Transcript Part 2

Hemoglobin
Hemoglobin
(Hb)
is
an
iron-containing
metalloprotein that functions to transport oxygen in
the red blood cells of vertebrates. The protein,
which accounts for 97% of the RBC’s dry content is
responsible for delivering oxygen from the lungs to
all other tissues in the body where it is taken up for
cell use.
Deepa John
Harini Chandra
1
2
Master Layout (Part 1)
This animation consists of 5 parts:
Part 1 – Structure of hemoglobin
Part 2 – Oxygen binding & structural changes
Part 3 – Factors affecting oxygen binding
Part 4 – Hemoglobin disorders
Propionate
Heme
Part 5 – Comparative study of hemoglobin & myoglobin
Fe-Protoporphyrin IX
Quaternary structure
of Hemoglobin
α1
β1
3
α2
β2
4
Pyrrole rings
Methyl
groups
Vinyl chains
5
Source: Biochemistry by Lubert Stryer, 6th edition (ebook)
1
2
Definitions of the components:
Part 1 – Structure of hemoglobin
1. Hemoglobin: Hemoglobin is the predominat metalloprotein in the red blood cells that
carries oxygen. Each hemoglobin molecule is made up of four heme groups
surrounding a globin group.
2. a, b subunits: Subunits that occur in the functional organization of macromolecules,
usually proteins.
3. Heme: Heme is a prosthetic group containing carbon, nitrogen and hydrogen atoms, with
a single Fe2+ ion at the center.
3
4. Prosthetic group: A tightly bound non – protein chemical compound that is required for
the activity of an enzyme.
5. Protoporphyrin IX: A metal-free porphyrin that combines with iron to form the heme of
iron-containing proteins. It is made up of four pyrrole rings linked by methene bridges
to form a tetrapyrrole ring. Four methyl groups, two vinyl groups, and two propionate
side chains are attached.
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1
Part 1, Step 1
Alpha globin gene cluster
Chromosome 16
Zeta 2
Zeta 1
Alpha 2
Adult Hemoglobin
heterotetramer (a2b2)
Alpha 1
5'
3'
α1
2
3
4
5
Beta globin gene cluster
Chromosome 11
epsilon
gamma
Expressed in fetal Hb
(a2g2)
delta
5'
Action
β1
α2
beta
β2
3'
Description of the action
Audio Narration
PLEASE REDRAW ALL FIGURES. First showHemoglobin is a heterotetramer composed of two alpha and
As shown in
the structure on top left with its labels. Next two beta chains. The alpha globin gene locus resides on
animation.
show the arrow appearing and the pink region chromosome 16 with each gene contributing to the synthesis
of the figure on right. Next the coloured boxes of the alpha globin chain. The beta globin gene locus resides
at the bottom left must appear followed by theon chromosome 11 and consists of all genes that are
arrow and the yellow region of the figure on expressed from the time of embryonic development of Hb to
right as depicted in the animation. Finally showthat of the adult beta globin gene. The globin chains are
the 2 regular arrows with the text box
synthesized by ribosomes in the cytosol.
‘expressed in fetal Hb’.
Source: Biochemistry by Lubert Stryer, 6th edition (ebook)
1
Part 1, Step 2
Propionate
Heme
Fe-Protoporphyrin IX
Quaternary structure
of Hemoglobin
α1
2
3
α2
β1
Methyl
groups
β2
Pyrrole rings
4
Action Description of the action
As shown
in
animation.
5
PLEASE REDRAW ALL FIGURES. First
show the structure on the left with its
labels. Next show the red box appearing
in the region indicated which must be
zoomed into to show the figure on the
right. The labels for these must then
appear after the figure has appeared.
Vinyl chains
Audio Narration
Every subunit of hemoglobin is bound to a prosthetic group known as
heme. This consists of a central iron atom in its ferrous state
surrounded by a complex organic ring structure known as
protoporphyrin. The heme group is essential for the oxygen binding
property of hemoglobin. The iron atom forms six coordination bonds,
four of which are to the nitrogen atom of porphyrin rings, one to a His
side chain in the globin subunit and the other being the binding site for
oxygen. Iron in its Fe3+ state does not bind to oxygen.
Source: Biochemistry by Lubert Stryer, 6th edition (ebook); Biochemistry by A.L.Lehninger, 4th edition (ebook)
Master Layout (Part 2)
1
2
This animation consists of 5 parts:
Part 1 – Structure of hemoglobin
Part 2 – Oxygen binding & structural changes
Part 3 – Factors affecting oxygen binding
Part 4 – Hemoglobin disorders
Part 5 – Comparative
study of hemoglobin & myoglobin
15o
α1
β1
β1
α1
Oxygen
binding
3
α2
β2
4
T state
deoxyhemoglobin
α2
β2
R state
oxyhemoglobin
O2 O2
O2 O2
5
Source: Biochemistry by Lubert Stryer, 6th edition (ebook); Biochemistry by A.L.Lehninger, 4th edition (ebook)
1
2
Definitions of the components:
Part 2 – Oxygen binding & structural changes
1. Cooperative binding: It is a form of allosteric binding in which ligand binding to
macromolecules having more than one binding site is carried out in a cooperative manner
such that binding of ligand at one site increases the affinity of another site for the ligand. In
tetrameric hemoglobin, binding of first oxygen molecule to one subunit brings about structural
changes which in turn positively influences the binding of the remaining subunits to oxygen.
2. Models for oxygen binding to Hb: Two models have been proposed by different groups
of scientists to explain the binding of oxygen to hemoglobin. These are the concerted and
sequential models:
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a) Concerted model: The first model proposed by Monod, Wyman and Changeux,
also known as the MWC model, assumes that each subunit can exist in at least two
conformational states and hypothesizes that all subunits make the transition from one
state to the other simultaneously. According to this model, each subunit of a
cooperatively binding protein is functionally identical. Binding of ligand can occur with
either conformation but with varying degrees of affinity for each. Binding of ligand to a
low affinity state makes the transition to the high affinity state more likely.
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b) Sequential model: The second model, proposed by Daniel Koshland in 1966,
postulates that binding of a ligand to one subunit can induce conformational changes in
the other subunits. Unlike the concerted model, it does not state that all subunits must
exhibit transition from one state to the other simultaneously, thereby making a larger
number of intermediate states more likely.
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1
Definitions of the components:
Part 2 – Oxygen binding & structural changes
3. Oxyhemoglobin: When all subunits of hemoglobin are bound to oxygen, it is known as
oxyhemoglobin and it transports oxygen to the various tissues of the body.
2
4. Deoxyhemoglobin: Hemoglobin in oxygen unloaded form is called deoxyhemoglobin.
5. T state: T stands for the tense state. It is one of the two quaternary forms of hemoglobin
that predominates in absence of oxygen. It has a lower affinity for substrates and, hence, lower
catalytic activity.
3
6. R state: R stands for relaxed state. One of the two quaternary forms of hemoglobin that
predominates when oxygen is bound. . It has a higher affinity for substrates and, hence, higher
catalytic activity.
7. Distal histidine: Distal histidine in vertebrate hemoglobins plays an important role in
oxygen binding and has been strongly conserved during evolution. It is considered to be
important in fine-tuning the ligand affinities of hemoglobin.
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8. Proximal histidine: The heme in hemoglobin is held in the cleft both by hydrophobic
interactions and by a covalent bond between the iron and a nitrogen atom of a nearby histidine
side chain. This histidine is referred to as the proximal histidine.
9. Sigmoidal plot: The binding of oxygen to hemoglobin displays marked sigmoidal behaviour,
which is indicative of cooperation between subunits. The binding of first oxygen causes a
conformational change in the other binding sites that makes it easier for oxygen to bind there.
This explains the initial up-swing in the sigmoidal curve. The "leveling out" at the top of the
curve is caused by saturation of the hemoglobin-oxygen binding sites.
1
Part 2, Step 1
Models of cooperative binding: Concerted (MWC) model
O2
2
O2 O2
O2 O2
O2 O2
O2
O2 O2
Low affinity
3
O2
O2 O2
O2 O2
O2
O2
O2
O2 O2
High affinity
4
Action Description of the action
Audio Narration
As shown PLEASE REDRAW ALL FIGURES. First show the Hemoglobin binds four molecules of oxygen per tetramer, one per heme, in a
four blank ovals and the four blank rectangles. Next cooperative manner. The first model for cooperative binding, proposed by
in
Animation. the ligand ‘O2 ‘should appear in the first oval and theMonod, Wyman and Changeux, also known as the MWC model, assumes
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colour should change. Then the down arrow should that each subunit can exist in at least two conformational states with all
appear followed by the four rectangles. Next ligand subunits making the transition from one state to the other simultaneously.
should appear in the first rectangle and the colour Each subunit of a cooperatively binding protein is functionally identical and
should change. Similarly ligand should occupy the binding of ligand can occur in either conformation but with varying degrees of
next oval and the colour should change and so on tillaffinity for each. Binding of ligand to a low affinity state makes the transition
to the high affinity state more likely.
all the ovals and rectangles are occupied by the
ligand as shown in animation.
Source: Biochemistry by A.L.Lehninger, 4th edition (ebook)
1
Part 2, Step 2
Models of cooperative binding: Sequential model
2
K1
O2
K2
O2
O2
3
4
O2
K4
O2 O2
Low affinity
the arrows below must appear and the same
must appear for the 2nd row. Then the figures in
the 3rd row must appear sequentially and so on
until all the figures have been displayed.
O2 O2
O2 O2
High affinity
Action Description of the action
As shown PLEASE REDRAW ALL FIGURES. First show
the blank circles and rectangles in the top row
in
Animation. appearing column-wise one after another. Next
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K3
Audio Narration
The second model for cooperative binding of oxygen to hemoglobin,
proposed by Daniel Koshland in 1966, postulates that binding of a
ligand to one subunit can induce conformational changes in the
other subunits. However, unlike the concerted model, it does not
state that all subunits must transition from one state to the other
simultaneously. Any conformational change brought about in one
subunit enhances the likelihood of a similar change as well as
binding of a ligand in the neighboring subunit. A larger number of
intermediate states are therefore more likely in the sequential
model.
Source: Biochemistry by Lubert Stryer, 6th edition (ebook)
1
Part 2, Step 3
Binding of oxygen to hemoglobin: Structural changes
Iron atom located slightly outside
plane of porphyrin ring
α1
β1
2
0.4 Ao
Iron
Porphyrin
His
3
β2
α2
T state
deoxyhemoglobin
4
Action Description of the action
As shown PLEASE REDRAW ALL FIGURES. First show
the figure on the left with its labels. Next, show
in
Animation. the green box appearing which must be
5
zoomed into to show the figure on the right. The
blue arrow must then appear which must
continuously flicker and the text box on top
must appear.
Audio Narration
Two major conformations of Hb have been deduced by X-ray
analysis – the T state which is stabilized in the absence of oxygen
and the R state which is relatively more stable in the presence of
oxygen. The pair of identical ab dimers of Hb are linked by several
ion pairs at the interface that stabilize it in its deoxy state. Many of
these interactions are disrupted upon binding to oxygen and new
ones are formed instead. The porphyrin ring in the T state is found
to be slightly puckered, thereby causing the iron atom to lie out of
plane and protrude onto the proximal histidine.
Source: Biochemistry by Lubert Stryer, 6th edition (ebook);
1
Part 2, Step 4
Binding of oxygen to hemoglobin: Structural changes
Iron moves into plane of heme &
pulls proximal histidine along with
it.
2
β1
α1
Bound O2
Iron
3
Porphyrin
His
α2
β2
R state
oxyhemoglobin
4
Action Description of the action
As shown PLEASE REDRAW ALL FIGURES. First show
the figure on the left with its labels. Next, show
in
Animation. the green box appearing which must be
5
zoomed into to show the figure on the right. The
blue arrow must then appear which must
continuously flicker and the text box on top
must appear.
Audio Narration
Upon binding to oxygen, one ab pair of subunits rotates with respect
to the other by around 15o. Several interactions at the ab interaface
are therefore disrupted and new ones formed. As Max Perutz rightly
postulated, there are changes in the position of many key amino
acid residues upon oxygenation. Binding of oxygen causes the iron
atom to move back into the plane of the porphyrin ring thereby
pulling the proximal histidine along with it. Once the transition from T
state to R state takes place in one subunit, the remaining subunits
also undergo the same transition more readily, thereby favouring the
cooperative binding to oxygen.
Source: Biochemistry by Lubert Stryer, 6th edition (ebook);
1
Part 2, Step 5
Effect of cooperative binding
Proten without
cooperativity
Lungs
Hb
2
3
Capacity for oxygen
delivery to tissues is
greatly increased due to
cooperative binding of
hemoglobin
Oxygen
uptake
Oxygen
release
– 66%
Oxygen
release
– 38%
Tissues
4
5
Action Description of the action
PLEASE REDRAW ALL FIGURES. First show the image of ‘lungs’,
As shown ‘tissues’ and the blue squares marked ‘Hb’. Small white circles
in
must then enter the blue squares with the label ‘oxygen uptake’.
Animation. These must then move down to the ‘tissues’ and majority of the
circles must be released onto the tissues. When this happens, the
red curve in the graph must appear. Next the green squares must
appear and fewer number of white circles must be taken up onto
these squares. Again, these must move down and only few
(~38%) of the circles must be released onto the tissues. This is
followed by appearance of the blue curve in the graph and then
the dialogue box above.
Source: Biochemistry by Lubert Stryer, 6th edition (ebook);
Audio Narration
Cooperative binding of oxygen to hemoglobin allows efficient
transfer of oxygen from lungs to tissues. The high partial
pressure of oxygen in lungs allows Hb to be saturated upto
98% due to the cooperative nature while saturation level in
tissues drops to 32% due to lower partial pressure. This
allows 66% of the oxygen taken up to be released. In case of
proteins showing no cooperativity, however, the maximum
amount of oxygen that can be delivered under similar
conditions is only around 38% due to less uptake and
relatively less release. This cooperative binding nature leads
to a sigmoidal binding curve for Hb allowing it to deliver 1.7
times more oxygen compared to non-cooperative proteins.
Master Layout (Part 3)
1
This animation consists of 5 parts:
Part 1 – Structure of hemoglobin
Part 2 – Oxygen binding & structural changes
Part 3 – Factors affecting oxygen binding
Part 4 – Hemoglobin disorders
Part 5 – Comparative study of hemoglobin & myoglobin
BOHR EFFECT
2
Effect of pH & CO2
Effect of 2,3-bisphosphoglycerate (BPG)
O2 O2
3
Fractional saturation
O2 O2
OxyHb
2,3-BPG
4
Oxygen partial pressure
5
Source: Biochemistry by A.L.Lehninger, 4th edition (ebook)
DeoxyHb
1
2
3
4
5
Definitions of the components:
Part 3 – Factors affecting oxygen binding
1. Bohr Effect: The effect of pH and CO2 concentration on the binding and release of
oxygen by hemoglobin is called the Bohr effect. Lowering the pH and raising the partial
pressure of carbon dioxide results in the release of O2 from oxyhemoglobin.
2. Effect of 2,3-bisphosphoglycerate: 2,3-bisphosphoglycerate is a highly anionic
compound that is present in RBCs at around the same concentration as Hb. 2,3-BPG binds
to a central pocket of the T form of Hb tetramer and stabilizes it by interacting with three
positively charged amino acids on each β-chain.
3. Fractional saturation: It is the fraction of active sites that are bound to the substrate and
is directly proportional to reaction velocity.
4. Oxygen partial pressure: It is the partial pressure of oxygen in the gas phase above the
solution.
1
Part 3, Step 1
Bohr effect
O2 O2
O2 O2
Raising partial pressure of carbon
dioxide
O2 O2
Lowering pH
Deoxyhemoglobin
Oxyhemoglobin
2
Exhaled
O2 O2
Peripheral tissues
Lungs
2CO2 + 2H2O
3
Carbonic anhydrase
2H2CO3
2HCO3- + 2H+
Hb + 4O2
O2
2H+ + 2HCO3-
4O2
Hb + 2H+
(buffer)
2H2CO3
Carbonic
anhydrase
2CO2 + 2H2O
4
Action Description of the action
PLEASE REDRAW ALL FIGURES. FirstThe effect of pH and CO2 concentration on the binding and release of oxygen
show the four circles on left top with ‘O2 by hemoglobin is called the Bohr effect. Lowering the pH and raising the partial
‘and its label. Next show the arrow and pressure of carbon dioxide results in the release of O2 from oxyhemoglobin. In
peripheral tissues, CO2 released from Krebs cycle and other cellular processes
the text followed by the four circles on
combines with water to form carbonic acid, which dissociates into protons and
the right with O2 leaving them. Next,
bicarbonate ions. Hb which has just released its bound oxygen into the tissues
show the two coloured boxes & their
acts as a buffer by binding protons and delivering them to the lungs. In the
labels on top. The reaction sequence lungs, the uptake of oxygen by hemoglobin releases protons that combine with
must then appear as shown in
bicarbonate ion, forming carbonic acid, which when dehydrated by carbonic
animation.
anhydrase becomes carbon dioxide, which is then exhaled.
Source : Harper's Biochemistry 26th ed
As
shown in
animatio
n.
5
Audio Narration
1
Part 3, Step 2
At lower pH, His 146 is
protonated which
favours the deoxyHB
conformation thereby
leading to release of O2
2
3
Fractional saturation
Chemical basis for Bohr effect
Oxygen partial pressure
Carbamate
4
Action
As shown in
animation.
5
Description of the action
PLEASE REDRAW ALL FIGURES. In the
figure on left, first show on the colored
circles with their bonds joined. The circle
with the label ‘added proton’ must appear
after that and the dotted line must be
formed between the white circle and the
pink circle and a thick line with the blue
circle on the left. Once that happens, the
graph must be shown followed by the
dialogue box with text.
Audio Narration
The chemical basis for the effect of pH has been found to lie in the
interactions of the various side chains in hemoglobin. In deoxyHb, the
b-His 146 forms a salt bridge with a lysine residue in the a subunit.
This interaction is however not possible when the His residue is
deprotonated, which occurs at high pH. Upon lowering the pH,
protonation of the His residue brings about salt bridge formation,
thereby favouring the deoxy form and leading to release of bound O2.
CO2 reacts with the terminal amino groups forming negatively charged
carbamate groups, thereby stabilizing the deoxyHb form.
Source: Biochemistry by Lubert Stryer, 6th edition (ebook); Biochemistry by A.L.Lehninger, 4th edition (ebook)
1
Part 3, Step 3:
Effect of 2,3-bisphosphoglycerate (BPG)
2,3-BPG bound Hb
His 143
Lys 82
His 2
b1 subunit
2
N
2,3-BPG
N
b2 subunit
3
Lys 82
His 2
2,3-BPG
His 143
O2 O2
O2 O2
Deoxyhemoglobin
Oxyhemoglobin
4
Action Description of the action
PLEASE REDRAW ALL FIGURES. First show the
As shown
figure on top left followed by the red box. This area
in
must be zoomed into to show the figure to its right.
animation. Then show the four coloured circles at the bottom with
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the label ‘oxyhemoglobin’. The small blue circle must
then bind to the centre of the bigger orange circles.
Once this happens, The O2 labels must leave and the
colour of the circ le must change from orange to white
with the label then showing ‘deoxyhemoglobin’.
Source: Biochemistry by Lubert Stryer, 6th edition (ebook);
Audio Narration
2,3-bisphosphoglycerate is a highly anionic compound that
is present in RBCs at around the same concentration as Hb.
2,3-BPG binds to a central pocket of the T form of Hb
tetramer and stabilizes it by interacting with three positively
charged amino acids on each β-chain. Transition from T to R
state requires bonds to be broken between Hb and 2,3-BPG.
This enhances the oxygen releasing capacity of Hb, without
which it would be an extremely inefficient transporter
molecule.
Master Layout (Part 4)
1
This animation consists of 5 parts:
Part 1 – Structure of hemoglobin
Part 2 – Oxygen binding & structural changes
Part 3 – Factors affecting oxygen binding
Part 4 – Hemoglobin disorders
Part 5 – Comparative study of hemoglobin & myoglobin
2
SICKLE CELL ANEMIA
Thalassemia
3
Normal red blood cell
4
5
Sickled red blood cell
α thalassemia
β thalassemia
1
2
3
Definitions of the components:
Part 4 – Hemoglobin disorders
1. Sickle cell anemia: Sickle-cell anemia is a genetic disease in which an individual has
inherited the allele for sickle-cell hemoglobin from both parents characterized by
abnormal, rigid, sickle shape (HbS) as compared to the normal flexible biconcave disk
shaped red blood cells (HbA). It results from a single amino acid substitution, a Val
instead of a Glu residue at position 6 in the two β chains. As a result of this change,
deoxyhemoglobin S has a hydrophobic patch on its surface, which causes the molecules
to aggregate into strands that align to form insoluble fibres. Sickle-cell disease may lead
to various acute and chronic complications, several of which are potentially lethal.
2. Thalassemia: This is another inherited genetic disorder due to deletions or mutations
in the globin genes which results in abnormalities and deficiencies in α-globin synthesis.
Signs and symptoms of thalassemias are due to lack of oxygen in the bloodstream. This
occurs because the body doesn't make enough healthy red blood cells and hemoglobin.
The severity of symptoms depends on the severity of the disorder.
3. Point mutation: A type of mutation resulting from substitution of a single nucleotide
base with another.
4
5
Part
4,
Step
1:
1
Sickle cell anemia
Sequence in normal hemoglobin (Hb A)
2
Nucleotide
CTG ACT CCT GAG GAG AAG TCT
Amino acid
Leu
Thr
Pro
Glu
Glu
Lys
Ser
Normal red blood cells
(HbA)
3
Sequence in mutant hemoglobin (Hb S)
Nucleotide
CTG ACT CCT GT
A G GAG AAG TCT
Amino acid
Leu
Thr
Pro
Val
Glu
Glu
Lys
Ser
Sickled red blood cells (HbS)
4
Action
Description of the action
Audio Narration
PLEASE REDRAW ALL FIGURES. First show the
A large number of mutations have been described in the globin
in the box as depicted in animation. Next, the image
for ‘sickled red blood cells’ must be shown which
must be zoomed into and the green sequences on
the left must be shown. The ‘A’ shown in red must
disappear and be replaced by ‘T’. Similarly in the
sequences below, ‘Glu’ must diappear and be
replaced by ‘Val’ as shown in animation.
the b chain. This change converts a glutamic acid residue to
valine in the corresponding amino acid sequence. Replacement of
the Glu residue by Val creates a “sticky” hydrophobic contact
point at position 6 of the β chain. These sticky spots cause
deoxyHbS molecules to associate abnormally with each other
leading to clumping of the cells. Their oxygen carrying capacity is
greatly reduced and these patients require frequent transfusions.
As shown in figure with ‘normal red cells’ on the right top which genes. The mutation causing sickle cell anemia is a single
animation. must be zoomed into to show the sequences shown nucleotide substitution of A to T in the codon for amino acid 6 of
5
Source: www.ntic.uson.mx
Part
4,
Step
2:
1
β- Thalassemia
2
Splice site
Normal
5’ C C T A T T G G T C T A T T T T C C A C C C T T A G G C T G 3’
β-Thalassemia
A G T C T A T T T T C C A C C C T T A G G C T G 3’
5’ C C T A T T G
Point mutation
resulting in
abnormality in β
globin synthesis
Splice site
α- Thalassemia
3
Zeta 2
5
Alpha 2
Description of the action
PLEASE REDRAW ALL FIGURES. First
show the sequence labeled as ‘normal’
followed by the sequence labeled as βThalassemia. Then the top right braces
and its text should appear.Next the red
letter G shoild be replaced by letter A in
the nucleotide sequence labeled as βThalassemia. Then the bottom right
braces and its text should appear. Then
the text on right should pop out. Next thre
bottom figure should be shown. Then the
red block should dissapear followed by
the text on right.
Source: www.sickle.bwh.harvard.edu
As shown in
animation.
Alpha 1
3'
5'
Action
4
Zeta 1
Deletion mutation
resulting in
abnormality in α
globin synthesis
Audio Narration
Thalassemia are the result of abnormalities in hemoglobin
synthesis. Deficiencies in β-globin synthesis result in the βthalassemias . Mutation of a single base from G to A in an
intron of the β-globin gene generates a new splice site.
The resulting mRNA contains a stop codon further
upstream and leads to premature translation termination
thereby producing aberrant protein. Deficiencies in αglobin synthesis due to inactivation of one or all the four αglobin genes results in the α-thalassemias.
Master Layout (Part 5)
1
This animation consists of 5 parts:
Part 1 – Structure of hemoglobin
Part 2 – Oxygen binding & structural changes
Part 3 – Factors affecting oxygen binding
Part 4 – Hemoglobin disorders
Part 5 – Comparative study of hemoglobin & myoglobin
Heme group
2
Iron atom
α1
β1
3
α2
4
Myoglobin
5
Source: Biochemistry by Lubert Stryer, 6th edition (ebook);
β2
Hemoglobin
1
2
3
4
5
Definitions of the components:
Part 5 – Comparative study of hemoglobin &
myoglobin
1. Myoglobin (Mb): Myoglobin is a globular protein having a single polypeptide chain
consisting of eight alpha helices linked by short polypeptide segments, with a total of 153
amino acid residues. It is found in muscle tissues of most mammals and plays a role in
oxygen binding. Myoglobin resembles the alpha subunit of hemoglobin and like
hemoglobin, it consists of a central heme group which enables it to bind oxygen.
Myoglobin was the first protein to have its X-ray crystallography structure determined in
1959 by John Kendrew.
1
Part 5, Step 1:
Structure of Myoglobin – determined by X-ray crystallography
Detector
2
X-ray
source
Electron
density map of
myoglobin
X-ray beam
Myoglobin
crystals
3
X-ray
diffraction
Iron atom
Pyrrole rings
Eight alpha helices
joined by turns
4
Action
Description of the action
Audio Narration
PLEASE REDRAW ALL FIGURES. First show
X-ray crystallography is a very useful visualization technique that facilitates the
coming out from the source and passing through
on of the crystals where it must get split into
several beams as shown and then strike the
detector. Once this happens, the figure shown on
right must appear after which the green regions
must be highlighted with labels followed by the
arrow mark and the figure on left.
studies. When a beam of X-ray was passed through the crystals of myoglobin,
some part of the beam was found to pass straight through while the others were
scattered in different directions. These scattered beams were detected by means
of an X-ray film which, after several spot intensity calculations, provided an
electron density map of the protein. The protein was found to consist of a single
polypeptide chain having eight alpha helices along with a heme group in the centre
similar to hemoglobin.
As shown in the figure marked ‘myoglobin crystals’, ‘X-ray
determination of three-dimensional coordinates of atoms in a protein. Myoglobin
animation. source’ and ‘detector’. Next show a beam of light was the first protein whose structure was determined by X-ray crystallography
5
Source: Modified from Biochemistry by Lubert Stryer, 6th edition (ebook);
1
Part 5, Step 2:
Structural similarity between alpha chain of Hb and Mb
Structural
homology
a-subunit
Hemoglobin
Myoglobin
2
Sequence
homology
3
Identical
residues
Conserved
substitution
4
Action
Description of the action
Audio Narration
PLEASE REDRAW ALL FIGURES. First show
Myoglobin, found largely in muscle tissues, has been found to be
which must flicker until the label ‘structural
homology’ is highlighted as depicted. Next,
show the arrows appearing and the box below
with the orange and yellow highlighted regions
& the corresponding labels. Please ensure that
this is redrawn with appropriate alignment as
shown.
with the recurring structure being known as the globin fold. The
hemoglobin chain having 141 amino acids and myoglobin having
153 residues have also been found to have very high sequence
homology. Despite the similarities, myoglobin functions largely as
an oxygen binding protein that stores a reserve supply of oxygen
in muscle tissues while hemoglobin serves to transport oxygen.
As shown in the blue and red structure on the left & right.
structurally similar to the alpha subunit of hemoglobin. The alpha
animation. Next show the double headed arrow appearing helix arrangement of both proteins has been found to be the same
5
Source: Modified from Biochemistry by Lubert Stryer, 6th edition (ebook);
1
Part 5, Step 3:
Oxygen binding curve of myoglobin
Myoglobin acts as a useful reserve
supply of oxygen in muscle tissues
but uses only 7% of its oxygen
All sites filled
carrying capacity.
Fractional saturation
2
3
OO
2 2 O2
O2
O2
1.0
0.5
P1/2 = 2 torr
0
25
50
75
pO2 (torr)
100
Myoglobin
All sites empty
4
5
Action Description of the action
As
shown
in
animati
on.
PLEASE REDRAW ALL FIGURES.
First show the figure on right and the
graph axes on the left with the labels.
Next, show the violet oxygen molecules
binding to the ‘myoglobin’ structure. As
the molecules bind, the green curve of
the graph must appear gradually.
Finally the labels on the graph must
appear followed by the text on top.
Audio Narration
Myoglobin binds strongly to oxygen and acts as an oxygen storage protein rather
than a transporter. It shows 50% saturation at a pressure as low as 2 torr and gets
saturated even under low oxygen pressure conditions that prevail in the muscle.
Myoglobin can use only 7% of the oxygen carrying capacity as opposed to
hemoglobin which can utilize nearly 90% of the carrying capacity. Unlike hemoglobin,
which has a sigmoidal oxygen binding curve, myoglobin has a hyperbolic curve
indicating that it binds to oxygen irrespective of the surrounding partial pressure of
oxygen in the tissues. It is this property that allows sea mammals such as whales
that have very high amounts of myoglobin in their muscle to remain underwater for
long periods of time.
Source: Modified from Biochemistry by Lubert Stryer, 6th edition (ebook);
1
Interactivity option 1:Step No: 1
2,3-BPG has been found to bind readily to adult hemoglobin but not to fetal hemoglobin which
has the composition, a2g2 (as shown in animation). The g subunit has less positive charge
compared to the adult b subunit, due to which the negatively charged 2,3-BPG does not bind
to it. This phenomenon is believed to be of evolutionary significance for which of the following
reasons?
2
3
4
α
β
β
α
β
β
α
BPG bound maternal Hb
Adult maternal
Hb – a2b2
α
γ
α
γ
γ
α
γ
α
Fetal Hb – a2g2
Fetal Hb – a2g2
Interacativity Type
5
2,3-BPG
α
Choose the correct
option.
Options
User has to choose one of
the four options after the
animation and graph have
been displayed. If a, b or d
are chosen, they must turn
red.
Boundary/limits
Results
User has to choose one of
the four options. If a, b or d
are chosen, they must turn
red with the remark ‘wrong
answer’. If user chooses (c
), it must turn green with
the remark ‘right answer’.
Interactivity option 1:Step No: 2
1.0
Fetal red cells
0.8
0.7
Maternal
red cells
0.6
0.4
0.2
100
50
Oxygen partial pressure
a) Allows proper development of the fetus.
b) To minimize the oxygen saturation of the fetus.
c) To allow for better oxygen saturation and transfer of oxygen from mother to fetus.
d) To prevent the toxic effects of 2,3-BPG from entering the fetus.
1
Interactivity option 2:Step No: 1
Drag and drop the statements given below into their correct column headings.
HEMOGLOBIN
2
MYOGLOBIN
90% oxygen
carrying capacity
Red blood cells
Muscles
7% oxygen
carrying capacity
Oxygen storage
protein
1 polypeptide
chain
3
Oxygen transport
protein
4 polypeptide
chains
4
Interacativity Type
5
Drag & drop.
Options
User must drag and drop the
statements given on the sides into
their correct columns. Every time the
user drags it correctly, a green tick
must appear. If user drags it under the
wrong column a cross must appear
and the statement must return to its
original position.
Results
The red statements must come under
the ‘hemoglobin’ column and the green
under the ‘myoglobin’ column as
shown in animation..
1
Questionnaire
1. Which of the following is the major form of hemoglobin produced in red blood cells during
normal adult life?
Answers: a) a2b2 b) a2g2 c) z2 e 2 d) a2e2
2
2. All of the following factors influence hemoglobin dissociation curve except ?
Answers: a) pH b) CO2 tension c) Temperature d) 2,3 BPG levels
3
4
3. The cause of sicke cell anemia is :
Answers: a) a deletion of the beta globin gene promoter b) a missense mutation in the coding
region of the beta globin gene c) a nonsense mutation in the coding region of the beta globin
gene d) the increased production of the alpha globin gene due to a duplication
4. Bohr effect explains the effect of following on the binding and release of oxygen by
hemoglobin:
Answers: a) CO2
b) pH
c) 2,3, BPG
d) both a and b
c) protein
d) steroid
5. What is the heme group?
5
Answers: a) lipid
b) tetrapyrrole
Links for further reading
Books:
Biochemistry by Lubert Stryer et al., 6th edition (ebook)
Biochemistry by A.L.Lehninger et al., 4th edition
Biochemistry by Harper, 26th edition (ebook)
Research papers:

The crystal structure of a tetrameric hemoglobin in a partial
hemichrome stateAntonio Riccio by Luigi Vitagliano, Guido di
Prisco,Adriana Zagari and Lelio Mazzarella