MYOGLOBIN and HEMOGLOBIN

Dr. Bonleon HEME

- Iron is held in the center by bonds to the 4
Structure - function relationship Nitrogens of the porphyrin ring
- Fe2+ can form 2 additional bonds
HEME Proteins - One on each side of the planar
- Myoglobin and Hemoglobin porphyrin ring
- Main function: Maintain O2 supply for - Fe2+
oxidative metabolism - Center of the planar tetrapyrrole
- Oxidation of Fe2+ -> Fe3+
Myoglobin - Destroys biologic activity
- Storage of O2 - Substituents at Beta-positions:
- Methyl
Hemoglobin - Vinyl
- Transport of O2 - Propionate

Hemeproteins Function:
- Group of specialized proteins that contain - Prosthetic group of myoglobin &
heme as a tightly bound prosthetic group hemoglobin
- Role of heme is dictated by the 3D - O2 binding
structure of the protein - O2 transport
- Electron transport
CYTOCHROME - Photosynthesis
- O2 carrier that is alternately oxidized &
reduced MYOGLOBIN
- 153-aminoacyl residue polypeptide
CATALASE - MW 17,000
- Part of the active site of the enzyme that - Present in heart and skeletal muscles
catalyzes the breaqkdown of hydrogen - Compactly folded, roughly spherical
peroxide - 75-80% of residues are present in 8 right
handed, 7-20 residue alpha-helices
HEMOGLOBIN & MYOGLOBIN - A-H helices (from amino terminal)
- 2 most abundant
- Serves to reversibly bind O2 Structure:
- A-H helices is terminated by:
HEME STRUCTURE - Presence of proline
- Complex of protoporphyrin IX & Ferrous - Beta bends and loops stabilized by
iron (Fe2+) hydrogen bonds and ionic bonds
- Cyclic tetrapyrrole
- 4 molecules of pyrrole Hyperbolic
- Linakage: alpha-methylene bridges - loads readily at PO2 of the lung capillary
- Planar network of conjugated double bed (100mmHg)
bonds = Deep Red

PAT
- Releases only a small fraction of its bound
O2 at the PO2 values in active muscles
(20mmHg) or other issues (40mmHg) ->
effective vehicle for delivery of O2
Binding sites of 90% saturation at 20mmHg
- PO2 in the lung capillary bed: 100mmHg
- PO2 of venous blood: 40mmHg
- PO2 of active muscles: 20mmHg
Strenuous exercise -> decrease PO2 on
muscle tissues to 5mmHg, myoglobin release
O2 for mitochondrial synthesis of ATP ->
permit continued muscular activity
Surface - POLAR
- charged amino acid
- Can form H bonds, with each other with
water
- Leucine, Valine, Phenylalanine and
Methionine
Interior - NON POLAR
- Packed closely together
- Stabilized by hydrophobic interactions
Exceptions:
- Proximal Histidine (His F8)
- Binds directly to the iron of the heme
- Distal Histidine (His E7)
- Does not directly interact with the heme
group
- Helps stabilize the binding of O2 to
Fe2+
- His E7 & His E8 - lie close to the heme iron
- Function: O2 binding
- Heme
- Lies in a crevie between helices E & F
- Globin
- Creates a special environment for the
heme that permits the reversible
binding of one O2 molecule
(oxygenation)
1. UNOXYGENATED MYOGLOBIN
- Iron lies outside the plane of the heme
ring
- Toward His F8
2. OXYGENATED MYOGLOBIN
- Iron moves within the planer
Oxygenation
- Accompanied by motion of the iron of HIs
F8 & of residues linked to His F8.
Function:
- O2 storage & transport to mitochondria
- O2 released during O2 deprivation
- O2 used for O2 synthesis of ATP
- To perform function: must bind O2 well at
low O2 tension when Hgb releases O2
O2 binds to myoglobin
- Fe2+ in heme can form 6 Ligand Bonds
- 4 bind iron to protoporphyrin ring
- 5th -> His residue in F alpha-helix (His F8)
-> proximal side of the protoporphyrin
plane
- 6th -> between O2 molecule & His E7 ->
distal side of the protoporphyrin ring
O2 binding site
- Bond between 1st O2 atom & Fe2+ is
perpendicular to the plane of the heme
ring
- Orientation: 2nd O2 away from the distal
histidine
PAT
ISOLATED HEME
- binds carbon monixde 25,000 times more
than O2
HINDERED ENVIRONMENT
- Apoproteins of myoglobin & hemoglobin
-> binding at a less favored angle reduces
the strength of heme-CO bond: 200x heme-
O2 bond
-> 1% of myoglobin typically present
combined with CO
HEMOGLOBIN
- Found exclusively in red blood cells
- Structurally and functionally more complex
than myoglobin
Functions:
- Transports O2 to the issues
- Returns CO2 and protons (pH) to the
lungs
- Bind O2 at relatively high PO2 in the lungs
- Carry CO2 from the tissues & release it to
the lungs
Structure:
- Tetramers
- Comprised of pairs of 2 different
polypeptide subunits:
- A2b2 (HbA; normal adult hgb)
- A2y2 (HbF; fetal Hgb)
- A2S2 (HbS; Sickle cell Hgb)
- A2d2 (HbA2; minor adult Hgb)
Differences with myoglobin
- Kind and number of amino acid present
- Alpha-polypeptide possesses 7 rather than
8 helical regions
Similarities with myoglobin
- Location of the hemer
- B-polypeptide has 8 helical regions
- Presence of amino acid with similar
properties at comparable locations
- Bind 4 Molecules of )2 per tetramer, one
per heme
COOPERATIVE O2 BINDING
- O2 binds to Hgb tetramer more readily if
other O2 molecules are already bound
- Permits Hgb to maximize quantity of:
- O2 loaded at PO2 of the lungs
- O2 released at PO2 of the peripheral
tissues
- Deoxyhemoglobin (not bound to O2)
- Iron is slightly out of plane secondary to
steric repulsion between proximal His &
N atoms or porphyrin ring
- Oxygenation -> iron atom moves into
the plane of the porphyrin ring so that it
can form a strong bond with O2
- Structural change
- Increase O2 binding activity constants
- Movement of iron into the plane of
protoporphyrin ring -> pulling of His F8
residue toward heme -> changes
interaction of F-helix with C-helix of
adjacent subunit.
- Change in structure transmitted to
adjacent subunit -> increase affinity
constant binding of O2 of adjacent
subunit -> binding of the 1st O2
cooperatively enhance the binding of
subsequent O2 to the other heme groups
in Hgb.
PAT
2 Forms of Hgb that binds O2 with
different affinities:
- Deoxyhemoglobin state: quaternary
conformation -> tight-taut form or T form
- Oxyhemoglobin: change in structure
induced by initial binding of O2 -> relaxed
or R form
- The trigger for the R to T transition of Hgb
is the movement of the iron in & out of the
plane of the porphyrin ring
T vs R
T towards the plane of the heme ring
- Taut or tense
- Deoxy form of Hgb
- The 2 aB dimers interact through a
network of ionic bonds & hydrogen bonds
that constrain the movement of the
polypeptide chains
- Low O2 affinity form
R 6. T (Taut) state - low affinity
Oxygenation Dissociation Curves
- Relationship between concentration, or
partial pressure of O2 (PO2) and quantity
of O2 bound
*SIGMOIDAL (Hgb)
- Easily releases at decrease PO2 levels -
tissues
- Loads at increase PO2 levels - Lungs
Concept of P50
- Measurement of O2 concentration
- PO2 that half-saturates a given Hgb
- Vary widely
- HbA & HbF - low
- Placenta - enable extraction of O2 from
mother’s blood -> undergoes change
Oxygenation of Hemoglobin
- Lead to conformational changes
1. Binding of O2 molecule to
deoxygenated Hgb shifts heme iron
2. Change transmitted to proximal His F8 &
to residues attached
3. Rupture of salt bridges between the
carboxyl terminal residues of all 4
subunits
4. One pair of a/B subunits rotates 15
degrees with respect to the other
5. Compacting the tetramer
7. R (relaxed) state - high affinity
8. Increase the affinity of the remaining
unoxygenated hemes for O2
BOHR EFFECT
- Describes relationship of Hgb’s O2 affinity
to PCO2 level & pH
- Dependent on cooperative interactions
between hemes of Hgb Tetramer
- Reciprocal coupling of proton & O2
binding
- Myoglobin - a monomer (no Bohr Effect)
- The oxygen dissociation curve shifts to the
left when blood pH increases, facilitating
oxygen uptake in the lung. At the same
time however, the release of oxygen to
target issues is decreased by that shift.
PAT
- The Hypoxic Ventilatory Response
increases gas exchange in the lung, thus
removing CO2 from the blood. This leads
to alkalosis of the blood, left shifting the
dissociative curve.
T <—> R
Influenced By:
- Protons
- CO2
- BPG
**the higher their concentration, the more
O2 must be bound to trigger the transition
BOHR EFFECT
- Increase H —> Decrease O2 binding
affinity of Hgb
- Increase PCO2 —> Decrease O2 Binding
Capacity
- Rightward shift on O2-dissociation curve
- Increase PCO2 & Decrease pH —>
promote release of O2
- Protons bind to specific sites in Hgb —>
shift from R to T
- Lungs: pH 7.4 & Increase PO2 —> Hgb
shift to R form with lower affinity for
protons —> protons released
- CO2 binds to the terminal amino groups
of Hgb subunit —> carbamate
- Binding of CO2 —> favors shift from R to
T form
- Lungs: Higher PO2 —> shift to R form —>
release of CO2 —> bind O2
- HYPERVENTILATION
- Decrease tissue concentration of CO2
levels —> less O2 delivered to tissues
- S/Sx: numbness, dizziness, muscle
cramps, etc.
Peripheral tissues:
- pH - Lower
- Stabilizes the T state
- Enhances the delivery of O2
Lungs
- Process is reversed
1. O2 binds to deoxygenated Hgb
2. Protons are released
3. Combined with bicarbonate
4. Carbonic acid
- Dehydration of H2CO3 (carbonic
anhydrase) —> CO2 is exhaled
- Binding of O2 drives exhalation of CO2
2,3 BIPHOSPHOGLYCERATE
- Found in RBCs at nearly the same
concentration as Hgb
- From glycolytic intermediate 1,3 BPG
- 1 molecule of 2,3 BPG: 1Hgb tetramer
- Stabilizes T (deoxygenated) state
- Peripheral tissues with low PO2 —>
synthesis of 2,3 BPG in the RBCs
- Additional salt bridges must be broken ->
R State
- Lower O2 affinity —> release O2
PAT
- Increase 2,3 BPG in RBCs —> shift to T
form —> release of O2
- O2 shortage in peripheral tissues —>
Increase 2,3 BPG
- Hypoxia —> Increase 2,3 BPG
- Storage of blood —> RBCs fail to maintain
high levels of 2,3 BPG —> Increase stored
blood’s affinity for O2 —> Decrease ability
to release O2 in tissues
MINOR HEMOGLOBINS
Fetal Hemoglobin
- A2y2
- HbF - major Hgb found in the fetus and
newborn
- HbA synthesis starts in the bone marrow at
about the 8th month of pregnancy and
gradually replaces HbF
- Physiologic conditions - HbF has higher
affinity for O2 than does the HbA as a
result of HbF’s binding weakly to 2,3 BPG
- Higher O2 affinity to HbF facilitates the
transfer of O2 from the maternal
circulation across the placenta to the RBC
of the fetus
Hemoglobin A2 (HbA2)
- A2d2
- Minor component of normal adult Hgb
- First appears about 12 weeks after birth
Hemoglobin A1C
- Physiologic conditions - HbA is slowly &
nonenzymatically glycosylated
- Extent of glycosylation dependent on the
plasma concentration of a particular
hexose
- Most abundant form of glycosylated Hgb
- Has glucose residues attached
predominantly to the NH2 groups of the N
terminal valines of the B-globin chains
- Increase amounts with DM
- Higher HbA of the diabetics have higher
glucose concentrations during the 120 day
lifetime of these cells
HEMOGLOBINOPATHIES
- Family D/o caused by:
- Production of a structurally abnormal
Hgb Molecule
- Synthesis of insufficient quantities of
normal HgB
- Rarely, Both
Sickle Cell (HbS), Hemoglobin C disease
(Hgb C)
- result from production of Hgb with an
altered amino acid sequence
- Most common inherited blood d/o in the
USA
- Homozygous recessive
- Single nucleotide alteration in the globin
gene
- A point mutation
- A molecule of HbS contains 2 normal
alpha-globin chains & 2 B-globin chains
(Bs)
- Nonpolar valine replaced the polar residue
Glutamate 6
Sickle Cell:
- “sticky patch:
- Distort RBCs into sickle shaped -> lysis
- a2Bs2
- Infant doesn’t begin showing symptoms
until sufficient HbF has been replaced by
HbS —> sickling
- Lifelong episodes of pain (crises), chronic
hemolytic anemia and increase
susceptibility to infections
- Begins in early childhood
PAT
- S/Sx: acute chest syndrome, stroke, and
splenic & renal dysfxn
- Lifetime of HbS - 20 days
- Heterozygotes (contain both HbS and
HbA)
- Sicke cell trait
- Don’t Show clinical symptoms
- Can have a normal life span
Sickling
- Low O2 tension —> HbS polymerizes
inside the RBCs —> form a gel —>
assembling into a network of fibrous
polymers —>
- Stiffen & distort the cell —> rigid,
misshaped RBCs —> sickled cells block
the flow of blood in the narrow capillaries
- O2 deprivation —> tissue anoxia - pain —
> death/ infarction of cells in the vicinity of
the blockage
Variables that increase Sickling
- Any variable that increases the proportion
of HbS in the deoxy state —> reduces the
affinity of HbS for O2
- Decreased O2 tension
- High altitudes or flying in a nonpressurized
plane
- Increased PCO2
- Decreased pH
- Increased concentration of 2,3 BPG in
RBCs
Advantages of Heterozygous State
- Less susceptible to malaria caused by
Plasmodium Falciparum
- Parasite cannot complete the intracellular
stage of its development
Hemoglobin C Disease
- Single amino acid substitution in the 6th
position of the B-globin chain
- Lysine is substituted for the glutamate
- Homozygous
- Mild, chronic hemolytic anemia
- No specific therapy
- Some B-globin chains have the sickle cell
mutation
- Other B-globin chains carry the mutation
found in HbC disease
- Compound heterozygote
- Doubly heterozygous
- HgB levels higher than in sickle cell
disease
- Remain well until they suffer an interactive
crisis
- Crisis often follows childbirth or surgery -
may be fatal
Methemoglobinemia
- Heme iron is ferric
- Can neither bind nor transport O2
- Cause: Side effect of sulfonamides,
hereditary, reduced enzyme activity
(Methgb reductase)

Treatment - Chocolate Cyanosis

- Adequate hydration - Brownish-Blue coloration of the skin and
- Analgesics membranes
- Aggressive antibiotic therapy - Chocolate-colored blood
- Hydroxyurea decreases frequency of - S/sx: are related to tissue hypoxia
painful crisis and reduces mortality - Anxiety, headache, dyspnea
- Transfusions - Rare - coma and death
- Complications - hemosiderosis, blood
borne infections, immunologic

PAT
Thalassemia
- caused by decrease production of normal
Hgb
- Imbalance in the synthesis of globin chains
- Most common in single gene d/o in
humans
- Causes: entire gene deletions, or
substitutions or deletions of one to many
nucleotides in the DNA
- Normally, synthesis of the alpha and beta
globin chains are coordinated —> each
alpha-globin chain has a B-globin chain
partner —> a2B2
B-Thalassemias
- Synthesis of B-globin chains is decreased
or absent
- A-globin chain is normal
- Cannot form stable tetramers —>
precipitate
- Premature death of cells
- B - Thalassemia Trait (B-Thalassemia
Minor)
- Only one defective B-globin gene
- B - Thalassemia Major
- Both genes are defective
- Physical manifestations appear only after
birth
- Minor - dont require specific tx
- Major - seemingly healthy at birth
- Become severley anemic
- Require regular BT during the 1st and
2nd year of life
- Tx life saving —> hemosiderosis occurs
between 15 & 25 years of life
A - Thalassemias
- A-globin chains - decreased or absent
- Each individual genome contains 4 copies
of the A-globin gene (2 on each
chromosome 16)
- Silent carrier - 1 out of 4 genes is defective
- No physical manifestations
- A-Thalassemia Trait - 2 out of 4 genes
- HbH - 3 out of 4 genes
- Mildly to moderatlely severe hemolytic
anemia
- Hydops fetalis - all 4 genes are defective
- Fetal death
- A-Globin genes are required for the
synthesis of HbF
HEMOGLOBIN M
- His F8 replaced by tyrosine
- Iron forms a tight ionic complex with the
phenolate anion of tyrosine that stabilizes
the Fe3+ form
- Favor the R state —> increase O2 affinity
—> failure to deliver adequate O2 to the
peripheral tissues —> tissue hypoxia —>
Polycythemia Vera
OTHERS:
- Myoglobinuria
- Massive crash injury
- Myoglobin released from damaged
muscle fibers colors the urine dark red
- Detected in plasma following MI
- Anemia
- Reduction in the number of RBCs or of
Hgb
- Impared synthesis or production
PAT