sdf sda f sdf

1. Lecture 4
Protein structure and function

2. Sample questions
Sample questions
Give brief definitions or unique descriptions of the following terms:
• (a) oligopeptide/peptide
• (b) quaternary structure
• (c) zwitterion
A short polymer of amino acids, usually up to ~50 residues
Spatial arrangement of subunits in a multi subunit protein
Molecule contain both a positive and a negative charged functional group

3. Protein structure review
• Primary structure refers to the amino acid sequence of a protein
(including the covalent bonds that link amino acids).
• Secondary structure refers to stable arrangements of amino acid
residues giving rise to recurring structural patterns.
• Supersecondary structure (or motif) refers to particularly stable
arrangements of several elements of secondary structure.
• Tertiary structure includes all aspects of the three-dimensional
structure of a protein
• Quaternary refers to the three-dimensional arrangement of two or
more polypeptide subunits.

4. Sample questions
Sample questions
• The Alpha Helix Is a Coiled Structure
Stabilized by ______ Hydrogen Bonds
• Beta Sheet Is Stabilized by ______Hydrogen
Bonds
A. Intrachain B. Interchain
A
B

5. Dihedral angles of
protein
• To avoid atomic collisions, only certain angles are
allowed.
• The angles in proteins are restricted to certain values

6. When phi angle is zero, no matter what the psi angle is,
there are atomic collisions between O1 and C2.

7. Ramachandran plot
All you need to know is:
• Ramachandran plot represents
sterically allowed conformations of
the protein backbone
• We can predict the protein
secondary structure from the phi
and psi angle.

8. Ramachandran plot
• Can be used to
check the quality of a
protein structure

9. Amino Acid Ionization State as a Function of pH

10. This equation tell us when there is
equal amount of A- and HA, pH = pKa
Henderson-Hasselbalch Equation
Log (1) = 0

11. Titration
• If sodium hydroxide is added to water.
• When the pH = pKa, there are equivalent amounts of the conjugate
acid and base.
pH
value
NaOH volume
HA
exhausted
[HA] = [A-]
A- showed
up

12. Titration of glycine
• The pKa of the α-COOH group is 2.4, whereas
that of the α-NH3 + group is 9.8.

13. Titration Curve for Alanine
pI (isoelectric point) = the pH at which the number of positive and
negative charges on a population of molecules is equal (i.e. no net charge).
pK1 carboxylic acid = 2
pK2 amino group = 10
pI = (pK1+ pK2)/2

14. Sample questions
Sample questions
• At a pH >pI of a given protein, that protein
becomes ______, at the pH<pI of that
same protein, it becomes _______.
• A. negatively charged (an anion)
• B. positively charged (a cation)
A
B
The isoelectric point (pI) is the pH at which a particular molecule or surface
carries no net electrical charge.

15. The Purification of Proteins Is an Essential First Step in
Understanding Their Function
• “Never waste pure thoughts on an impure protein”.
• Proteins Must Be Released from the Cell to Be
Purified
In the first step, a homogenate is formed by disrupting the cell
membrane, and the mixture is fractionated by centrifugation.
• Proteins Can Be Purified According to Solubility,
Size, Charge, and Binding Affinity

16. Salting Out (by solubility)
• Most proteins are less soluble at high salt concentrations, an effect
called salting out.
• The salt concentration at which a protein precipitates differs from
one protein to another. Hence, salting out can be used to fractionate
proteins.
• Dialysis can be used to remove the salt if necessary.

17. Gel-Filtration Chromatography
(by size)
• The sample is applied to the top of a column consisting of porous beads
• Large molecules flow more rapidly through this column and emerge first
• Small molecules can enter these beads and exit slowly.

18. • The negatively charged SDS (sodium dodecyl sulfate)-protein complexes
migrate in the direction of the anode, at the bottom of the gel.
• The polyacrylamide gel separates proteins according to size, with the
smallest moving most rapidly.
Polyacrylamide Gel
Electrophoresis (by size)

19. Ion-Exchange Chromatography
(by charge)
• Positively charged proteins (cationic proteins) can be separated on
negatively charged columns.
• Conversely, negatively charged proteins (anionic proteins) can be
separated by chromatography on positively charged columns.
• Proteins that have a low density of net positive charge will tend to
emerge first, followed by those having a higher charge density.

21. Affinity Chromatography
(by Binding Affinity)
• For example, the plant protein concanavalin A can be purified by
passing a crude extract through a column of beads containing
covalently attached glucose residues.
• Affinity chromatography is a powerful means of isolating
transcription factors, proteins that regulate gene expression by
binding to specific DNA sequences. A protein mixture is percolated
through a column containing specific DNA sequences attached to a
matrix; proteins with a high affinity for the sequence will bind and be
retained.

22. Quantification of the purification
protocol by Electrophoretic Analysis

23. Sample questions
Sample questions
• Consider the following ion exchange chromatography test at pH 6.0. A
mixture of proteins were applied to the column in a low [NaCl] buffer.
Proteins retained by the cation exchange column were eluted by gradually
increasing the NaCl concentration within the buffer. What can be concluded
regarding the pI and relative charge of the protein(s) in each chromatogram
peak?

24. Short summary
• Proteins can be purified according to
solubility, size, charge, and binding affinity
• Proteins have an UV absorption peak
at 280 nm

25. Hemoglobin, a molecule to
breathe with
• Blood can carry very little
oxygen in solution.
• Hemoglobin is required to
carry oxygen around.
• Hemoglobin is found in red
blood cells
• Each red blood cell can carry about
one million molecules of oxygen
• Hemoglobin is 97% saturated when
it leaves the lungs
• Under resting conditions is it about
75% saturated when it returns.

26. • The main function of red blood cell
• Transfer of O2 from lungs to tissue
• Transfer of CO2 from tissue to lungs
• To accomplish this function red cells has
hemoglobin (Hb)
• Each red blood cell has 640 million
molecules of Hb
Introduction

27. The α2β2 Tetramer of Human
Hemoglobin
• The structure of the two identical α subunits (red) is similar to but
not identical with that of the two identical β subunits
• The molecule contains four heme groups
• Bind up to four oxygen molecules.
The iron-containing oxygen-transport metalloprotein
in the red blood cells of all vertebrates
In mammals, the protein makes up about 96% of the
red blood cells' dry content.

29. Hydrophobic interactions between
subunits

30. Electrostatic interactions
between subunits

31. Tertiary folding gives rise to at least 3 functionally important
characteristics of the hemoglobin molecule
• 1- Polar or charged side chains tend to be directed to the
outside surface of the subunit and, conversely, non-polar
structures tend to be directed inwards. The effect of this
is to make the surface of the molecule hydrophilic and
the interior hydrophobic.
• 2- An open-toped cleft in the surface of the subunit
known as heme pocket is created. This hydrophobic
cleft protects the ferrous ion from oxidation.
• 3- The amino acids, which form the inter-subunit bonds
responsible for maintaining the quaternary structure, and
thus the function.

32. Structure of Heme
•Heme contains:
 conjugated system of double
bonds → red colour
 4 nitrogen (N) atoms
 1 iron cation (Fe2+
)
→ bound in the middle of
tetrapyrrole skelet by
coordination covalent bonds
• At the core of the molecule is porphyrin
ring which holds an iron atom.
• An iron containing porphyrin is termed a
heme.
• This iron atom is the site of oxygen
binding.
• The name hemoglobin is the
concatenation of heme and globin

33. Types of hemoglobin
 Adult Hb (Hb A)
•HbA1 (2 α and 2 β subunits) is the major form of Hb in adults and in children over 7
months.
•HbA2 (2 α, 2 δ) is a minor form of Hb in adults. It forms only 2 – 3% of a total Hb
A.
 Fetal Hb (Hb F) = 2 α and 2 γ subunits
- in fetus and newborn infants Hb F binds O2 at lower tension than Hb A → Hb F
has a higher affinity to O2
•After birth, Hb F is replaced by Hb A during the first few months of life.
 Hb S – in β-globin chain Glu is replaced by Val
= an abnormal Hb typical for sickle cell anemia

34. Fetal Hb vs. adult Hb
Pressure units:
1 mmHg = 1 Torr
1 mmHg = 133.22 Pa
1 Pa = 0.0075 mmHg

36. Hb A Hb A2 Hb F
structure 2β2
22 2γ2
Normal % 96-98 % 1.5-3.2 % 0.5-0.8 %
Adult hemoglobin

37. Hemoglobin
• Hemoglobin is a remarkable molecular
machine that uses motion and small
structural changes to regulate its action.
• Oxygen binding at the four heme sites in
hemoglobin does not happen
simultaneously.
• Once the first heme binds oxygen, it
introduces small changes in the structure
of the corresponding protein chain.

38. Hemoglobin
• These changes nudge the neighboring chains
into a different shape, making them bind oxygen
more easily.
• Thus, it is difficult to add the first oxygen
molecule, but binding the second, third and
fourth oxygen molecules gets progressively
easier and easier.
• This provides a great advantage in hemoglobin
function.
• When blood is in the lungs, where oxygen is
plentiful, oxygen easily binds to the first subunit
and then quickly fills up the remaining ones.
Hb must bind oxygen in lungs and release it in capillaries

39. Models for Allosteric Behavior
• Monod, Wyman, Changeux (MWC) Model:
allosteric proteins can exist in two states:
R (relaxed) and T (taut)
• In this model, all the subunits of an
oligomer must be in the same state
• T state predominates in the absence of
substrate S (Oxygen in our case)
• S binds much tighter to R than to T

40. More about MWC
• Cooperativity is achieved because S
binding increases the population of R,
which increases the sites available to S
• Ligands such as S are positive homotropic
effectors
• Molecules that influence the binding of
something other than themselves are
heterotropic effectors

42. • Adjacent subunits' affinity for oxygen
increases after the first oxygen binding.
• This is called positive cooperativity

43. Hemoglobin
• When oxygen level is low, hemoglobin releases
its bound oxygen. As soon as the first oxygen
molecule drops off, the protein starts changing
its shape.
• This prompts the remaining three oxygens to be
quickly released.
• In this way, hemoglobin picks up the largest
possible load of oxygen in the lungs, and
delivers all of it where and when needed.

44. Process of O2
binding to Hb
•Hb can exist in 2 different forms: T-form and R-form.
•T-form (T = tense, or taut) has a much lower oxygen affinity than the R-form.
The subunits of Hb are held together by electrostatic interactions. The binding
of the first O2 molecule to subunit of the T-form leads to a local conformational
change that weakens the association between the subunits → R-form
(„relaxed“) of Hb.
•Increasing of oxygen partial pressure causes the conversion of T-
form to R-form.
• T ↔ R
• Hb + ↑pO2 ↔ HbO2

45. • When a first oxygen binds to Fe in heme of Hb,
the heme Fe is drawn into the plane of the
porphyrin ring
• This initiates a series of conformational changes
that are transmitted to adjacent subunits

46. Heme Binding Site After O2 Binds
•O2 binds at distal side of Heme
•Fe2+
moves into porphyrin plane

47. Heme Prosthetic Group
Prosthetic Group =
non-polypeptide
unit of a protein
that can function
without the protein

48. Hemoproteins
• Hemoglobin (Hb)
• Myoglobin (Mb)
• Cytochromes
• Catalases (decomposition of 2
H2O2 to 2 H2O and O2)
• Peroxidases

49. Myoglobin
Mb is a monomeric heme protein
• Mb polypeptide "cradles" the heme group
• Fe in Mb is Fe2+
- ferrous iron - the form that binds
oxygen
• Oxidation of Fe yields 3+ charge - ferric iron
-methemyoglobin does not bind oxygen
• Myoglobin provides muscle tissue with an oxygen
reserve AND facilitates oxygen movement in
muscle
One of the first proteins characterized by
X-Ray Crystallography (Kendrew, 1959)

50. Myoglobin and hemoglobin
monomer are structurally similar

51. Compare hemoglobin and
myoglobin
A classic example of allostery
• Hemoglobin and myoglobin are oxygen
transport and storage proteins
• Compare the oxygen binding curves for
hemoglobin and myoglobin
• Myoglobin is monomeric; hemoglobin is
tetrameric
• Mb: 153 aa, 17,200 MW
• Hb: two alphas of 141 residues, 2 betas of 146

53. -sigmoidal curve is shallow at low oxygen
concentrations, low concentrations indicates that all
molecules are in the T state
-sigmoidal curve is steep when molecules increase
at the R state and then it plateaus when the sites
are filled

54. Binding of Oxygen by Hb
The Physiological Significance
• Hb must be able to bind oxygen in the lungs
• Hb must be able to release oxygen in
capillaries
• If Hb behaved like Mb, very little oxygen
would be released in capillaries
• The sigmoid, cooperative oxygen binding
curve of Hb makes this possible!

55. The Conformation Change
The secret of Mb and Hb!
• Oxygen binding changes the Mb conformation
• Without oxygen bound, Fe is out of heme plane
• Oxygen binding pulls the Fe into the heme plane
• Fe pulls its His F8 ligand along with it
• The F helix moves when oxygen binds
• Total movement of Fe is 0.029 nm - 0.29 A
• This change means little to Mb, but lots to Hb!

57. The energy in the formation of the Fe-O2 bond
formation drives the T→ R transition.
Hemoglobins O2 -binding Cooperativity derives from
the T → R Conformational shift.
•The Fe of any subunit cannot move into its heme plane
without the reorientation of its proximal His so as to prevent
this residue from bumping into the porphyrin ring.
•The proximal His is so tightly packed by its surrounding
groups that it can not reorient unless this movement is
accompanied by the previously described translation of the F
helix across the heme plane.
•The F helix translation is only possible in concert with the
quaternary shift.

58. Molecular Shift at the Heme Group
after Oxygen Binding
Blue: deoxy
Red: oxy

59. Fe at the Heme Plane after Oxygen Binding

60. A Quaternary Structure Change
One alpha-beta pair moves relative to the other by 15 degrees upon oxygen binding
This massive change is induced by movement of Fe when oxygen binds

61. Conformational Changes
• The movement of the histidine causes the
movement of the F-helix. This causes a
corresponding rearrangement of the other
helices in the protein subunit.
• The conformational change of the subunit
causes it to move away from its partner in the
αβ pair. Movement of one subunit in the αβ
pair causes a corresponding conformational
adjustment in the paired subunit, enhancing
the ability of the latter to bind oxygen.

62. Hemoglobin: Axis of
Symmetry Channel View
α2 β2
α2
α1
α1
β2
β1
β1
The Oxy form (red) forms a more condensed channel along the
interface as compared to the Deoxy form (blue)

63. The reactivity of the heme group is different in the
presence or absence of the polypeptide
• CO binds 25,000 times as strongly as O2 in the
isolated heme, but only 200 times as strongly as O2
in myoglobin or hemoglobin
Heme
O2
Histidine

64. • CO Binding in Myoglobin
and Hemoglobin
– CO is a poison because it
displaces O2
– CO prefers linear
coordination, O2 bent
– Distal His forces bent
coordination of CO
– Let O2 compete with CO
• CO produced in body takes
1% Hb
• Without Distal His, CO >
99% Hb
The reactivity of the heme
group is different in the
presence or absence of
the polypeptide
1
1
25,000
N Fe O
O
N Fe C O
200
N Fe C
O
ISOLATED HEME
ISOLATED HEME
HEME WITH POLYPEPTIDE
ENVIRONMENT

66. Hb-oxygen dissociation curve
Hb-oxygen dissociation curve

67. Hb-oxygen dissociation curve
Hb-oxygen dissociation curve
 The normal position of curve depends on
The normal position of curve depends on
 Concentration of 2,3-DPG
Concentration of 2,3-DPG
 H
H+
+
ion concentration (pH)
ion concentration (pH)
 CO
CO2
2 in red blood cells
in red blood cells
 Structure of Hb
Structure of Hb
When oxygen affinity is increased, the dissociation curve is shifted
Leftward, and the value is reduced.
Conversely, with decreased oxygen affinity, the curve is shifted to the
right.

68. Hb-oxygen dissociation curve
Hb-oxygen dissociation curve
 Right shift (easy oxygen delivery)
Right shift (easy oxygen delivery)
 High 2,3-DPG
High 2,3-DPG
 High H
High H+
+
 HbS
HbS
 Left shift (give up oxygen less readily)
Left shift (give up oxygen less readily)
 Low 2,3-DPG
Low 2,3-DPG
 HbF
HbF
Effects of carbon dioxide. Carbon dioxide affects the curve in two ways:
first, it influences intracellular pH (the Bohr effect),
second, CO2 accumulation causes carbamino compounds to be generated through chemical interactions.
Low levels of carbamino compounds have the effect of shifting the curve to the right,
while higher levels cause a leftward shift.

69. The Bohr Effect
Competition between oxygen and H+
• Discovered by Christian Bohr
• Binding of protons diminishes oxygen binding
• Binding of oxygen diminishes proton binding
• Important physiological significance
• Hemoglobin releases H+
when it binds O2
• Hemoglobin binds H+
when it releases O2
• HbO2 + H+
HbH+
+ O2

70. Bohr Effect II
Carbon dioxide diminishes oxygen binding
• Hydration of CO2 in tissues leads to proton
production
• These protons are taken up by Hb as
oxygen dissociates
• The reverse occurs in the lungs

71. Carbon Dioxide
(CO2)
• Transport of carbon dioxide by red
cells, unlike that of oxygen, does not
occur by direct binding to heme.
• In aqueous solutions, carbon dioxide
undergoes a pair of reactions:
1. CO2
+ H2
O H2
CO3
2. H2
CO3
H+
+ HCO3

72. (CO2)
• Carbon dioxide diffuses freely into the red cell where the
presence of the enzyme carbonic anhydrase facilitates
reaction 1.
• The H+
liberated in reaction 2 is accepted by deoxygenated
hemoglobin, a process facilitated by the Bohr effect.
• The bicarbonate formed in this sequence of reactions diffuses
freely across the red cell membrane and a portion is
exchanged with plasma Cl-
, a phenomenon called the
"chloride shift." the bicarbonate is carried in plasma to the
lungs where ventilation keeps the pCO2
low, resulting in
reversal of the above reactions and excretion of CO2
in the
expired air.
• About 70% of tissue carbon dioxide is processed in this way.
Of the remaining 30%, 5% is carried in simple solution and
25% is bound to the N-terminal amino groups of
deoxygenated hemoglobin, forming carbaminohemoglobin.

73. Physiological Bohr Effect

74. Hb and the Bohr Effect (cont)
• An equivalent amount of H+
released (about
0.31 H+
per oxygen bound) is picked up by Hb
as acid (H+
) produced from tissue metabolism.
• At the lungs, when oxygen rebinds, the H+
released is converted to carbon dioxide and
exhaled. About 40% of acid produced by the
tissues is buffered in this way by Hb

75. Hemoglobin and the Bohr Effect
• The Bohr Effect is the direct result of the conformational
changes that occur in Hb during oxygen binding.
• About 50% of the effect is caused by a change in pKa of His
146 (on beta subunit).
• In the T-state, the His is H-bonded to Asp 94. When Hb shifts
from the T to R conformation, the H-bond to Asp 94 is
disrupted, allowing a proton on the His to readily dissociate.
• The remainder of the effect results from similar pKa changes of
other ionizable groups as a consequence of the T to R
transition. This is the O2 bind/H+
release portion of the effect

78. Bohr Effect Summary
• The change in oxygen affinity with pH is known
as the Bohr effect.
• Hemoglobin oxygen affinity is reduced as the
acidity increases.
• Since the tissues are relatively rich in carbon
dioxide, the pH is lower than in arterial blood;
therefore, the Bohr effect facilitates transfer of
oxygen.
• The Bohr effect is a manifestation of the acid-
base equilibrium of hemoglobin.

79. 2,3-diphosphoglycerate
(2,3-DPG)
• This compound is synthesized from glycolytic intermediates.
• In the erythrocyte, 2-3-DPG constitutes the predominant
phosphorylated compound, accounting for about two thirds of the
red cell phosphorus.
• The proportion of 1,3-DPG pathway appears to be related largely to
cellular ADP and ATP levels; when ATP falls and ADP rises, a
greater proportion of 1,3-DPG is converted through the ATP-
producing step.
• This mechanism serves to assure a supply of ATP to meet cellular
needs.
• In the deoxygenated state, hemoglobin A can bind 2,3-DPG in a
molar ratio of 1:1, a reaction leading to reduced oxygen affinity
and improved oxygen delivery to tissues.

81. Physiological Function of DPG
• The presence of DPG in the erythrocyte creates a
dynamic competition for oxygen binding to deoxy-Hb.
DPG bound to deoxy-Hb reversibly stabilizes the
deoxy conformation.
• As [DPG] increases, the greater the percentage of
oxygen that will be released by Hb at the peripheral
tissues. This process occurs naturally due to the
differences in oxygen pressures between the lung and
periphery, plus the presence of DPG further promotes
this oxygen release.
• This is one mechanism to increase oxygen delivery to
tissues during exercise and/or at high altitudes

83. 2,3-Bisphosphoglycerate
An Allosteric Effector of Hemoglobin
• In the absence of 2,3-BPG, oxygen binding to
Hb follows a rectangular hyperbola!
• The sigmoid binding curve is only observed in
the presence of 2,3-BPG
• Since 2,3-BPG binds at a site distant from the
Fe where oxygen binds, it is called an allosteric
effector

84. 2,3-BPG and Hb
• Where does 2,3-BPG bind?
– "Inside"
– in the central cavity
• What is special about 2,3-BPG?
– Negative charges interact with 2 Lys, 4 His,
2 N-termini
• Fetal Hb - lower affinity for 2,3-BPG, higher
affinity for oxygen, so it can get oxygen from
mother

85. 2,3 Diphosphoglycerate
(DPG)
2,3 Diphosphoglycerate (DPG) (or called 1,3 bisphosphoglycerate
(BPG)) binds tightly to the channel interface of deoxyHb, a binding
stabilized by interactions with positively charged sidechains. DPG
has a poor binding affinity for oxyHb due to the compacted channel

86. • When oxygen is unloaded by the hemoglobin
molecule and 2,3 DPG is bound, the molecule
undergoes a conformational change becoming what is
known as the ""Tense, Taut" or "T" form.
• The resultant molecule has a lower affinity for
oxygen.
• As the partial pressure of oxygen increases, the 2,3,
DPG is expelled, and the hemoglobin resumes its
original state, known as the "relaxed" or "R" form, this
form having a higher oxygen affinity.
• These conformational changes are known as
"respiratory movement".
• The increased oxygen affinity of fetal hemoglobin
appears to be related to its lessened ability to bind
2,3-DPG.
• The increased oxygen affinity of stored blood is
accounted for by reduced levels of 2,3-DPG.

87. • Changes in 2,3-DPG levels play an important
role in adaptation to hypoxia. In a number of
situations associated with hypoxemia, 2,3-DPG
levels in red cells increase, oxygen affinity is
reduced, and delivery of oxygen to tissues is
facilitated.
• Such situations include abrupt exposure to high
altitude, anoxia due to pulmonary or cardiac
disease, blood loss, and anemia.
• Increased 2,3-DPG also plays a role in
adaptation to exercise. However, the compound
is not essential to life; an individual who lacked
the enzymes necessary for 2,3-DPG synthesis
was perfectly well except for mild polycythemia

88. Hb and Carbon Dioxide
Transport
• Some of the dissolved CO2 in the blood
reacts with amino groups on Hb (and other
proteins). This reaction occurs with N-
terminal amino groups in the NH2 form;
• At blood pH, the side chain groups are in
the NH3
+
form and will not react. About 15%
of tissue-produced CO2 is bound by
protein in this way (termed carbamino-
CO2)

89. Interactions between DPG,
Carbamino-CO2 and Bohr Effect
• The carbamino-CO2 can be bound to some of
the same groups involved in DPG binding,
thus diminishing DPG binding; the reverse
can occur, DPG can block CO2
• CO2 also binds to some of the groups
involved in the Bohr effect; thus bound CO2
can diminish the H+
buffering capacity of
hemoglobin (again the reverse effect can
occur, bound H+
blocking CO2

91. Methemoglobinemia
• In order to bind oxygen reversibly, the iron in the heme
moiety of hemoglobin must be maintained in the
reduced (ferrous) state despite exposure to a variety of
endogenous and exogenous oxidizing agents.
• The red cell maintains several metabolic pathways to
prevent the action of these oxidizing agents and to
reduce the hemoglobin iron if it becomes oxidized.
Under certain circumstances, these mechanisms fail and
hemoglobin becomes nonfunctional.
(mět'hē-mə-glō'bə-nē'mē-ə)

93. Methemoglobinemia
• At times, hemolytic anemia supervenes as
well. These abnormalities are particularly
likely to occur
(1) if the red cell is exposed to certain oxidant drugs
or toxins
(2) if the intrinsic protective mechanisms of the cell
are defective or
(3) if there are genetic abnormalities of the
hemoglobin molecule affecting globin stability or
the heme crevice.

94. Sickle Cell Anemia
• The most common form of sickle cell anemia is
caused by a single amino acid substitution of valine for
glutamate at position 6 on the β-subunit of
hemoglobin.
• This defect is an autosomal (non-sex chromosome)
recessive inherited disease, meaning both parents
must be heterozygous carriers to produce a
homozygous child.
• Even heterozygous carriers can experience sickle cell
symptoms after vigorous exercise or unpressurized
travel at high altitudes.

95. Sickle Cell Anemia (cont)
• The Glu to Val mutation in sickle cell hemoglobin
(termed Hb-S) reduces the solubility of
deoxyhemoglobin and allows formation of fibrous
polymeric filaments of deoxyhemoglobin that
precipitate in the red blood cells.
• This precipitation leads to an ultrastuctural deformity
of the red blood cell, the "sickle" shape, which gives
these cells a tendency to get hung up and accumulate
in the narrow capillaries (thus leading to the
associated peripheral pain and many other
complications).

96. Subunit interactions
that promote
polymerization of
deoxy-HbS

97. Sickle Cell Anemia (cont)
• Upon exposure of oxygen at the lungs, the HbS
filaments immediately dissolve.
• Thus, situations that slow down flow of blood through
the capillaries (normally 0.5 to 2 secs) can lead to the
periodic sickle cell anemia “crises”. Conditions like
oxygen deprivation (such as high altitudes or
strenuous exercise) and dehydration can contribute to
slower capillary passage of erythrocytes. The longer
times allow deoxy-HbS polymerization to occur, and
thus further contribute to the blockage of the affected
area.

98. Other Types of Hemoglobin
Mutations (Examples)
• Higher O2 affinity, β143 His to Gln; 2,3DPG binding
decreased
• Lower O2 affinity, β102 Asn to Thr; T-form stabilized
• Methemoglobinemias, β63 His to Tyr; increased
stability for Fe3+ caused by Tyr residue α-helix
disruptors, β63 His to Pro; Helix E disrupted, heme
pocket opened, MetHb forms
• Decreased stability of Hb, β43 Phe to Val; heme
pocket destabilized

99. Sickle Cell Anemia -
Structure/Function Concepts
• Hb-S polymerization illustrates how one amino acid
change from a charged to non-polar residue can lead
to powerful, cumulative hydrophobic interactions.
• These are the same forces that hold the Hb monomer
cores together, while the salt bridges and hydrogen
bonds stabilize the surface interactions between
subunits.
• Loss of the charged surface Glu allows more stable
hydrophobic interactions to form in the deoxy-Hb
conformation.

100. Summary of Hemoglobin
Summary of Hemoglobin
 Oxygen delivery to the tissues
Oxygen delivery to the tissues
 Reaction of Hb & oxygen
Reaction of Hb & oxygen
 Oxygenation not oxidation
Oxygenation not oxidation
 One Hb can bind to four O
One Hb can bind to four O2
2 molecules
molecules
 β
β chain move closer when oxygenated
chain move closer when oxygenated
 When oxygenated 2,3-DPG is pushed out
When oxygenated 2,3-DPG is pushed out
 β
β chains are pulled apart when O
chains are pulled apart when O2
2 is unloaded, permitting
is unloaded, permitting
entry of 2,3-DPG resulting in lower affinity of O
entry of 2,3-DPG resulting in lower affinity of O2
2

101. Short Summary
• Cooperativity of oxygen binding to
hemoglobin
• Modulators of oxygen binding to hemoglobin
and the Bohr Effect
• Blood pH, and partial pressure of carbon
dioxide influence hemoglobin saturation by
altering hemoglobin's three-dimensional
structure, thus changing its affinity for oxygen.

102. Sample questions
Sample questions
• What is the prosthetic group that hemoglobin and
myoglobin's oxygen binding ability depend on?
• Define cooperativity relative to binding oxygen
• What are the two states of the hemoglobin quaternary
structure? And what are their characteristics?
Heme
binding of oxygen at one site increases chances
of binding oxygen at the other sites; loss of an
oxygen at one site increases the chances of
losing oxygen at the other sites
T state = taut (deoxy form)
R state = relaxed (oxygenated form)

103. Derivatives of hemoglobin
 Oxyhemoglobin (oxyHb) = Hb with O2
 Deoxyhemoglobin (deoxyHb) = Hb without O2
 Methemoglobin (metHb) contains Fe3+
instead of
Fe2+
in heme groups
 Carbonylhemoglobin (HbCO) – CO binds to Fe2+
in heme in case of CO poisoning or smoking. CO has
200x higher affinity to Fe2+
than O2.
 Carbaminohemoglobin (HbCO2) - CO2 is non-
covalently bound to globin chain of Hb. HbCO2
transports CO2 in blood (about 23%).