Protein Classification• Simple – composed only of amino acid

residues

• Conjugated – contain prosthetic groups(metal ions, co-factors, lipids, carbohydrates)Example: Hemoglobin – Heme

Protein Classification• One polypeptide chain - monomeric protein• More than one - multimeric protein• Homomultimer - one kind of chain• Heteromultimer - two or more different

chains

(e.g. Hemoglobin is a heterotetramer. It has two alpha chains and two beta chains.)

Protein ClassificationFibrous –1) polypeptides arranged in long strands or

sheets2) water insoluble (lots of hydrophobic AA’s)3) strong but flexible4) Structural (keratin, collagen)

Globular –1) polypeptide chains folded into spherical or

globular form2) water soluble3) contain several types of secondary structure4) diverse functions (enzymes, regulatory

proteins)

Protein Function

• Catalysis – enzymes• Structural – keratin• Transport – hemoglobin• Trans-membrane transport – Na+/K+ ATPases• Toxins – rattle snake venom, ricin• Contractile function – actin, myosin• Hormones – insulin• Storage Proteins – seeds and eggs • Defensive proteins – antibodies

Globular Proteins Myoglobin/Hemoglobin

Hemeproteins: group of specialized proteins that contain

heme group as a tightly bound prosthetic group.

prosthetic group: is a non-protein compound that is

permanently associated with protein

The role of heme group is dependent on the environment

created by the three-dimensional structure of the protein.

e.g. heme in cytochrome electron carrier,

enzyme catalase active site

Myoglobin and hemoglobin Oxygen carrier

First protein structures determined

Oxygen carriers

Hemoglobin transport O2 from lungs to tissues

Myoglobin O2 storage protein

Myoglobin/Hemoglobin

Structure and function of Hemoglobin Mb and Hb subunits structurally

similar

myoglobin hemoglobin

•8 alpha-helices•Contain heme group•Mb monomeric protein•Hb heterotetramer (α22)

- Hb found exclusively in red blood cells-transport O2 from lungs to capillaries of tissues and transfer CO2 from tissues to lungs -composed of 4 polypeptides held together by non-covalent interaction-each subunit is similar to myoglobin and contains a heme group

The interior of the myoglobin is composed of NON-polar a.a. they packed together stabilized by hydrophobic interaction.Charged a.a are located at the surface.

-Myoglobin is O2 binding protein found in almost all mammals mainly in muscles and heart -its main function is to store O2 for periods where energy demands is high, it also increases the rate of transport of oxygen within the muscle cells.- Compact structure, 80% of its polypeptide chain is α-helix that labeled A to h that terminated by Proline or by -bends

porphyrine

protoporphyrine IX

Structure of HemeHeme is a complex of protoporphyrine IX (4 pyrrole rings linked by methene bridges) and ferrous iron (Fe+2) Ferrous ion has 6 coordination bonds: 4 with the N of pyrrole rings and 2 are perpendicular one with N of histidine and the other is with O2

Heme = Fe++ bound to tertapyrrole ring (protoporphyrin IX complex)

Heme non-covalently bound to globin proteins through His residue

O2 binds non-covalently to heme Fe++, stabilized through H-bonding with another His residue

Heme group in hydrophobic crevice of globin protein

Heme group

Heme = Fe++ bound to tertapyrrole ring (protoporphyrin IX complex)

Heme non-covalently bound to globin proteins through His residue

O2 binds non-covalently to heme Fe++, stabilized through H-bonding with another His residue

Heme group in hydrophobic crevice of globin protein

Distal histidine: stabilizes the binding of O2 to heme

Proximal histidine

Distal histidine: stabilizes the binding ofO2 to heme

Proximal histidine

Heme

Ferrous ion

Oxygen

Oxygen binding Oxygen binding to Heme groupto Heme group

Hb tetramer can be described as two identical dimers, (α)1 and (α)2The interaction between α and subunits is strong (hydrophobic, ionic and hydrogen interactions )The interaction (α)1 and (α)2 is weak interaction (primary hydrophobic). The two dimers can move with respect to each other two conformations according to the presence or absence of O2

T-form (Taut or tense): the deoxy form of Hb. The two α dimers interact through a network of ionic bonds and hydrogen bonds that constrain the movement of the dimer. This form is the low O2 affinity form of HbR-form (relaxed form): the binding of O2 causes rupture of some ionic bonds and H-bonds the polypeptide chains have more freedom of movement. The R-form is the high O2 affinity form of Hb.

Oxygen binding to MyoglobinOxygen binding to Myoglobin

Deg

ree o

f satu

rati

on

Concentration of Oxygen (Partial pressure)

Myglobin has one heme group bind only one oxygen molecule.Hemoglobin has 4 heme groups bind to 4 oxygen molecules O2 dissociation curve has hyperbolic shape

Oxygen dissociation curve

Myglobin has higher O2

affinity than of

Hemoglobin

P50 of Mb is about 1

mmHg and for Hb is 26

P50 is O2 Partial

pressure needed to half

saturation of the Mb of

Hb

Oxygen Oxygen transpotransport rt proteinproteinssEfficient O2

transport protein should bind to O2 at high partial pressure (loading in lung) and release it (low affinity) at low Partial pressure of (unloading in the tissue)

•Mb has hyperbolic O2 binding curve

•Mb binds O2 tightly. Releases at very low

pO2

•Hb has sigmoidal O2 binding curve

•Hb high affinity for O2 at high pO2 (lungs)

•Hb low affinity for O2

at low pO2 (tissues)

Oxygen Binding CurvesOxygen Binding Curves

tissues

lungs

Strong-binding

Weak-binding

Transition from weak to strong binding

OO22 Binding to Hb shows positive Binding to Hb shows positive cooperativitycooperativity

O2 Binding to Hb shows sigmoidal shape, low binding affinity at low con of Oxygen and high affinity at higher concooperative binding by the four subunit of Hb The binding of one O2 molecule at one heme group increases the oxygen affinity of the remaining heme groups in the same hemoglobin molecule. The affinity of hemoglobin for the last O2 bound is 300 times greater than its affinity for the first O2

O2 affinity increases as each O2 molecule bindsIncreased affinity due to conformation changeDeoxygenated form = T (tense) form = low affinityOxygenated form = R (relaxed) form = high affinity

Increasing affinity

for O2

Hb

Hemoglobin is efficient in delivering the O2 to the tissues from lung, myglobin which has hyperbolic O2-dissociation curve is unable to do that

Cooperative O2 Binding to HbCooperative O2 Binding to Hb

Myoglobin-Oxygen bindingMyoglobin-Oxygen binding

Allosteric InteractionsAllosteric Interactions

• Allosteric interaction occur when specific molecules bind a protein and modulates activity

• Allosteric modulators or allosteric effectors bind reversibly to site separate from functional binding or active site

• Modulation of activity occurs through change in protein conformation

• 2,3 bisphosphoglycerate (BPG), CO2 and

protons are allosteric effectors of Hb binding of

O2

CO2 + H2O H2CO3 HCO3 + H+

Most of the CO2 produced in metabolism is hydrated and transported

as bicarbonate ion. the hydration of CO2 by the zinc-dependent

enzyme carbonic anhydrase.

How is COHow is CO22 Exported? Exported?

Binding of Hemoglobin to CO2Binding of Hemoglobin to CO2

Some CO2 is carried as carbamate bound to the uncharged α-amino group

Carbon dioxide is transported in the form of a carbamate on the amino terminal residues of each of the polypeptide subunits.

Direct binding of CO2 to Hb stabilizes the T- form (deoxy) of Hb resulting in a decrease in its affinity for oxygen

The formation of a carbamate also results in release of a proton into solution indirectly induces the Bohr effect

Amino Terminus Carbamate on Amino Terminus

O

C

O

C C

R

H

O

ProteinH2N C C

R

H

O

ProteinHNC

O

O

H+

Bohr EffectBohr Effect Increased CO2 leads to

decreased pH CO2 + H2O <-> HCO3- + H+

At decreased pH several key a.a’s protonated, causes Hb to be converted to T-conformation (low affinity)HbO2 + H HbH + O2.

Deoxy form of Hb has higher affinity of H than O2

Protonation of some amino acids stabilizes the deoxyhemeglobin (T-form)

HCO3- combines with N-terminal α-amino group to form carbamate group.

Carbamation stabilizes T-conformation

Bisphosphoglycerate (BPG)Bisphosphoglycerate (BPG)

2,3-Bisphosphoglycerate is an important allosteric effecter of hemoglobin Binding of BPG to Hb causes low O2 affinity

One molecule binds at the interface of all four subunits, and makes contacts with the β

-subunits. BPG binds in the cavity between β-Hb subunits

Its binding stabilizes the deoxyhemoglobin state so stabilizes T-conformation. This promotes oxygen dissociation from oxyhemoglobin.

2,3-BPG concentration increases in response to chronic

hypoxia as in pulmonary obstruction or to high altitude or

chronic anemia. 2,3-BPG is present in erythrocytes at about 5 mM (at sea

level), At high altitudes it is present at 8 mM. Shifts the

curve to the right increase the O2 delivery to the tissue. At high altitudes, wherein the partial pressure of oxygen is

low, one would want hemoglobin to give up more of its

bound oxygen to the tissues Fetal Hb (α22) has low affinity for BPG, allows fetus to

compete for O2 with mother’s Hb (α2 β2) in placenta Role of 2,3-BPG in transfused blood

2,3-Bisphosphoglycerate2,3-Bisphosphoglycerate

Formation of methemoglobin:Formation of methemoglobin: • oxidation of the heme component of Mb and Hb into ferric (Fe+3)

state form metmyglobin and methmoglobin• The oxidized heme can't bind the O2• This oxidation can result from drugs or toxins or from inherited

defects • Occasional oxidation of heme is corrected by the enzyme NADH-

cytochrome b5 reductase that found in the red blood cell.• Methemoglobin binds strongly to CN (poison that inhibits the

cytochromal electron transport), so in the case of the cyanide poisoning amyle nitrite is taken which able to oxidize the heme group sequestering the CN

Carbon monoxide binding to HbCarbon monoxide binding to Hb• CO binds tightly to one or more of heme iron forming carbon

monoxyhemoglobin (HbCO) and hemoglobin is shifted to R-form causing the remaining heme with high O2 affinity shifts the O2-binding curve to the hyperbolic (left) inability of affected hemoglobin to deliver O2 to the tissue

Types of HemoglobinsTypes of HemoglobinsThere are 4 different types of hemoglobins known:

The most common is Hb A that form 90% of total Hb and consists of α2 β2Hb F (α22) less than 2%

Hb A2 (α22) 2-5%Hb A1C (α2 β2-glucose) 3-9 %

Fetal hemoglobin (Hb F): tetramer α22 • Hb F is major Hb in the fetus and newborn. During the last month of

pregnancy , it accounts for 60 % of the total Hb. • In the first few weeks of pregnancy embryonic Hb is synthesized Hb

Gower1 (ζ2ε2) after that the liver starts HbF synthesis. After the

development of the bone marrow the Hb A is synthesized at about the eighth month of the pregnancy and gradually replaces the Hb F

Binding of the 2,3-BPG to HbF (Binding of the 2,3-BPG to HbF (αα222)2) • HbF has higher affinity for O2 than dose HbA, bec it has lower binding

affinity to 2,3-BPG and this facilitates the transfer of O2 from maternal circulation across the placenta to the red blood cells of the fetus

2 globin chains (HbF) lack some positively charged amino acids found that found in the β globin (Hb A) reduce the 2,3BPG binding higher

affinity to O2

Hemoglobin A2 (Hb A2 (Hemoglobin A2 (Hb A2 (αα222))2))

• Hb A2 is a minor component of normal

adult hemoglobin, appear firstly about

12 week after the birth and can form

about 2% of the total Hb

Hemoglobin A1cHemoglobin A1c

• Under physiologic conditions HbA is

slowly and non-enzymatically

glycosylated

• The extent of glycosylation is

dependent on the plasma level of

particular hexoses

• The most abundant glycosylated Hb is

HbA1c which has glucose unit that

covalently linked to amino group of N-

terminal valines of the beta chain

• In the case of Diabetes mellitus, the

amount of HbA1c will increase

HemoglobinopathiesHemoglobinopathiesDefined as a family of disorders caused either by production of

structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin or rarely both

Sickle- cell anemia (HbS) Hemoglobin C disease (HbC)

Thalassemia

Sickle- cell anemia (Hemoglobin S disease “HbS”)Sickle- cell anemia (Hemoglobin S disease “HbS”)• a glutamate residue is replaced by valine residue in the β-chains.

This results in two fewer negative charges for the tetrameric structure.

• The substitution of a hydrophobic amino acid for a hydrophilic one makes the resulting molecule “sticky.” This is because a

hydrophobic patch has been created, which causes molecules to stick together at this point. This causes aggregation to occur in

deoxyhemoglobin. • Subsequent to strand formation, several strands can assemble to

form an insoluble fiber, which is what gives sickled cells there shape.

• People with sickle cell anemia suffer from repeated crises brought on by physical exertion.

HemoglobinopathiesHemoglobinopathies

Sickle- cell anemia (Hemoglobin S disease “HbS”)Sickle- cell anemia (Hemoglobin S disease “HbS”)

Hemoglobin C disease HbC is a hemoglobin variant having a single substitution in the sixth

position of the β-globin chain. In this case lysine is substituted. Patients have a relatively mild chronic hemolytic anemia and they

don't suffer from infractive crises Hemoglobin SC disease

in this disease some β-globin chains have sickle-cell mutation and other β-globin chains carry mutation found in HbC

Thalassemias Thalassemia is a hereditary hemolytic disease in which an imbalance

in the synthesis of globin chains occurs Normally the synthesis of α-chains and β-chains are coordinated so

that each α-globin has its β-globin in the thalassemia the synthesis of either α- or β-globin chain is

defectiveα-thalassemia: defect in the synthesis of the α-globin and there are 4

different levels of this type β-thalassemia: β-globin is decreased or absent, there are 2 different

level of this type

The EndThe End

2,3-bisphosphoglycerate binding to hemoglobin stabilizes the T state.

The negative charges on 2,3-bisphosphoglycerate interact with positive charges on hemoglobin (shown in blue)

Shown here is the R state of hemoglobin, to which oxygen has a greater affinity. Notice how the binding site for BPG collapses.

2,3-bisphosphoglycerate Binding to Hemoglobin

Sickle Cell Anemia• Sickle cell anemia was the first condition for which a genetic

mutation was correlated with a physiological response. This is a homozygous recessive condition, in which offspring must inherit both of the mutated genes in order to develop the disease fully.

• There are more than 300 different genetic variants of hemoglobin that are known.

• In the case of sickle cell disease, a valine residue is substituted for a glutamate residue in the b chains. This results in two fewer negative charges for the tetrameric structure.

• The substitution of a hydrophobic amino acid for a hydrophilic one makes the resulting molecule “sticky.” This is because a hydrophobic patch has been created, which causes molecules to stick together at this point. This causes aggregation to occur in deoxyhemoglobin.

• Subsequent to strand formation, several strands can assemble to form an insoluble fiber, which is what gives sickled cells there shape.

• People with sickle cell anemia suffer from repeated crises brought on by physical exertion. The hemoglobin content of their blood is about 1/2 of normal erythrocytes, and the sickled cells can block capillaries, causing severe pain.

Mutations in a- or b-globin genes can cause disease state

• Sickle cell anemia – E6 to V6

• Causes V6 to bind to hydrophobic pocket in deoxy-Hb

• Polymerizes to form long filaments

• Cause sickling of cells

• Sickle cell trait offers advantage against malaria

• Fragile sickle cells can not support parasite

Structure and function of HemoglobinStructure and function of Hemoglobin-found exclusively in red blood cells-transport O2 from lungs to capillaries of tissues and transfer CO2 from tissues to lungs -composed of 4 polypeptides held together by non-covalent interaction-each subunit is similar to myoglobin and contains a heme group

Oxygen Binding CurveOxygen Binding Curve

Oxygen Binding CurveOxygen Binding Curve