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Hemoglobin: Portrait of a Protein in Action

Copyright 2007 by W. H. Freeman and Company

Erythrocytes (Red cells)

Hemoglobin and Myoglobin

These are conjugated proteins. A simple protein has only a polypeptide chain. A conjugated protein has a non-protein part in addition to a polypeptide component. Both myoglobin and hemoglobin contain heme. Myoglobin 17000 daltons (monomeric) 153 amino acids Hemoglobin - 64500 daltons ( tetrameric) a-chain has 141 amino acids b-chain has 146 amino acids

Hemoglobin O2 carrying capability

Erythrocytes/ml blood: 5 billion Hemoglobin/red cell: ( 5 x 109 )

280 million ( 2.8 x 108 )

O2 molecules/hemoglobin: 4
O2 ml blood: (5 x 109)(2.8 x 108)(4) = (5.6 x 1018) or (5.6 x 1020) molecules of O2/100 ml blood

A single subunit of Hemoglobin, an a2b2 tetramer

Myoglobin, monomeric

3o structure overlap: myoglobin, a-globin and b-globin

a-Globin (blue)
b-Globin (violet)

Myoglobin (green)

Aromatic Heme

Iron in Hemoglobin binding O2

Iron in Myoglobin binding O2

Resonance in Iron binding O2

Hemoglobin, a2b2 tetramer

O2 binding: Hemoglobin & Myoglobin

P50 = 2 torr P50 = 26 torr

O2 transport capability, a comparison

Resting state vs exercise

O2 Binding Changes 4o Structure

Decreasing O2 affinity
2,3-bisphosphoglycerate (2,3-BPG)
Lowers the affinity of oxygen for Hemoglobin

2,3-bisphosphoglycerate (2,3-BPG)
The binding pocket for BPG contains 4 His and 2 Lys

Binding of bisphosphoglycerate

The Bohr Effect

Bohr Effect:
Lowering the pH decreases the affinity of oxygen for Hb

Loss of O2 from Hemoglobin

CO2 combines with NH2 at the N-terminus of globins

Carbamate formation
Covalent binding at the N-terminus of each subunit

Combined Effects
CO2 , BPG and pH are all allosteric effectors of hemoglobin.

CO2 & Acid from Muscle

CO2 & Hemoglobin Blood Buffering

Metabolic oxidation in cells uses oxygen and produces CO2 . The pO2 drops to ~20 torr and oxygen is released from incoming HbO2-. HbO2- <===> Hb- + O2 Release is facilitated by CO2 reacting with the Nterminus of each hemoglobin subunit, by non-covalent binding of BPG and the Bohr effect.

Events at Cell sites

The localized increase in CO2 results in formation of carbonic acid which ionizes to give bicarbonate and H+. CO2 + HOH <===> H2CO3
carbonic anhydrase

H2CO3 <===> HCO3- + H+

pKa = 6.3

The increase in [H+] promotes protonation of Hb-. HHb <===> Hb- + H+ pKa = 8.2

Events at Cell sites

The predominant species in this equilibrium at pH 7.2 is HHb. So, O2 remains at the cell site, HHb carries a proton back to the lungs and bicarbonate carries CO2 . Charge stability of the erythrocyte is maintained via a chloride shift, Cl- <==> HCO3- .

Events at Lung sites

Breathing air into the lungs increases the partial pressure of O2 to ~100 torr. This results in O2 uptake by HHb to form HHbO2. HHb + O2 <===> HHbO2 Ionization of HHbO2 then occurs and HbO2carries O2 away from the lungs.

HHbO2 <===> HbO2- + H+

pKa = 6.6

So, the predominant species at pH (7.4) is HbO2-.

Events at Lung sites

The localized increase in [H+] from hemoglobin ionization serves to protonate HCO3- .

H2CO3 <===> HCO3- + H+

H2CO3 <===> CO2 + HOH
carbonic anhydrase

pKa = 6.3

The resulting H2CO3 decomposes in presence of carbonic anhydrase and CO2 is released in the lungs. Charge stability of the erythrocyte is maintained again via a chloride shift, HCO3- <==> Cl-.

Sickle Cell due to Glu 6 Val 6