Hemoglobin and Myoglobin 2010

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.


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.


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.


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


• 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.


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.


In oxymyoglobin the 6th coordinate position is


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.


Each heme residue contains one central coordinately


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.


• 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.


• 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.


• 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.


• 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.


• 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


• 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


• 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 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.

Hemoglobin and Myoglobin 2010

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