LECTURE 9

PROTEINS
HAEMOGLOBIN
Chromoproteins

• They are heteroproteins with a coloured prosthetic group.

• Function of the nature of the prosthetic group, the chromoproteins can be
classified as:
• Porphyrinic chromoproteins – their prosthetic group is the heme, in
different variants (heme is a derivative of the porphyrinic nucleus). The
main proteins in this group are:
• Hemoglobin
• Myoglobin
• Cytochroms
• Chlorophylls
• Porphyrinic enzymes: catalase, peroxidase.
• Non porphyrinic chromoproteins – the prosthetic group is different of
heme. The main proteins in this group are:
• Caroteno-proteins – for example visual pigments;
• Flavoproteins – for example enzymes with enzymatic cofactor
derivatives of vitamin B2 (FMN and FAD).
Hemoglobin (abbreviated Hb) structure

• MW 64,500; iron represents 0.338%

• is roughly spherical;
• it is a tetrameric protein containing four heme prosthetic groups, one
associated with each polypeptide chain.
• Adult hemoglobin, A1, contains two types of globins, two α-chains (141
residues each) and two β-chains (146 residues each). Although fewer than
half of the amino acid residues in the polypeptide sequences of the α and β
subunits are identical, the three-dimensional structures of the two types of
subunits are very similar. Furthermore, their structures are very similar to
that of myoglobin.
• Both polypeptides are members of the globin family of proteins. Each
globin contains several α-helices:
• 7 segments, noted A – G in globin α
• 8 segments, noted A – H in globin β.
• In both types of α-helix structures C, E, F, and G are constituted
predominantly of hydrophobic amino acids and delimitate a space as a
pocket, where the prosthetic group – heme – is found.
• Heme is the prosthetic group of hemoglobins, in the ration 1 : 1 with the
proteic part – the globin.
• As structure, heme is a derivative of the porphyrin nucleus, the
protoporphyrin III (9), bound to the ion Fe2+.
• Heme is a cyclic tetrapyrrole consisting of four molecules of pyrrole linked
by α-methylene bridges. This planar network of conjugated double bonds
absorbs visible light and colours heme deep red.
• The substituents at the β-positions of heme are methyl (M - 1,3,5,8), vinyl
(V – 2,4), and propionate (Pr -6,7) groups
• One atom of ferrous iron (Fe2+) resides at the centre of the planar
tetrapyrrole.
• Other proteins with metal-containing tetrapyrrole prosthetic groups include
the cytochromes (Fe and Cu) and chlorophyll (Mg).
• Oxidation and reduction of the Fe and Cu atoms of cytochromes is
essential to their biologic function as carriers of electrons.
•
• By contrast, oxidation of the Fe2+ of myoglobin or hemoglobin to Fe3+
destroys their biologic activity
The bond heme - globin
• Bonds of Fe2+
• Bonds between the substituents of porphyrin ring and side chains of amino
acids residues in globin structure.
• Bonds of Fe2+: Fe2+ ion forms 6 bonds of which 4 with protoporphyrin (coordinative)
and 2 with Hys residues from the globin constitution, so binding the two
components.
• These bonds are permanent and coplanar, Fe2+ and the 4 pyrrole rings are situated
in the same plane.
• The 2 bonds with globin are coordinative bonds with nitrogen atoms of the 2
hystidine residues: proximal Hys and respectively distal Hys,
• The bond with proximal Hys is permanent and the bond with distal Hys is optional,
depending of the oxygen binding.
• ⇒ two structures:
• one without oxygen – deoxygenated Hb,
• a structure with oxygen – oxygenated Hb.

• Because one Hb molecule contains 4 heme molecules, it will bind 4 oxygen

molecules.
 H O H O

catenã globularã
Globular chain N CH C N CH C Globular
catenã chain
globularã

N His proximalã N
Proximal Hys

N N

Fe2+ Fe2+

N N
plan
Heme plan
Heme
N N
hem
plane hem
plane

Although Fe2+ is coplanar with protoporphyrin in deoxygenated form, the

binding to proximal Hys trigger it up the heme plane, resulting the tensioned
(taut) state T
 H O

N CH C Globular chain
catenã globularã

N
Proximal Hys
His proximalã
N

planplane
Heme
Fe2+
hem

O2

Distal Hys
His distalã
N

N CH C catenã globularã
Globular chain
H O

The oxygen binding permits the realization of the bond between Fe2+ and
distal Hys, and Fe2+ is replaced in the heme plane. This structure, realized
by oxygen binding, is called relaxed state, R .
Bonds between the substituents of porphyrin ring and side chains of amino
acid residues in globin
• These bonds are weaker, being non covalent.
• hydrophobic bonds between methyl and vinyl substituents of heme and the
hydrophobic side chains of amino acids from globin structure. Because
heme is found in a cavity (pocket) of globin, these bonds expel water from
this zone.
• ionic bonds: ionized groups –CH2-CH2-COO- from heme and -NH3+ groups
from globin structure.
• All these bonds attach tightly heme to globin and, in plus, hinder the
oxidation of Fe2+ of heme.
• In the case of free heme, this oxidation takes place immediately, and the
highest drawback is that a strong bond Fe3+ - O2 is formed, fact that would
hinder the oxygen release to tissues.
• the binding Fe2+ - O2 is weak and reversible, fact that permits the oxygen
release to tissues when the conformation modifies from the R state to T
state (Hb-O2 → Hb).
• This conformational modification induced by oxygen molecule at the level
of the whole Hb molecule, shows that oxygen is an allosteric factor.
 O2

Cooperativity phenomenon
Simplified model of hemoglobin passing TConformaţia
conformation O2
from the T conformation into the R T

conformation
O2
O2 O2
O2
O2

O2

O2

O2

O2 O2

Conformaţia
R conformation
R

O2 O2
• The quaternary structure of hemoglobin features strong interactions
between unlike subunits.
•
• The α1β1 interface (and its α2β2 counterpart) involves more than 30
residues, and its interaction is sufficiently strong that although mild
treatment of hemoglobin with urea tends to cause the tetramer to
disassemble into αβ dimers, these dimers remain intact.
•
• The α1β2 (and α2β1) interface involves 19 residues.

• Hydrophobic interactions predominate at the interfaces

• There are also many hydrogen bonds and a few ion pairs (sometimes
referred to as salt bridges).
Functional properties of hemoglobin:

Oxygen transport from lungs to tissues

Facilitation of CO2 and H+ transport from tissues to lungs

Role in maintaining of acid-base equilibrium, the system Hb

– O2 having a high buffering capacity
Oxygen transport
• Hb is found in erythrocytes and not free in blood because these
cells have no nucleus and mitochondria, so that they will not
consume oxygen during oxidizing processes, needed to obtain
energy. They gain energy by anaerobic glycolysis.
• Hb structure permits very important physiological adaptations:
• It ensures a quantitative transport – 4 oxygen molecules /
Hb molecule
• Oxygen releasing to tissues is regulated just by their
energetic needs (pO2 ↑ concentraGon CO2 ↓)
• It permits the permanent existing of a oxygen reserve
quantity in blood, quantity that ensures the surviving for 2-3
minutes in the conditions of breathing stop, or a medium
without oxygen (in diving)
• A first question is why oxygen needs a transporter. To answer,
blood performances of oxygen transport with and without Hb
must be compared.

• At 37°C, 1 litre of plasma may dissolute only 2.3 ml of oxygen.

• Mean time, 1 litre of blood that contains 150 g Hb can
transport 200 ml of oxygen, which corresponds to 1.34 ml
oxygen / g Hb;
• ⇒ that means a quantity 87 times higher.
• In the absence of Hb blood should circulate 87 times more
quickly, with a velocity of 18.6 m/sec, fact that would need a
high pressure pump, flowing turbulences and specially, an
insufficient contact with lungs to take over the oxygen.
• Structural differeneces between Hb and Myo→different
oxygen binding capacity of the two proteins
• Two evolutionary related proteins
• Nearly identical structures for oxygen binding

O=O
O-
Fe 2+ O

Fe 3+
• All this properties are possible due to the
quaternary structure of Hb that makes
possible a cooperative effect at the oxygen
binding. They can be explained by comparison
with a similar molecule, myoglobin that has a
monomer tertiary structure.

• If the oxygen saturation (expressed as

percent, %) is represented graphically as a
function of the partial pressure of oxygen,
two different curves are obtained, a sigmoid
for hemoglobin and a semi-hyperbola for
myoglobin. In myoglobin case, the oxygen
saturation curve has a hyperbola shape
because, being a monomeric structure, there
is no cooperative effect.

• Myoglobin affinity for oxygen is much higher,

adapted to its role to extract oxygen from
blood and to transmit it intracellular to
mitochondrion.
EFFECT ON THE O2 TRANSPORT FROM LUNGS
TO TISSUE

• HB (FROM 98% SAT. (LUNGS) TO 32% IN TISSUES (TRANSPORT -66%)

• MYO (FROM 98% SAT. (LUNGS) TO 91% IN TISSUES (TRANSPORT - 7%)

• NON COOPERATIVE EFFECT : 63% (LUNGS) TO 25% IN TISSUES (transport 38%)

• !!! Hb approx. 10 times more effective in O2 transport when compared to MYO

• !!! Hb approx. 2 times more effective than non cooperative protein
EFFECT ON RELEASING O2 DURING EXERCISES
• LUNGS (100 TORR) SAT. IS 98%
21% O2 RELEASE
• REST MUSCLE (40 TORR) SAT. IS 77%
45% O2 RELEASE

• MUSCLE DURING EXERCISES (20 TORR) SAT. IS 32%

• !!!! THE O2 RELEASE IS DONE ACCORDINGLY WITH THE NEEDS

• Allosteric mechanism by which oxygen manifests the positive
cooperative effect is confirmed by the action of other allosteric
factors as: H+, CO2, Cl- and 2,3-bisphosphoglycerate (BPG).
• These do not interact with heme, so they do not intervene
directly in the binding Fe2+ - O2, but interact with components
of globins, producing conformational modifications, which
favour the reverse transiGon R → T, being negative allosteric
effectors by reducing the Hb affinity for oxygen.
• A special mention must be presented for 2,3-
bisphosphoglycerate. This is a compound contained by red
blood cells in high quantities, the molar ratio 2,3-
bisphosphoglycerate – Hb being 1:1.
• Unlike oxygen, an effector with homotrope action, BPG is a
heterotrope effector.
 O O
C O

H C O P O

H C H O

O P O

2,3-bisphosphoglycerate BPG binding to

hemoglobin cavity
It is considered that in T conformation there is a cavity large enough in which the negative
BPG forms strong bonds with NH3+ groups of the N-ends of β-globins.
In oxygenated form, R, this cavity diminishes, and BPG cannot enter that easy. Because
BPG prefers T conformation, it will s6mulate the transi6on R → T. In lungs → a high
oxygen pressure → oxygen will pass hemoglobin from conforma6on T → R, even if
BPG is present

In tissue hypoxia conditions (anaemia, cardio-pulmonary deficiency, high altitude), BPG

concentration increases.
Temperature
• < 37°C, the binding oxygen – Hb is much stronger, oxygen being tightly
bound.
• Temperature increase loosens this bond, oxygen being easier released.
• This phenomenon has physiological explanations.
• ⇒ T ↑ (fever, intense physical effort) ⇒ oxygen quantity that is released to
tissues will increase.

00 100 200 300 370 430

100
O2 saturation, %

50

20

20 40 60 80 100
O2 partial pressure, mmHg
Carbon monoxide

• incomplete combustion, smoke, exhaust gases, heme catabolism ⇒ CO

•
• CO binds to Fe2+ from Hb heme ⇒ competitive inhibitor for oxygen.

• CO affinity for Hb is 200 times higher that for O2 ⇒ very small quantities of CO will
replace O2 in Hb ⇒ produce fatale intoxications.

• binding CO – heme is reversible

• ⇒ The only remedy in the case of acute intoxication is administration of oxygen at

high pressure, so that to replace CO, by quantity

• In case of smokers, CO from cigarette blocks approximate 4 – 8% of Hb in the Hb –

CO form. Hemoglobin loses the oxygen transport capacity, smokers having a reduced
oxygenation of tissues.
• ⇒ smoking is forbidden to patients with reduced tissue oxygenation (i.e.
myocardial ischemia)
Glucose

• contact of Hb from RBC with blood glucose ⇒ formation, non

enzymatically, of keto-amine bonds between the carbonyl group of glucose
and terminal amino groups of β-globins.

• s6mulates transi6on R → T, reducing the affinity of hemoglobin for oxygen.

• Physiologically, 4-6% from total HbA1 is glycated (HbA1c).

• In diabetes mellitus, in the condition of maintaining of high glucose

concentration in blood, HbA1c percentage increases to 15-20%, ⇒ ⇒
reduces dramatically oxygen transport to tissues, especially at extremities
level.
• Possibility of diagnosis, control of diet effect, drug effect
H+ as allosteric negative effector (Bohr effect)
CO2 transport from tissues to lungs
(Bohr effect)
• tissue metabolism → CO2
• CO2 is eliminated at lung level by respiration.
Transport of CO2 from tissues to lungs is done using three ways:
• Dissolution in plasma, circa 10%
• Formation of chemical bonds with Hb (carbamate - hemoglobin)
+
→ 2H + + Hb - NH - COO -
CO 2 + Hb - NH 4 

• Conversion of N-terminal groups from + to – charge facilitates the transition R → T.

• In lungs, at high pO2 , Hb is oxygenated and CO2 is eliminated by respiration.
• Transport as HCO3- (Bohr effect)
Bohr effect (transport of CO2 as HCO3-)
In extralungs tissues:

ERYTHROCYTE
ERITROCIT
ŢESUT
TISSUE
PLASMA
PLASMĂ

O2 O2

HbH(+)
HbO2

HCO3 HCO3 H+
Cl Cl H2CO3
H2O H2O
CARBONIC
Anhidrază
carbonică
ANHYDRASE

CO2 CO2 + H2O

CO2 effect on Hb to peripheral tissues level

• CO2 + H2O ⇔ H2CO3 - Zn-enzyme carbonic anhydrase.

• HCO3- can cross the erythrocyte membrane by a special transport

exchanging Cl_, but H+ cannot.
• H+ will be neutralized in erythrocytes by the reaction:

• HbO2 + H+ ⇔ HHb+ + O2
(R) (T)
• CO2 + H2O ⇔ H+ + HCO3-

• HbO2 + CO2 + H2O ⇔ HHb+ + HCO3- + O2

• ⇒ Hb releases oxygen to tissues, attaching instead protons

⇒ Bohr effect:
• [H+] ↑ (pH ↓) ⇒ liberation of oxygen
• pO2 ↑ (in lungs) ⇒ liberation of H+.
In lung capillaries:
• pO2 ↑ ⇒ liberation of CO2 that is expired
ERYTHROCYTE
ERITROCIT
PLASMA
PLASMĂ

+
HCO3 HCO3 H
Cl Cl H2CO3
Anhidrază
CARBONIC
carbonică
ANHYDRASE

RESPIRATION
RESPIRAŢIE CO2 CO2 + H2O

CO2 liberation at lung level

Maintaining of acid – base equilibrium

• cellular catabolism → CO2 → H+ → pH modification

CO2 + H2O ⇔ HCO3- + H+

• Hb neutralizes (buffers) this excess of protons:

• 1. Buffering the ionisable groups at pH = 7.2 in erythrocytes

• - 4 amino-terminal groups of globins
• - nitrogen in imidasol heterocycle of Hys residues (38 of Hys / Hb)

• 2. Bohr effect - H+ resulted from CO2 + H2O ⇔ HCO3- + H+ → HHb+ →

lungs where HCO3- + H+ ⇔ CO2 + H2O;
• CO2 will be eliminated by respiration.
 Types of hemoglobins

• In cytogenia, a spectrum of hemoglobins - differ by the types of globins that form hemoglobin
structure.
• There is a succession of Hbs, in the order:
Hb Gower 1 (ε2ε2)
Hb Gower 2 (α2ε2) HbF (α2γ2) HbA (α2β2)
Hb Portland (ε2δ2) fetal stage adult

• At birth: HbF 75% and 25% HbA.

• HbA is, at its turn, of two types:

HbA1 (α2β2) – 97-98%
in adult stage
HbA2 (α2δ2) – 2-3%
• HbA becomes important at 4 months after birth.
• HbF has higher affinity for oxygen than HbA from the mother’s
blood;
• ⇒ foetus extracts oxygen from mother’s blood, foetus having
no contact with atmospheric air.

• HbF : in the cavity that binds 2,3-BPG, Hys with + charge is

replaced by Ser (non-ionisable).
• ⇒ reduces BPG binding ⇒ increases oxygen binding.
• 15-20% of HbF is acetylated at the N-terminal ends (HbF1), fact
that completely cancels the BPG binding.
• P50HbF = 20 mmHg,
• P50HbA = 26.8 mmHg
• ⇒ oxygen transfer from mother’s blood to the foetus blood
 Modified hemoglobins

Methemoglobins – Fe2+ → Fe3+ (oxidation)

• ⇒ irreversible binding of oxygen, loosing the capacity of oxygen releasing to tissues.
• Normally, 0.4-1% metHb, as a consequence of different oxidizing agents.
• metHb-reductase ⇒ Fe3+ → Fe2+.
• Methemoglobin has a brown color.
Sulfhemoglobin – S atom in porphyrinic ring
• ⇒ presence of certain drugs (Sumatriptan – head akes) or soluble sulfides.
• Sulfhemoglobin has a green color.
• The subunit sulfur-globin does not bind oxygen, but influences the other subunits in the sense of
oxygen releasing.
Pathologic hemoglobins – hemoglobinopathies

• genetic diseases ⇒ Hb synthesis ⇒ abnormal structure (substitution of an amino acid in a globin

with another amino acid, or the absence of one or more amino acids)
• ⇒ diverse pathologies ⇒ hemoglobinopathies.

• Hundreds of such mutation have been identified, most of them benign. Many of them include
endemic characteristics of certain populations.
• In some cases, the presence of such mutations ⇒ severe diseases, which can be divided into two
categories:

• 1) qualitative anomalies; i.e. sickle cell anemia (falciform)

• 2) quantitative anomalies; i.e. thalasemia
Sickle cell anemia

• mutation in globin β: Glu in position 6 ⇒ Val

• ⇒ HbS - modification of the entire Hb molecule
• ⇒ tendency to aggregate with other HbS molecules ⇒ polymers.
• ⇒ modification red blood cell from disc to sickle.
• ⇒ rigid, cannot pass through capillaries, blocking them.
• ⇒ frequent and strong pains in bones
• ⇒ On long term, this obstruction of capillary circulation will affect the
other organs (kidneys, heart, lungs) generating a chronic anaemia.

• identified in foetal stage by amniocentesis and DNA analysis at the level of

the gene that encodes β-globin.
• At adults - electrophoresis of plasma Hb
• Sickle cell anaemia is spread in equatorial (over 20% of population), due to
an adaptive advantage.
• ⇒ induces resistance to parasites that produce malaria; their life cycle
includes an obligatory stage in erythrocytes.
Thalasemias

• a family of genetic disease (Mediterranean aria and Asia)

• blocking synthesis of α-globin → α-thalasemias
β-globin → β-thalasemias.

• In case of α-thalasemias, there are 2 types:

- The majority of globins are β → HbH-anemia
- Only globin β → tetramers β4, resulGng hydrops fetalis
• In β-thalasemias - a large variety of mutations, which modify globin β, or
block its synthesis.

• In case of major thalasemia (Cooley anaemia) - globin synthesis is

completely blocked ⇒ fatal, the treatment consisting only in transfusions.

• hemoglobinopathia with HbF persistence - β globin synthesis is blocked,

but the synthesis of globin γ continues, the adult having HbF.
•
Porphyrinic chromoproteins
Myoglobin
• red pigment of muscle (at dolphins).
• similar structure to Hb, but is monomeric
(153 amino acids + 1 heme).
• tertiary structure - very similar to β-globin
• Myoglobin monomers interact with water,
so preventing their association.
• Affinity for oxygen is much higher, P50 = 1
mmHg.
• Oxygen binding gives a saturation curve
with hyperbola shape (similar to enzymes
saturation).
• In muscle cells - role of intermediary
between the venous oxygen (20-40 mmHg)
and the mitochondrial one (< 1-5 mmHg).
• represents the oxygen deposit of muscle
cell.
Cytochromes

• hemoproteins in which the heme Fe can transfer, reversibly, one electron, passing
in the states Fe2+ ⇔ Fe3+. metal participates effectively to the catalytic act.
• The electrons transport - cytochromes Fe3+ ⇔ Fe2+.
Two types of cytochromes:
• Cytochromes in respiratory chain in mitochondria (internal membrane):
a / a3, b, c, c1. (different binding of heme to globin or by differences in heme
structure.

• Cytochromes P450 - in microsomes or mitochondrion

• final acceptor of electrons,
• oxidation of different substances as: steroids, eicosanoides, drugs or other
exogenous
• Cytochromes P450 - the main detoxifying systems of the organism.