Hemoglobin and Myoglobin Atif H. Khirelsied B.Sc., M.Sc., Ph.D. Biochemistry Faculty of Medicine and Health Sciences International University of Africa
Apr 21, 2015
Hemoglobin and Myoglobin
Atif H. Khirelsied B.Sc., M.Sc., Ph.D. Biochemistry
Faculty of Medicine and Health SciencesInternational University of Africa
Hemoglobin and MyoglobinHemoglobin and Myoglobin
• Hemoglobin and myoglobin are hemeproteins
whose physiological importance is principally
related to their ability to bind molecular oxygenrelated to their ability to bind molecular oxygen.
Hemoglobin
Hemoglobin is the oxygen-carrier in the blood stream
HemoglobinHemoglobin
Our blood stream contains about 150 g/L of
hemoglobin (Hb) which is so effective as an oxygenhemoglobin (Hb), which is so effective as an oxygen‐
carrier that the concentration of O2 in the blood stream
reaches 0.01 M the same concentration as air.
Hemoglobin and MyoglobinHemoglobin and Myoglobin
Once the Hb‐O2 complex reaches the tissue that
th O l l t f dconsumes oxygen, the O2 molecules are transferred
to another protein myoglobin (Mb) which stores p y g ( )
oxygen in the muscle tissue.
MyoglobinMyoglobin
• Is a monomeric hemoprotein.
• Serves as an intracellular storage for oxygen in muscles.
• Releases oxygen during periods of oxygen deprivation.
MyoglobinMyoglobin
Myoglobin is responsible of the characteristic red color of muscle tissueof muscle tissue
Myoglobin structureMyoglobin structure
• The tertiary structure of• The tertiary structure of myoglobin is a typical globular protein.protein.
• It has a very high proportion• It has a very high proportion (75%) of α‐helix.
• Comprises 8 α‐helices, designated A to H connected by short nonA to H, connected by short non helical regions.
MyoglobinMyoglobin
• Myoglobin is a typical Myoglobin is a typicalglobular protein.
• The heme prosthetic group embedded in a g phydrophobic pocket.
HemoglobinHemoglobin
HemoglobinHemoglobin
Hb and Mb are only slightly related in primary sequence.
Although most amino acids are different between the two proteins, the amino acid changes are generally conservative.
The secondary structures of Mb and the subunits of Hb are virtually identical.
HemoglobinHemoglobin
Both Hb and Mb proteins are largely alpha‐helical,
and the helices fit together in a similar way.
One O2 molecule is bound to each protein
molecule by a coordinate covalent bond to an iron
atom (Fe(II)) in the heme group
Heme
Heme is a planar molecule
Heme
Heme is a planar molecule
containing four pyrrole groups,
whose nitrogens form
d l b d hcoordinate covalent bonds with
four of the iron's six availablefour of the iron s six available
positions.
Heme
The fifth position is used to form a coordinate covalent bond with the side chain of a single histidine in the protein (proximal histidine).
The sixth and last position is used for oxygen binding.
Histidine F8
Hemoglobin and MyoglobinHemoglobin and Myoglobin
Hemoglobin and MyoglobinHemoglobin and Myoglobin
• The interior of the molecule is predominantly
h d h bi hil h f i h d hilihydrophobic while the surface is hydrophilic.
• Each Mb or Hb subunit contains one heme
prosthetic group inserted into a hydrophobic cleft
i h iin the protein.
Hemoglobin and Myoglobin
d h b b h lHydrophobic interactions between the tetrapyrrolering and hydrophobic amino acid on the interior of h l f l b l h hthe cleft strongly stabilize the heme protein conjugate.
A nitrogen atom from a histidine R group located b h l f h h i i di dabove the plane of the heme ring is coordinated with the iron atom provides further stabilization.
Hemoglobin and Myoglobin
In oxymyoglobin the 6th coordinate position is
Hemoglobin and Myoglobin
In oxymyoglobin the 6th coordinate position is
occupied by the oxygen, whose binding is
stabilized by a second histidine residue.
CO binds coordinately to heme iron in a mannerCO binds coordinately to heme iron in a manner
similar to that of oxygen, but is much stronger
causing asphyxia of carbon monoxide poisoning.
Hemoglobin and Myoglobin
Each heme residue contains one central coordinately
Hemoglobin and Myoglobin
Each heme residue contains one central coordinately bound iron atom in the Fe2+, or ferrous, state.
The oxygen carried by Mb is bound directly to the ferrous iron atom.
Oxidation of the iron to the Fe3+, ferric, state renders the molecule incapable of normal oxygen binding. p yg g
Hemoglobin
Hemoglobin is a tetrameric hemeprotein found in RBCs , transporting oxygen from the lungs to body tissues.
Each subunit of a hemoglobin has a heme prosthetic group identical to that of Mb.
The peptides are designated α, β, γ, and δ which are arranged into various forms of functional hemoglobins.
HemoglobinHemoglobin
• The secondary and tertiary structure of Hb
subunits are similar, with extensive homology insubunits are similar, with extensive homology in
amino acid composition.
• The quaternary structure of Hb results in• The quaternary structure of Hb results in
physiologically important allosteric interactions
between the subunits.
HemoglobinHemoglobin
• Exhibits sigmoidal curve of O2 binding, allosteric
proteins in which O is a positive homotropicproteins in which O2, is a positive homotropic
effector.
• When O2 binds to the first subunit of
deoxyhemoglobin it increases the affinity of thedeoxyhemoglobin it increases the affinity of the
remaining subunits for O2 .
The curve of oxygen binding to Hb is sigmoidal
Hemoglobinand myoglobin oxygen saturation curves
Hemoglobin
Binding of O2 to iron of deoxy‐Hb pulls the iron atom g 2 y pinto the plane of the heme.
Since the iron is also bound to histidine F8, this residue is also pulled toward the plane of the heme p pring.
The resulting conformational change is transmitted throughout the peptide backbone causing a significant change in tertiary structure of Hb.
HemoglobinHemoglobin
• The latter changes in subunit interaction are
transmitted from the s rface to the heme bindingtransmitted, from the surface, to the heme binding
pocket of a second deoxy subunit and result in p y
easier access of O2 to the iron atom of the second
heme and thus a greater affinity of the hemoglobin
molecule for a second O moleculemolecule for a second O2 molecule.
HemoglobinHemoglobin
• The configuration of low affinity, deoxygenated
hemoglobin (Hb) is known as the taut (T) statehemoglobin (Hb) is known as the taut (T) state.
• The structure of the fully oxygenated high affinity
form of hemoglobin is known as the relaxed (R)
statestate.
Hemoglobin and MyoglobinHemoglobin and Myoglobin
• In the context of the affinity of Hb for O2 there are
four primary regulators each of which has afour primary regulators, each of which has a
negative impact. These are
o CO2
o hydrogen ion (H+)
o chloride ion (Cl‐)o chloride ion (Cl )
o 2,3‐bisphosphoglycerate.
Hemoglobin
• In the high pO2 of the lungs there is sufficient O2 to
Hemoglobin
In the high pO2 of the lungs there is sufficient O2 to
overcome the inhibitory nature of the T state.
h b d d l f f• The O2 binding induces alteration from T to R form
• Several amino acid side groups on the surface of Hb
will dissociate protons.
Hemoglobin
h d f l l
Hemoglobin
• The dissociation of protons plays an important role in
the expiration of the CO2 that arrives from the tissuesthe expiration of the CO2 that arrives from the tissues
• Because of the high pO2, the pH of the blood in the
l ( ) i ffi i l l hlungs (~7.4 ‐ 7.5) is not sufficiently low enough to
exert a negative influence on hemoglobin binding O2.exert a negative influence on hemoglobin binding O2.
Hemoglobin
• When o hemo lobin rea hes the tiss es the pO2 is
Hemoglobin
• When oxyhemoglobin reaches the tissues the pO2 is
sufficiently low, as well as the pH (~7.2), that the T y , p ( ),
state is favored and the O2 released.
4O2 + Hb↔ nH+ + Hb(O2)4
Hemoglobin and Myoglobin
• In the tissues, CO2, H+ and Cl‐ exert their negative ff Hb bi di f O
g y g
effects on Hb binding of O2
• Metabolizing cells produce CO2 which diffuses into the blood and enters the circulating RBCs.
• Within RBCs the CO2 is rapidly converted to carbonic acid through the action of carbonic anhydrase as shown below:
CO2 + H2O ↔ H2CO3 ↔ H+ + HCO32 2 2 3 3
Hemoglobin and Myoglobin
• The bicarbonate ion produced in this dissociationThe bicarbonate ion produced in this dissociation reaction diffuses out of the RBC and is carried in the blood to the lungs.
• This effective CO2 transport process is referred to as i h d i t tisohydric transport.
A i t l 80% f th CO d d i• Approximately 80% of the CO2 produced in metabolizing cells is transported to the lungs in this way.way.
• A small percentage of CO2 is transported in the blood s a pe ce age o O2 s a spo ed e b oodas a dissolved gas.
Hemoglobin
• In the tiss es the H+ disso iated from arboni a id
Hemoglobin
• In the tissues, the H+ dissociated from carbonic acid
is buffered by Hb which exerts a negative influence y g
on O2 binding forcing its release to the tissues.
• Within the lungs the high pO allows for effective O• Within the lungs the high pO2 allows for effective O2
binding by Hb leading to the T to R state transition g y g
and the release of protons.
Hemoglobin
• The protons combine with the H CO ⎯ bicarbonate
Hemoglobin
• The protons combine with the H2CO3 bicarbonate that arrived from the tissues forming carbonic acid which then enters the RBCswhich then enters the RBCs.
• Through a reversal of the carbonic anhydrasereaction, CO2 and H2O are produced.
• The CO diffuses out of the blood into the lungThe CO2 diffuses out of the blood, into the lung alveoli and is released on expiration.
Hemoglobin
• In addition to isohydric transport, about 15% of CO2
g
2
is transported to the lungs bound to N‐terminal
amino groups of the T form of Hb.
CO2 + Hb‐NH2 <‐‐> H+ + Hb‐NH‐COO‐
• This reaction, forms what is called carbamino‐
hemoglobinhemoglobin.
Hemoglobin
• This reaction also produces H+, lowering the pH in
g
p g ptissues where the CO2 conc. is high.
• The formation of H+ leads to release of the bound O2to the surrounding tissues.
• Within the lungs, the high O2 content results in O2binding to Hb and release of H+binding to Hb and release of H .
• The released H+ promotes the dissociation of the• The released H promotes the dissociation of the carbamino to form CO2 which is then released with expiration.
Bohr effect
h f f b d b d
Bohr effect
• The conformation of Hb and its oxygen binding are
sensitive to hydrogen ion conc.sensitive to hydrogen ion conc.
• These effects of [H+] are responsible for the Bohr
ff i hi h i i h d ieffect in which increases in hydrogen ion
concentration decrease oxygen binding by Hb.concentration decrease oxygen binding by Hb.
Bohr effect
Bohr effectBohr effect
In the lungs, the uptake of oxygen by hemoglobin
l h b h b breleases protons that combine with bicarbonate ion,
forming carbonic acid, which when dehydrated byforming carbonic acid, which when dehydrated by
carbonic anhydrase becomes carbon dioxide, which
then is exhaled.
The chloride shift
• Diff sion of HCO‐ o t of RBCs is o pled to Cl‐ ion
The chloride shift
• Diffusion of HCO‐3 out of RBCs is coupled to Cl‐ ion
movement into the RBCs to maintain electrical
neutrality.
• In this way Cl‐ plays an important role in bicarbonate• In this way, Cl plays an important role in bicarbonate
production and diffusion and thus also negatively p g y
influences O2 binding.
The chloride shift
The role of 2 3 bisphosphoglycertaeThe role of 2,3-bisphosphoglycertae
• 2,3‐bisphosphoglycerate derived from the glycolysis
allosterically effects oxygen binding properties of Hballosterically effects oxygen binding properties of Hb.
• Binding of 2.3‐BPG to a cavity in the center of the
deoxygenated T form of Hb stabilizes the T state.
The role of 2 3 bi h h l t2,3-bisphosphoglycertae
• Increased 2,3‐BPG
concentration fa orsconcentration favors
conversion of R form Hb to
T form Hb and decreases
the amount of oxygen
bound by Hb at anybound by Hb at any
oxygen concentration. yg
Effect of temperature on Hb binding affinity
Fetal hemoglobin (Hb F)Fetal hemoglobin (Hb-F)
Hb-F binding affinity
• Hb. molecules differing in subunit composition have
g y
different 2,3‐BPG binding properties with different allosteric responses to 2,3‐BPG.
• For example, HbF binds 2,3‐BPG much less avidly than HbA
• HbF in fetuses binds oxygen with greater affinity than the mothers HbA, giving the fetus preferential access to oxygen.
Hb-F binding affinity
Hb-F binding affinityg y
Hemoglobin variants
The hemoglobinopathies
Structural abnormalities in proteins result in various types ofproteins result in various types of diseases.
The substitution of a hydrophobic amino acid (Val) for (Glu) in the βamino acid (Val) for (Glu) in the β‐chain of Hb‐A results in sickle cell anemia (HbS)anemia (HbS).
HbS polymerizes withinHbS polymerizes within erythrocytes, leading to the characteristic sickle shape
Atif Hassan Khirelsied
characteristic sickle shape.
Sickle cell anemia
Sickle cell anemia
Sickle cell is an autosomal recessive trait
The sickle-cell heterozygote is resistant to malaria and the frequency yg q yof the S allele closely matches the world-wide distribution of malaria.
Hemoglobin CHemoglobin C
• Hemoglobin C (HbC) is an abnormal hemoglobin with substitution of a glutamic acid residue for a lysine residue at the 6th position of the β‐globin chain.
• This mutated form reduces the normal plasticity of host erythrocytes causing a hemoglobinopathy os e y ocy es caus g a e og ob opa yresulting in mild hemolytic anemia
Hemoglobin ChristchurchHemoglobin Christchurch
• In normal hemoglobin, amino acid # 71 of the β‐subunits is buried deep in the hydrophobic core of the protein and is phenylalanine (hydrophobic).
• In Hb Christchurch amino acid #71 is serine (hydrophilic).( yd op c)
• Hb Christchurch is unstable and forms Heintz Bodies• Hb Christchurch is unstable and forms Heintz Bodiesin red blood cells.
Hemoglobin ChristchurchHemoglobin Christchurch
Hemoglobin K WoolwichHemoglobin K Woolwich
• In normal hemoglobin, amino acid 132 of the β subunit is on the surface of the protein and is lysine.
• In Hb Woolwich, amino acid 132 is glutamineIn Hb Woolwich, amino acid 132 is glutamine
• Hb Wool ich is non f nctional b t the protein• Hb Woolwich is non‐functional but the protein molecules do not form Heintz bodies. They are freely soluble just like normal hemoglobin but theyfreely‐soluble just like normal hemoglobin, but they do not bind oxygen.
Hemoglobin K Woolwichg
Review questions
In humans, oxygen is effectively delivered to the i b f h f l ll i
q
tissues because of the presence of several allostericmodulators. Name three of these modulators and explain how their presence allows oxygen to beexplain how their presence allows oxygen to bedelivered to the tissues.
Review questions
• Describe the Bohr effect in terms of oxygen saturation
q
curve.
W it ti hi h th l ti hi f• Write an equation which expresses the relationship of hemoglobin to [H+] for the oxygenation‐deoxygenationprocess and explain in some detail where the [H+] comeprocess and explain in some detail where the [H+] come from when one mole of O2 combines with deoxyhemoglobindeoxyhemoglobin.
• What is the physiological significance of the Bohr effect p y g gat the tissue and lung?
Review questionsq
Regarding the reaction of CO2 with hemoglobin, which of the following statements is true?
) l d b b h da) It is catalyzed by carbonic anhydrase. b) It accounts for the “chloride shift.” c) It gives rise to methemoglobinc) It gives rise to methemoglobin. d) It results in the binding of 2,3‐diphosphoglycerate
(DPG)(DPG). e) It is one of the mechanisms of isohydric transport
Review questionsq
More than half of the CO2 transported from peripheral tissues to the lung is in what form?
A. bicarbonate (HCO3‐) B. bound to hemeC. bound to 2,3‐diphosphoglycerate (DPG) D. bound to myoglobinE. bound to carbonic anhydrase
Review questionsq
The tertiary structure (three‐dimensional structure) of a protein is determined by:
A. its amino acid composition. B. its amino acid sequence. C. its isoelectric point. D. the number of proline residues in the molecule. E. whether the protein is an enzyme.