobin
dissociation curve, the T form would represent
by a shift to the right
➢ R form – relaxed form , 2-3DPG is expelled,
permits O2 binding, salt bridges are broken and
b chains move closer together. Increase affinity
to O2
Note: When plotted with oxyhemoglobin
dissociation curve, the R form would be
represented by a shift to the left
Globin Chain structure
➢ Primary and secondary structure
➢ Primary globin structure – specific sequence
of Aa residues . Alpha chains are numbered 1(N
terminal) to 141(C terminal) chain. The rest like
β, , 𝜻 are numbered 1-146
➢ The secondary globin structures are divided
into 8 separate helical segments designated as
A-H which give the rigidity to structures. There
are also seven non helical segments
(NA,AB,CD,EF,FG,GH,&HC) that lie between the
eight helical segments and provide flexibility
and allows bending of globin chain to tertiary
structure
Note: Heme molecule inserted in pockets
between E and F helices.
Heme production and structure
➢ Formation of protoporphyrin IX and iron
➢ Tetramethenetetrapyrrole – cyclic
compound consists of 4 pyrrole rings (A,B,C&D)
➢ Connected by 4 methene bridges (α, β ,𝜸,
)
Formation of PIX and heme
➢ Protoporphyrin IX is the last compound
leading to production of heme
➢ Produced Nucleated RBC - begins in
mitochondria
➢ Intermediate steps happen in cytoplasm
➢Go back to mitochondria for the Final
reaction – ferrochelatase (enzyme that catalyze
insertion of ferrous iron into Protoporphyrin IX
molecule
➢ Iron is of Fe+3 form then reduced to Fe2+
carried by transferrin (Heme molecule is form)
➢ Heme molecule release to cytoplasm and
join with globin
➢ 4heme+4globin=hemoglobin molecule
➢Rate of protoporphyrin synthesis is
proportional to the rate of globin synthesis -
synchronized
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➢ In mitochondria there are Remnant of
protoporphyrin – free erythrocyte
protoporphyrin (FEP) –
-elevated if iron supply is diminished
- can be used for certain disorders
Iron metabolism for heme synthesis
➢ Iron – present throughout the body , stored
for future use, most are in intracellular space of
the liver and bone marrow
➢ Stored form is ferritin (combination of iron
and protein apoferritin)
➢ If apoferritin is unavailable iron is stored as
hemosiderin
Sources, recycling and locations of iron
➢ Sources is of Fe+3 or Fe+2
➢ Reduced form is absorbed (Fe+2)
➢ Reduction is accomplished by acid pH of the
stomach and reducing substances
➢ Most are absorbed in duodenum and
jejunum . Few in the stomach
➢ Only 10% of dietary iron is absorbed
Note: only 10% is absorbed because we already
have iron stores in the body. When RBC is
disrupted, it will release iron which is readily
absorbed by the body.
➢ Absorbed by mucosal cells oxidized to Fe+3
then stored temporarily to cells as ferritin
➢ When saturated it will stop absorption and
the unabsorbed iron is excreted to feces
➢ During RBC destruction – iron are recycled
but with chronic destruction or acute blood loss
iron maybe lost
Iron transport
➢ When iron is needed, Fe+3 is released by
intestinal mucosal cells then attaches to
transferrin as (ferritin-transferrin)
➢ Transferrin can transport 2 atoms of iron and
delivers it to hemoglobin, myoglobin ,
cytochrome and other iron storage site
➢Developing erythrocytes have
ferric-transferrin membrane receptors –
complex with ferric transferrin forming vacuoles
➢ Iron is synthesized in mitochondria the
remainder is stored as crystalline aggregates of
ferritin in cytoplasm
➢ Ferritin in nucleated RBC – sideroblasts
Ferritin in mature RBC - siderocytes
Note: To demonstrate in peripheral blood
smear, Prussian blue staining is used.
Assembly of hemoglobin molecule
➢Iron+globinchains+protoporphyrinIX+ 2-3DPG
= hemoglobin
➢ Globin chain is completed in ribosome – α
and β forms αβ dimer which 2 heme binds
between E and F helices (secondary structure)
➢ Two of these dimers quickly form a2b2 Hb
dimers and assume the final three dimensional
(quarternary) structure
➢ 2-3 DPG is inserted on the central cavity
(between 2 beta chains)
Location of hemoglobin during function and
degradation
➢ Hb formed in bone marrow
➢Developing erythrocytes
(prorubricyte-shortly after release)
➢ RBC destruction – spleen and RE system
release iron , globin and biliverdin(non cyclic
porphyrin derivative)
➢ Iron and globin are recycled
➢ Biliverdin - degraded and excreted
Functions of hemoglobin
1. transport of molecular O2 from lungs to
tissues
2. transport of CO2 from tissues to the lungs
3. buffering of the blood to prevent changes in
pH that may be incompatible to life
Oxygen transport​ – heme-heme interaction
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➢the binding of one molecule causes an

increased affinity of the other heme groups for
O2.
Note: Hemoglobin molecule holds 4 02
molecules.
The more O2 bound by a Hb molecule the
greater the affinity for O2. The affinity coincides
with the expulsion of 2-3DPG (conversion to R
form)

Oxygen transport mechanism

Po2 (mmHg)
➢ Hb(O2)4 – fully oxygenated hemoglobin
Partial pressure of oxygen
(4- four O2 molecules)
● Normal curve - (center black curve)
➢ Alveolar capillaries of lungs– O2 transport
sigmoid/s-shaped.
➢ Atmospheric air 21% O2 – sea level
● -Partial pressure of oxygen between 20
149mmhg
to 60 is very steep.
➢ PO2 in alveoli is 100mmHg, venous blood –
Indication: Hb can release O2 rapidly
PO2 is 40mmHg
-When it reaches above 60 mmHg,
➢ Minute distance of O2 in alveoli and capillary
curve will be flatten
network of the lungs make transport very rapid
Indication: Hb reached 100% saturation
➢ Hb becomes 95% saturated with O2
of O2, found in alveoli of lungs
➢ 70kg man – 250mL of O2/min
➢ Hyperbaric oxygen-2.3x higher than
atmospheric pressure – treatment for carbon
monoxide poisoning
➢ Breathing pure o2 raises Po2 more than
600mmHg

Oxyhemoglobin dissociation curve

➢ Hb can bind to large quantities of O2 but is
also willing to release O2 when needed.
➢ Movement bet T and R form
➢ Represented by oxyhemoglobin dissociation
curve

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➢ Hemoglobin derivatives and their associated

disorders
1. methemoglobin
2. sulfhemoglobin
3. carboxyhemoglobin

Hemoglobin transport of CO2

➢ 70% of CO2 is carried in the plasma – in a
form of bicarbonate (HCO3)
➢ 30% carried by erythrocytes – 25% as HCO3,
2% nonionized CO2 and the remainder as 3 %
carbamino hemoglobin (Hb-NH-COO)
➢ Shift to the left (R form) – Hb has increase
➢ This reaction is not only for the removal of
affinity for O2 and does not readily release it to
CO2 but also acts as a buffer
the tissues conditions :
➢ Co2 – shift to right
❖ decrease body temperature
➢ Nonoxygenated blood has higher affinity to
❖ decrease erythrocyte 2-3DPG
CO2 than oxygenated blood (Bohr effect)
❖ decrease CO2 conc
❖​ increase in blood pH (alkaline)
Hemoglobin derivatives and their associated
eg : hyperventilation - large amounts of CO2 is
disorders
lost (breathes excessively)
1. methemoglobin and methemoglobinemia
➢ Shift to right (T form) – decrease affinity of
2. sulfhemoglobin and sulfhemoglobinemia
Hb to O2. conditions
3.carboxyhemoglobin
❖ increase in body temperature
&carboxyhemoglobinemia
❖ increase conc of erythrocyte 2-3DPG
❖ inc CO2
Methemoglobin
❖ ​decrease in ph (acidic)
➢ Hi
❖ necessitate delivery of O2 to tissues
➢ Gentle oxidation of Fe+2 to Fe+3
eg. Exercise, renal failure
➢ Heme can't bind O2
- During exercise muscle tissue temp
➢ Normally – 0.5-3% are converted to Hi daily
rises
but cant cause problems due to
- During renal failure, there is build-up in
NADH-methemoglobin reductase (diaphorase)
Hydrogen ions which leads to decreased
production – heme undergo reduction back to
pH
normal – protection of heme
➢ Nonoxygenated Hb binds more excess H
➢ Clinical manifestation are generally mild
ions than oxygenated Hb. Increased amounts of
➢ Rarely – inherited –Hb M
non oxygenated Hb are made available by the
➢ Clinical manifestations: Cyanosis (bluish skin
decrease in the affinity of Hb caused by the
color)
decrease in pH – Bohr effect
➢ Respond to methylene blue except for Hb M
➢ Binding of H ion to Hb - buffers
➢ Some inherited forms –
➢ Hb transport of carbon dioxide
NADH-methemoglobin reductase enzyme
➢ Hb role in acid base balance
deficiency (diaphorase deficiency) may respond
to methylene blue

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➢ Acquired forms – antimalarial drugs, oxidant ➢ Low levels – dizziness,nausea, headache,

drugs , sulfonamides, aniline dyes, nitrate rich vomiting and confusion
water may respond with MB ➢ Higher levels – asphyxiated (50-70%)
➢ Hb M not responsive but treatment & is not –unconsciousness, respiratory failure and death
necessary to be treated ➢ 80% - rapidly fatal
➢ S/S – Cyanosis – methemoglobin level ➢ 0.5% levels in non smokers
exceeds 10% of the total Hb, cosmetic problem, ➢ 5% in smokers
more than 35% - hypoxia, shortness of breath, ➢ Test
dizziness,headaches and tachycardia, 70% -fatal ➢ Lab test: One spot test – 0.5ml whole blood+
➢ Lab findings : PS – heinz bodies , 1ml NaOH – blood with 20%
➢ Diaphorase enzyme screening test – enzyme Carboxyhemoglobin will give cherry red color
assays Normal blood: brown color
➢ Methemoglobin quantitation – ➢ Other tests: Gas chromatography,
Methemoglobin absorbance 630to 635nm after spectrophotometry,
measuring the absorbance at 632nm add KCN Treatment:
(potassium cyanide) – convert methemoglobin ➢ CO – hyperbaric O2 treatment
to cyanohemoglobin which does not absorb at
632nm. The difference of absorbance before Abnormal heme Synthesis
and after addition of KCN is directly The porphyrias
proportional to methemoglobin conc. ➢ Inherited or acquired
➢ Accumulation of porphyrin precursors or one
Sulfhemoglobin or more porphyrin(ogens) in the bone marrow (
➢ Another derivative formed during oxidative erythrogenic porphyrias ) in liver (hepatic
denaturation of hemoglobin by addition of porphyria)
sulfur ➢ CEP – hemolysis , photosensitivity
➢ ​Retain iron in ferrous form
➢ O2 affinity is reduced to 1/100 of normal
➢ Cyanotic appearance, benign
➢ In vitro – H2S (hydrogen sulfide) + Hb
➢ In vivo – drugs and chemicals (acetanilid,
phenacetin, sulfonamides )
➢ Once formed cannot be converted back to
normal (irreversible)
➢ Removal of offending agent

Carboxyhemoglobin
➢ CO (carbon monoxide)
➢ Normally at 1% level of the total hemoglobin
➢ 200x more affinity than O2
➢ Asphyxiation
➢ Car exhaust, coal gas ( charcoal)
➢ Colorless and odorless
➢ Skin turns cherry red