Dr.S.Chakravarty MBBS,MD

HEMOGLOBIN-STRUCTURE AND FUNCTIONS

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Learning objectives:

• At the end of the session a student will be able to :-

– Describe the properties of globular proteins

– Describe the structure and function of haemoglobin and myoglobin

– Analyse the oxygen dissociation curve and factors affecting oxygen dissociation curve

– Differentiate the features of fetal and adult haemoglobin

– List the various hemoglobin variants and hemoglobin derivatives

– Describe the formation of Glycated hemoglobin and its use in monitoring diabetes

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Features of Globular proteins:

• Globular nature - tertiary structure.

• Good water solubility – hydrophobic core and hydrophilic surface.

• catalytic/regulatory/transport role i.e. a dynamic metabolic function

• Exists as colloids in the medium.

USMLE Concept!!—what type of amino acids will be present in the core? Valine, arginine or

glutamic acid ?

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Hemoproteins = Heme containing proteins

• HEME + PROTEINS

Eg.Hemoglobin • (Porphyrin ring + Iron) + Globin

chains • (protoporphyrin IX + Iron)

SOME IMPORTANT HEMOPROTEINSPROTEIN FUNCTIONHEMOGLOBIN TRANSPORT OF OXYGENMYOGLOBIN STORAGE OF OXYGEN IN THE

MUSCLECYTOCHROME C INVOLVEMENT IN E.T.ChainCYTOCHROME P450 METABOLISM OF XENOBIOTICSCATALASE DEGRADATION OF HYDROGEN

PEROXIDETRYPTOPHAN PYRROLASE OXIDATION OF TRYPTOPHAN

Imp. Concept :- Many drugs will induce cyt.p450-Induction of heme synthesis. ( Look into Porphyrias later!!)

Porphyrins

• Porphyrins are cyclic compounds formed by linkage of four pyrrole rings joined together by methenyl

bridges (=HC-)bridges.

• Characteristic property :- They form compelxes with metal ions bound to the nitrogen atom of the pyrrole rings.

– Eg. Hemoglobin with Iron. Chlorophyll with magnesium

.

NATURAL PORPHYRINS HAVE SUBSTITUENT SIDE CHAINS

• THERE ARE CHARACTERISTIC SIDE CHAINS SUBSTITUTED FOR THE 8 HYDROGENS.

Simplified Representation of Heme

Porphyrins

• Porphyrins are cyclic compounds formed by linkage of four pyrrole rings joined together by methenyl

bridges (=HC-)bridges.

• Characteristic property :- They form compelxes with metal ions bound to the nitrogen atom of the pyrrole rings.

– Eg. Hemoglobin with Iron. Chlorophyll with magnesium

.

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Fe2+ bound to 4 Ns, other 2 bonds perpendicular to plane of ring available for bonding

In Hb, one of these attached to N terminus of His, other binds O2.

N

N

N

N

O2

His

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Usmle loves proximal and distal Histidine!!

• Proximal histidine (F8 chain) – binds iron of heme

• Distal histidine (E7 chain) – stabilizes oxygen after binding to iron

NATURAL PORPHYRINS HAVE SUBSTITUENT SIDE CHAINS

• THERE ARE CHARACTERISTIC SIDE CHAINS SUBSTITUTED FOR THE 8 HYDROGENS.

Normal Hemoglobin Structure

Porphyrin ring

M V

V

M

M

M

P

P

Simplified Representation

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Quaternary structure:• TETRAMERIC

SRTUCTURE– chain- 141

AMINO ACIDS– β chain- 146

AMINO ACIDS

• Joined by– H bonds– Salt bridges– Van Der Walls

forces

• Iron must exist in Fe2+ state

A salt bridge is actually a combination of two noncovalent interactions: hydrogen bonding and electrostatic interactions

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2,3BPG

HEME

HEME

HEME

HEME

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Binding sites of various molecules on Hb:

• Oxygen and carbon monoxide – Fe2+

• 2,3 BPG – crevice in T-form of Hb

• Carbon-di-oxide – amino terminal end of globins.

• H+ ions – histidine 146 of beta subunit.

USMLE concept!!

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Oxygenation is accompanied by conformational changes

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Change from T form to R form

The α2β2 (green)rotates 15 degrees relative to α1β1

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Reasons for Co-operativity

• The Iron moves into the plane of the heme on oxygenation

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T structure to R structure

• b

Breaking of salt bridges as more Oxygen is added. The binding of oxygen to one heme residue increases the affinity of remaining Heme residues.

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OXYGEN DISSOCIATION CURVE

• P50 is the partial pressure of O2 that half-saturates a given Hb

USMLE concept!

MyoglobinRETAINS OXYGEN

HemoglobinDELIVERS OXYGENtissues

x x

At point X Myoglobin can attain half saturation at a lower partial pressure of oxygen = more affinity!

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Oxygen dissociation curves

Hb F

Hb A

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Questions on p50

• P50 is the partial pressure of O2 that half-saturates a given Hb

• REMEMBER :- lower values of P50 corresponding to high affinity and vice versa!!

• Low P50High affinity = less shift to right or shift to left !

• High P50 Low affinity = more shifting of ODC to right!

Concept for USMLE!

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Questions on p50

• REMEMBER :- lower values of P50 corresponding to high affinity and vice versa!!

• Low P50High affinity = less shift to right

• High P50 Low affinity = more shifting of ODC to right!

Concept for USMLE!

Answer :-Polycythemia !! Decreased p50 = increased affinity

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Shifts of the Oxygen Dissociation Curve

Protons!! :- oxygen saturation is increasing

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Sources of protons that lower the pH in the tissues:

• Production of lactic acid in the tissues:

• Production of carbonic acid due to increased production of carbon dioxide in the tissues.

• Co2 + H20 ------- > H2CO3 ------ > H+ + HCO3-

(Carbonic anhydrase)

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Bohr effect – transport of CO2

• Carbon dioxide +water carbonic acid dissociates into protons and bicarbonate ions.

• Deoxyhemoglobin acts as a buffer by binding protons and delivering them to the lungs.

• In the lungs, the uptake of oxygen by hemoglobin releases protons that combine with bicarbonate ion, forming carbonic acid, which when dehydrated by carbonic anhydrase becomes carbon dioxide, which then is exhaled.

Increased concentrations of CO2 and H+ promote the release of O2 from hemoglobin in the blood.

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Chloride shift or Hamburger effect• Cl- ions move into RBCS as HCO3- ions to maintain

electrical neutrality.This is CHLORIDE SHIFT!!• Causes RBC to swell in venous blood .

Why oxygenation disrupts salt bridges ?

Presence of “salt bridge” in T form (his 146 with asp 94 )

No ionic interaction inR form ( breaking of salt bridges)

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The Haldane effect what happens in the Lungs !!

• The Haldane effect describes how oxygen concentrations determine hemoglobin's affinity for carbon dioxide. For example, high oxygen concentrations enhance the unloading of carbon dioxide. The converse is also true: low oxygen concentrations promote loading of carbon dioxide onto hemoglobin. In both situations, it is oxygen that causes the change in carbon dioxide levels.

The Bohr effect, on the other hand, describes how carbon dioxide and H+ affect the affinity of hemoglobin for oxygen. High CO2 and H+ concentrations cause decreases in affinity for oxygen, while low concentrations cause high affinity for oxygen

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Role of 2,3 bisphosphoglycerate

Crevice where 2,3 BPG binds Absent crevice

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+

+

-ve

+

+

+

+

-ve

BPG preferentially binds to deoxy- haemoglobin &

stabilizesT-form

-ve

-ve

BPG Binds Hb in a pocket b/w Beta chains.On oxygenation this pocket collapses

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Adult Hb Types

• Hemoglobin A (α2β2) - The most common with a normal amount over 95%

• Hemoglobin A2 (α2δ2) - δ chain synthesis begins late in the third trimester and in adults, it has a normal range of 1.5-3.5%

• Hemoglobin F (α2γ2) - In adults Hemoglobin F is restricted to a limited population of red cells called F-cells. – However, the level of Hb F can be elevated in persons with

Chronic hemolytic anaemias like sickle-cell disease and beta-thalassemia .

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Hb F

α2 γ2

Greater affinity for oxygen

This greater affinity for oxygen is explained by the lack of fetal hemoglobin's interaction with 2,3-bisphosphoglycerate (2,3-BPG or 2,3-DPG).

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Modifications of hemoglobin: Hemoglobin derivatives

• Carbamino Hb• Carboxy Hb• Met Hb• Sulf Hb• Glycated Hb

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Carbamino hemoglobin:

• Carbon-di-oxide binds to Hb to form caramino hemoglobin.

• The N-terminal amino group Val forms carbamino complex with CO2

RNH2+CO2 RNHCOOH

• Transport of carbon dioxide in blood:1. HCO3 – 75%

2. Bound to Hb – 15%3. Dissolved in plasma – 10%

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Carboxy hemoglobin: CARBON MONOXIDE POISONING

• Carbon monoxide affinity(approx 250:1) compared to oxygen .

• PROBLEM NO UNLOADING OF OXYGEN TO TISSUES• Earlier days :- Burning in fireplace with windows closed• Tobacco smoking (through carbon monoxide inhalation)

raises the blood levels• Pollution from Automobiles• Sleeping inside the car with engine on in a Closed garage• Cherry red –appearance of blood.

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Carbon monoxide poisoning:

• Decreases hemoglobin saturation

• Shifts oxygen dissociation curve to the left

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Methemoglobin

• Iron in the heme group is in the Fe2+ (ferrous) state, not the Fe3+ (ferric) of normal hemoglobin.

• Normally 1-2% exists as met-Hb

• Chocolate brown color of blood.

• Hb cannot bind to - oxygen

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Met-hemoglobin reductase

About 20% of the reducing activity is directly due to NADPH also.A glutathione dependent Met-Hb reductase is also described.

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Methemogobinemia

1. Congenital causes:– Cytochrome b5 reductase (methhemoglobin reductase)

deficiency– G6PD deficiency – def of NADPH– Hemoglobin M – Pyruvate kinase deficiency – def of NADH

2. Acquired causes: – Environmental – aromatic amines, nitrates,

chlorobenzene.– Drugs – sulpha drugs, nitroglycerine, chloroquine.

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Clinical features:

• shortness of breath, cyanosis, mental status changes, headache, fatigue, exercise intolerance, dizziness and loss of consciousness.

Lab analysis :- Absorption spectra at 633nm Treatment:IV methylene blue.

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Cyanmet-Hb

• Cyanide poisoning

• Lab Hb estimation by Drabkin’s reagent-cyanmetHb method

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Treatment of cyanide poisoning • Cyanide has affinity for heme proteins mainly cytochrome oxidase inhibition of

cellular respiration .

• Treatment:-The United States standard cyanide antidote kit first uses a small inhaled dose of amyl nitrite, followed by intravenous sodium nitrite, followed by intravenous sodium thiosulfate.

1. Amyl nitrite + sodium nitrite forms met Hb. Cyanide has more affinity for met Hb forming cyanmeth Hb.

2. Sodium thiosulfate reacts with cyanmeth Hb to form sodium thiocyanate (water soluble)

3. Methylene blue to revert Met Hb.

4. Vit B12 in the form of Hydroxycobalamin – forms cyanocobalamin – water soluble

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Glycation and glycosylation:

• Attachment of sugars to protein:

• Glycation – Nonenzymatic, slower process

• Glycosylation – enzymatic and fast process

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Glycated hemoglobin

• Glycation is a haphazard process that impairs the functioning of biomolecules – irreversible.

• Sugars are highly reducing substances.• Binds to Hb of RBCs – stays until the death of RBCs.• Indicates the level of blood glucose. • Used in monitering Diabetes mellites.

Heme

“Distal” histidine

“Proximal” histidine

Structure of Myoglobin

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Structure of Myoglobin

• Single polypeptide chain• α-helical structure• 80% of peptide chains folds into 8 subunits- A to H• Terminated by β-bends or proline.

• Heme sits in the crevice surrounded by non-polar amino acids except proximal and distal histidine which are polar.

• MORE AFFINITY FOR OXYGEN!! – RETAINS OXYGEN FOR A LONGER TIME ( DEEP SEA WHALES )– CAN GIVE MORE OXYGEN DURING STRENOUS EXERCISE

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Competitive binding to heme

Hb F

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Thank you