Chapter 7: Hemoglobin:

Portrait of a Protein in Action

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition

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) -chain has 141 amino acids-chain has 146 amino acids

Hemoglobin O2 carrying capability

• Erythrocytes/ml blood: 5 billion ( 5 x 109 )

• Hemoglobin/red cell: 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 tetramer

Myoglobin, monomeric

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

-Globin (blue)

-Globin (violet)

Myoglobin (green)

Aromatic Heme

Iron in Hemoglobin binding O2

Iron in Myoglobin binding O2

Resonance in Iron binding O2

Hemoglobin, 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

Allosteric Proteins

• There are two limiting models of allosterism:

•Monod, Wyman & Changeux: Two State, concerted

•Koshland, Nemethy & Filmer: One State, sequential

• Allosteric effectors (modulators) bind to a protein at a site separate from the functional binding site (modulators may be activators or inhibitors)

• Oxygen binding and release from Hb are regulated by allosteric interactions

• Hemoglobin cooperativity behaves as a mix of the above two models.

Concerted, two state modelMonod, Wyman & Changeux

R-state vs T-state Binding

Sequential, one state model

Koshland, Nemethy & Filmer

Decreasing O2 affinity

2,3-bisphospho-glycerate (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

Carbamate:

• 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 pHare all allostericeffectors 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 N-terminus 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 HbO2-

carries 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+ pKa = 6.3

H2CO3 <===> CO2 + HOH carbonic anhydrase

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

Binding relationships

The binding of O2 to myoglobin can be shown by the equilibriuim:

Mb + O2 <===> MbO2 (1)

The dissociation constant for the loss of O2 is: [Mb][O2]

Keq = KD = -------------- (2) [MbO2]

Define the fraction of sites, Y, occupied by O2 as: [MbO2] sites bound

Y = --------------------- = ----------------- (3) [Mb] + [MbO2] total sites

Binding relationships

Substituting from equation (2) into (3): [MbO2] 1

Y = ---------------------------- = ------------K [MbO2] K ---------- + [MbO2] ---- + 1 O2 O2

or: [O2] pO2 pO2 Y = ---------- = ----------- = ------------

K + O2 K + pO2 p50 + pO2

Evaluating K at Y = 0.5 gives K = p50 for O2

End of Chapter 7

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition