09.02.

2015

Chromoproteins
Hemoglobin, • Chromoproteins contain a colored non-
protein part (from Greek “chroma” – dye)
myoglobin Chromoproteins possess a number of unique
biological properties: they are involved in the
fundamental process of vital activity such as
photosynthesis, respiration of cells and whole
organism, transport of oxygen and carbon
dioxide, oxidation-reduction reactions, light
and color perceptibility and others.

Hemoglobin Heme is an inorganic prosthetic

• Hemoglobin (Mr 64,500; abbreviated
Hb(αα β β)) is roughly spherical, with a
diameter of nearly 5.5 nm), a water
soluble globular protein which is
composed of Heme and Globin. :
group that mediates reversible
binding of oxygen by hemoglobin.
Globin is the protein that surrounds
and protects the heme molecule,
invludes:
two β polypeptide chains (146 AA)
two α polypeptide chains (141AA),

Hemoglobin
function is to carry
Oxygen and Carbon
dioxide around in the
blood
This is facilitated
by the presence of
the heme group
which contains a
Fe2+ ion, onto which
the oxygen
molecules binds.

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Heme The heme group is present in

myoglobin, hemoglobin, and many
other proteins, designated heme
proteins.
Heme consists of a complex organic
ring structure, protoporphyrin IX, to
which is bound an iron atom in its
ferrous (Fe2+ ) state.

Porphyrins, of which
protoporphyrin IX is only one
example, consist of four pyrrole
rings linked by methene bridges,
with substitutions at one or more
of the positions denoted X.

The iron atom of

heme has six
coordination
bonds:
four in the plane
of heme, and
bonded to, the flat
porphyrin ring
system, and
two perpendicular
to it.

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O2 binds reversibly to hemoglobin Fe2+

(oxyhemoglobin)
CO2 in the blood reacts (reversibly)
with amino groups on Hb forming
carbamino-CO2 (carbhemoglobin)
CO binds tightly to the Hb Fe2+ forming
Carbon Monoxyhemoglobin
(carboxyhemoglobin)
Hemoglobin transports H+ and CO2 in
addition to O2.

Myoglobin Has a Single Binding Site for

Oxygen

• Myoglobin (Mr 16,700; abbreviated Mb)

is a relatively simple oxygen-binding
protein found in almost all mammals,
primarily in muscle tissue.
• It facilitates oxygen diffusion in muscle.

•Myoglobin is
particularly
abundant in the • Myoglobin is a single polypeptide of
muscles of 153 amino acid residues with one
diving mammals
molecule of heme.
such as seals
and whales, • It is typical of the family of proteins
where it has an called globins, all of which have similar
oxygen-storage primary and tertiary structures
function for
prolonged
excursions
undersea.

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•The polypeptide
is made up of
eight -helical
segments
connected by
bends
•About 78% of the
amino acid
residues in the
protein are found
in these helices

• Oxygenation of myoglobin and

hemoglobin chain(ether α- or β- )is
accompanies by movement of the iron
atom, consequently movement of His
F8 and residues covalently linked to
the His F8 , towards the plane of the
ring.
• This motion brings about a new
conformation of the protein.

Molecular Shift at the Heme Group after

Conformational Changes in Hemoglobin upon
Oxygen Binding
binding of O2
• The heme iron atom in deoxyHb is slightly
out of the plane of the porphryin ring and
bound to a histidine imidazole side chain. Blue: deoxy
Red: oxy
• When oxygen binds to the heme on the side
opposite the histidine, the iron atom’s
electron cloud becomes slightly smaller, and
the iron moves into the plane of the
porphryin ring, also pulling the histidine side
chain in the same direction

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Quaternary conformational changes in the

Conformational Changes oxygen bound (red) and deoxyhemoglobins
• The movement of the histidine causes the (blue)
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 aβ pair.
• Movement of one subunit in the aβ pair
causes a corresponding conformational
adjustment in the paired subunit,
enhancing the ability of the latter to bind
oxygen.

Hemoglobin Binds Oxygen Cooperatively Transport of

Oxygen and Carbon
Thus, filling the first oxygen binding site Dioxide by
in hemoglobin increases the affinity of Hemoglobin
the remaining sites for oxygen.
The binding of O2 to
hemoglobin causes
Conversely, losing an oxygen from rupture of some of
hemoglobin makes it easier for the the ionic and
protein to lose its remaining oxygen hydrogen bonds.
molecules.

The tertiary configuration of low

affinity, deoxygenated hemoglobin
(Hb) is known as the taut (T) state.

Conversely, the quaternary structure

of the fully oxygenated high affinity
form of hemoglobin (HbO2) is known
as the relaxed (R) state.

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Oxygen-binding curves of both hemoglobin

and myoglobin.

• The myoglobin curve is hyperbolic while that

of hemoglobin is sigmoidal.
• The latter indicates cooperativity as
mentioned above.
• Myoglobin, with its hyperbolic binding curve
for oxygen, is relatively insensitive to small
changes in the concentration of dissolved
oxygen and so functions well as an oxygen-
storage protein.
• The affinity for oxygen by Myoglobin 25
times fold

• Hemoglobin, with its multiple subunits • Interactions among the subunits in

and O2-binding sites, is better suited to
oxygen transport.
• Interactions between the subunits of a
multimeric protein can permit a highly
sensitive response to small changes in
ligand concentration.
hemoglobin cause conformational
changes that alter the affinity of the
protein for oxygen.
• The modulation of oxygen binding
allows the O2-transport protein to
respond to changes in oxygen demand
by tissues.

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• Hemoglobin must bind oxygen
efficiently in the lungs, where the pO2
is about 13.3 kPa (90-100 mmHg), and
release oxygen in the tissues, where
the pO2 is about 4 kPa(20-40 mmHg).
• Myoglobin, or any protein that binds
oxygen with a hyperbolic binding
curve, would be ill-suited to this
function
• Hemoglobin solves the problem by
undergoing a transition from a low-
affinity state (the T state) to a high-
affinity state (the R state) as more O 2
molecules are bound.
• As a result, hemoglobin has a hybrid S-
shaped, or sigmoid, binding curve for
oxygen.
• A single-subunit protein with a single
ligand-binding site cannot produce a
sigmoid binding curve— even if
binding elicits a conformational
change— because each molecule of
ligand binds independently and cannot
affect the binding of another molecule
• In contrast, O2 binding to individual
subunits of hemoglobin can alter the
affinity for O2 in adjacent subunits.
09.02.2015
• A protein that bound O2 with high
affinity would bind it efficiently in the
lungs but would not release much of it
in the tissues.
• If the protein bound oxygen with a
sufficiently low affinity to release it in
the tissues, it would not pick up much
oxygen in the lungs.
• The first molecule of O2 that interacts
with deoxyhemoglobin binds weakly,
because it binds to a subunit in the T
state.
• Its binding, however, leads to
conformational changes that are
communicated to adjacent subunits,
making it easier for additional
molecules of O2 to bind.
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• In effect, the T n R transition occurs
more readily in the second subunit
once O2 is bound to the first subunit.
• The last (fourth) O2 molecule binds to a
heme in a subunit that is already in the
R state, and hence it binds with much
higher affinity than the first molecule
• CO2, H+, and a special
metabolite of erythrocytes—
2,3-bisphosphoglycerate
(BPG)— act as heterotropic
effectors of hemoglobin.
09.02.2015
Oxygen transport
• Most tissues are constantly dependent on a
supply of molecular oxygen (O2) to maintain
their oxidative metabolism.
• 0,3ml of oxygen / 100 ml of blood is
transported in soluble state (1,5% of all
transported oxygen).
• Due to its poor solubility, O2 is bound to
hemoglobin for transport in the blood.
Hb deliver 600 L of oxygen to tissues and
promotes excretion of 500L of carbon
dioxide
• This not only increases the oxygen
transport capacity, but also allows
regulation of O2 uptake in the lungs and
O2 release into tissues.
• Oxygen is transported as HBO2.
• BPG is synthesized from 1,3
bisphosphoglycerate, an intermediate of
glycolysis, and it can be returned to
glycolysis again by breakdown into 2–
phosphoglycerate, with loss of an Pi.
• BPG binds selectively to deoxy–Hb, thereby
increasing its amount of equilibrium
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• The presence of BPG in the
erythrocyte creates a dynamic
competition for oxygen binding to
deoxy-Hb.
• BPG bound to deoxy-Hb reversibly
stabilizes the deoxy-conformation.
• As [BPG] increases, the greater
the percentage of oxygen that will
be released by Hb at the
peripheral tissues.
09.02.2015
• This process occurs naturally due to
the differences in oxygen pressures
between the lung and periphery, plus
the presence of BPG further promotes
this oxygen release.
• This is one mechanism to increase
oxygen delivery to tissues during
exercise and/or at high altitudes
• Imagine that this person is quickly
transported to a mountainside at an
altitude of 4,500 meters, where the pO2
is considerably lower.
• The delivery of O2 to the tissues is now
reduced.
• However, after just a few hours at the
higher altitude, the BPG concentration
in the blood has begun to rise, leading
to a decrease in the affinity of
hemoglobin for oxygen.
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• This adjustment in the BPG level has

only a small effect on the binding of O2
in the lungs but a considerable effect
on the release of O2 in the tissues
• As a result, the delivery of oxygen to
the tissues is restored to nearly 40% of
that which can be transported by the
blood.
• The situation is reversed when the
person returns to sea level.

• The BPG concentration in erythrocytes

also increases in people suffering from
hypoxia, lowered oxygenation of
peripheral tissues due to inadequate
functioning of the lungs or circulatory
system

• The result is increased O2 release

at constant pO2.
• In the diagram, this corresponds to a right
shift of the saturation curve.
• CO2 and H+ act in the same direction as
BPG.
• Their influence on the position of the curve
has long been known as the Bohr effect.

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• The effects of CO2 and BPG are

additive.
• In the presence of both effectors, the
saturation curve of isolated Hb is similar
to that of whole blood.

• Bohr effect: acidity increased O2 releasing in tissues

• H+ + HbO2 → HHbO2 → HHb + O2 ( into tissues)
• pCO2
• ↑ pCO2 → ↑H2CO3 → ↑H+
• CO2 enhances O2 releasing into tissues.

• ↑t° promotes HHbO2 dissociation

When the oxygen concentration is

Hemoglobin releases H+ when it binds
high, as in the lungs, hemoglobin binds
O2
O2 and releases protons.

Hemoglobin binds H+ when it releases

When the oxygen concentration is low, O2
as in the peripheral tissues, H is bound
and O2 is released. HbO2 + H+ HHb + O2

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Physiological Bohr Effect
• In lungs:
• CO2 removal by lungs could lead to shift of
pH to alkaline region, but simultaneously the
following reaction occurs:
• HHb + O2 → HHbO2 → H+ + HbO2
that counteracts alkalinization
09.02.2015
• HHb plays a role of weak acid
• HHbO2 – is the more strong acid then HHb.
• This buffer works with respiratory function of
blood.
• In tissues:
CO2 released by metabolic reactions:
CO2 + H2O→ H2CO3 → H+ + HCO3‫־‬
• That lead to acidation, but
H+ + HbO2 → HHbO2 → HHb + O2
that counteracts acidation
• Hb released O2 to tissues and binds H+
HHb + O2 ↔ HHbO2 ↔ H+ + HbO2
Hemoglobinopathies
• The reason of hemoglobinopathies is
the alteration in AA sequence.
• Classical example is sickle cell anemia
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Sickle Cell Anemia HbS Amino acid Substitution in HbC and HbS

HbS

• VHLTPVEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRF
FESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLK
GTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPP
VQAAYQKVVAGVANALAHKYH

Normal Hemoglobin HbA

• VHLTPGEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRF
FESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLK
GTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPP
VQAAYQKVVAGVANALAHKYH

• In HbS, the nonpolar amino acid valine has

replaced the polar surface residue Glu6 of
the β subunit, generating a hydrophobic
“sticky patch” on the surface of the β subunit
of both oxyHbS and deoxyHbS.
• Both HbA and HbS contain a complementary
sticky patch on their surfaces that is
exposed only in the deoxygenated, T state.
• Thus, at low PO2, deoxyHbS can polymerize
to form long, insoluble fibers.

• Binding of deoxy- HbA terminates fiber

polymerization, since HbA lacks the second
sticky patch necessary to bind another Hb
molecule.
• These twisted helical fibers distort the
erythrocyte into a characteristic sickle
shape, rendering it vulnerable to lysis in the
interstices of the splenic sinusoids.
• They also cause multiple secondary clinical
effects. A low PO2 such as that at high
altitudes exacerbates the tendency to
polymerize.

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Hb Electrophoresis for HbA, HbS and HbC

• Sickle-cell anemia occurs in individuals

homozygous for the sickle-cell allele of the
gene encoding the subunit of hemoglobin.
• Individuals who receive the sickle-cell allele
from only one parent and are thus
heterozygous experience a milder condition
called sickle-cell trait; only about 1% of their
erythrocytes become sickled on
deoxygenation.
• These individuals may live completely
normal lives if they avoid vigorous exercise
or other stresses on the circulatory system.
• Sickle-cell anemia is a life-threatening
and painful disease.
• People with sickle-cell anemia suffer
from repeated crises brought on by
physical exertion.
• They become weak, dizzy, and short of
breath, and they also experience heart
murmurs and an increased pulse rate.

•The hemoglobin •An even more

content of their serious consequence
blood is only about is that capillaries
half the normal become blocked by
value of 15 to 16 the long, abnormally
shaped cells, causing
g/100 mL, because
severe pain and
sickled cells are interfering with
very fragile and normal organ
rupture easily; this function—a major
results in anemia factor in the early
death of many people
with the disease.

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About 30% of Jamaican patients with Sickle • Without medical treatment, people with sickle-cell
Cell develop ulcers in comparison to 1% of anemia usually die in childhood.
Americans • Nevertheless, the sicklecell allele is surprisingly
common in certain parts of Africa.

•Investigation into the

persistence of an • Natural selection has resulted in an allele
allele that is so population that balances the deleterious
obviously deleterious effects of the homozygous condition against
in homozygous the resistance to malaria afforded by the
individuals led to the heterozygous condition.
finding that in
heterozygous • Even heterozygous carriers can experience
individuals, the allele sickle cell symptoms after vigorous exercise
confers a small but or unpressurized travel at high altitudes.
significant resistance
to lethal forms of
malaria.

Sickle cell Trait are resistant to Malaria,
theories…
1. The carriers of Sickle Cell have some abnormal
Hemoglobin, and when they come in contact
with the Malaria parasite they become sickled.
Then those cells go through the spleen, which
eliminates the cells because of their sickle
shape, so the Malaria would be eliminated as
well.
2. The Sickle Cell trait causes the malaria to stay
in the body for an extended period of time, so it
is able to build up a defense to it.
3. Because oxygen concentration is low in the
spleen, and because infected cells often get
trapped in the spleen, it is possible that they are
destroyed in the spleen
4. The Malaria parasite produces an acid when it
is inside of the red blood cells.
• This causes the red blood cells to polymerize,
and the cells will sickle.
• These sickled cells are then destroyed when
the blood cells go through the spleen.

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• Homologos proteins are present in organism
• They arose in evolution due to exchange of a
• Their conformation usually contains the same
• All vertebrates have an immune system
capable of distinguishing molecular
“self” from “nonself” and then
destroying those entitatives identified
as nonself.
• In this way, the immune system
eliminates viruses, bacteria, and other
pathogens and molecules that may
pose a threat to the organism.
• Immunity is brought about by a variety of
leukocytes (white blood cells), including
macrophages and lymphocytes, all
developing from undifferentiated stem
cells in the bone marrow.
• Leukocytes can leave the bloodstream
and patrol the tissues, each cell
producing one or more proteins capable
of recognizing and binding to molecules
that might signal an infection.
09.02.2015
Immunoglobulins
have close conformation but differ in
polypeptide structure for example myoglobin
and hemoglobin.
particular amino acids in the chain.
amount of α and β structures and have the
same turns and kinks. Homologos proteins are
numerous and combined to protein family
(immunoglibulins, hemoglobin, albumins and so
on).
• On a physiological level, the response
of the immune system to an invader is
an intricate and coordinated set of
interactions among many classes of
proteins, molecules, and cell types.
• However, at the level of individual
proteins, the immune response
demonstrates how an acutely sensitive
and specific biochemical system is
built upon the reversible binding of
ligands to proteins.
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• The proteins at the heart of the

•The immune response consists of two
complementary systems, the humoral and
cellular immune systems.
•The humoral immune system (Latin humor,
“fluid”) is directed at bacterial infections
and extracellular viruses (those found in
the body fluids), but can also respond to
individual proteins introduced into the
organism.
•The cellular immune system destroys host
cells infected by viruses and also destroys
some parasites and foreign tissues.
humoral immune response are soluble
proteins called antibodies or
immunoglobulins, often abbreviated Ig.
•Immunoglobulins bind bacteria, viruses,
or large molecules identified as foreign
and target them for destruction.
•Making up 20% of blood protein, the
immunoglobulins are produced by B
lymphocytes, or B cells, so named
because they complete their
development in the bone marrow.

• The agents at the heart of the cellular

immune response are a class of T
lymphocytes, or T cells (so called
because the latter stages of their
development occur in the thymus),
known as cytotoxic T cells (TC cells,
also called killer T cells).

• The fundamental structure of

immunoglobulins was first established
• Recognition of infected cells or
by Gerald Edelman and Rodney Porter.
parasites involves proteins called T-
cell receptors on the surface of TC • Each chain is made up of identifiable
cells. domains; some are constant in
sequence and structure from one Ig to
• Receptors are proteins, usually found
the next, others are variable.
on the outer surface of cells and
extending through the plasma • The constant domains have a
membrane; characteristic structure known as the
immunoglobulin fold, a well-conserved
• They recognize and bind extracellular
structural motif
ligands, triggering changes inside the
cell.

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• There are three of these constant

domains in each heavy chain and one
in each light chain.
• The heavy and light chains also have
one variable domain each, in which
most of the variability in amino acid
residue sequence is found.
• The variable domains associate to
create the antigen-binding site.

• Immunoglobulin G (IgG) is the major

class of antibody molecule and one of
the most abundant proteins in the
blood serum.
• IgG has four polypeptide chains: two
large ones, called heavy chains, and
two light chains, linked by noncovalent
and disulfide bonds into a complex of
Mr 150,000.

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• The heavy chains of an IgG molecule

interact at one end, then branch to
interact separately with the light
chains, forming a Y-shaped molecule.
• At the “hinges” separating the base of
an IgG molecule from its branches, the
immunoglobulin can be cleaved with
proteases.
• Cleavage with the protease papain
liberates the basal fragment, called Fc
because it usually crystallizes readily,
and the two branches, called Fab, the
antigen-binding fragments.
• Each branch has a single antigen-
binding site.

The overall structures of IgD and IgE are

similar to that of IgG

• In many vertebrates, IgG is but one of

five classes of immunoglobulins. Each
class has a characteristic type of
heavy chain, denoted α, δ, ε, γ, and μ
for IgA, IgD, IgE, IgG, and IgM,
respectively.
• Two types of light chain, κ and λ, occur
in all classes of immunoglobulins.

• IgM occurs either in a monomeric,

membrane bound form or
• in a secreted form that is a cross-
linked pentamer of this basic
structure.
• Monomeric – Is a receptor recognizing
antigen.
• Pentamer is secreted in primary
immune response and effective against
viruses and bacteria

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IgA, found principally in secretions such
as saliva, tears, and milk, can be a
monomer, dimer, or trimer.
• The IgG described above is the major
antibody in secondary immune
responses, which are initiated by
memory B cells.
• As part of the organism’s ongoing
immunity to antigens already
encountered and dealt with, IgG is the
most abundant immunoglobulin in the
blood.
• It composes 75% of all immunoglobulins.
09.02.2015
• Microorganisms attached to IgA
can’t bind to mucosa surface and
participate in contamination
• IgM is the first antibody to be made by
B lymphocytes and is the major
antibody in the early stages of a
primary immune response.
• Some B cells soon begin to produce
IgD (with the same antigen-binding site
as the IgM produced by the same cell),
have monomeric structure and plays
role as a surface receptor for antigen
recignizing.
• When IgG binds to an invading bacterium
or virus, it activates certain leukocytes
such as macrophages to engulf and
destroy the invader, and also activates
some other parts of the immune
response.
• Yet another class of receptors on the cell
surface of macrophages recognizes and
binds the Fc region of IgG. When these
Fc receptors bind an antibody-pathogen
complex, the macrophage engulfs the
complex by phagocytosis.
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IgG is the only passing placental barrier

and providing antenatal defense of fetus
and passive immunity of newborns in fist
weeks of life

• IgE plays an important role in the

allergic response, interacting with
basophils (phagocytic leukocytes) in
the blood and histamine-secreting
cells called mast cells that are widely
distributed in tissues.
• This immunoglobulin binds, through its
Fc region, to special Fc receptors on
the basophils or mast cells.

They also produce the symptoms

• In this form, IgE serves as a kind of
normally associated with allergies.
receptor for antigen. Pollen or other allergens are recognized
• If antigen is bound, the cells are as foreign, triggering an immune
induced to secrete histamine and response normally reserved for
other biologically active amines that pathogens.
cause dilation and increased
permeability of blood vessels.
• These effects on the blood vessels are
thought to facilitate the movement of
immune system cells and proteins to
sites of inflammation.

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• The binding specificity of an antibody • Specificity is conferred by chemical

is determined by the amino acid complementarity between the antigen
residues in the variable domains of its
and its specific binding site, in terms
heavy and light chains.
of shape and the location of charged,
• Many residues in these domains are nonpolar, and hydrogen-bonding
variable, but not equally so. groups.
• Some, particularly those lining the
antigen-binding site, are
hypervariable— especially likely to
differ.

• For example, a binding site with a

negatively charged group may bind an
antigen with a positive charge in the
complementary position.
• In many instances, complementarity is
achieved interactively as the structures of
antigen and binding site are influenced by
each other during the approach of the
ligand.
• Conformational changes in the antibody and /or
the antigen then occur that allow the
complementary groups to interact fully. This is an
example of induced fit.

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