Hemoglobin and MyoglobinBecause of its red color, the red blood pigment has been of interest since antiquity.

•First protein to be crystallized (1849)•First protein to have its mass accurately measured•First protein to be studied by ultracentrifugation•First protein to associated with a physiological

condition•First protein to show that a point mutation can cause

problems•First proteins to have X-ray structures solved•Theories of cooperativity and control explain

hemoglobin function

Hemoglobin Function2,2 dimer: each monomer is structurally similar to myoglobin

•transports oxygen from lungs to tissues

•O2 diffusion alone is too poor for transport in larger animals

•Solubility of O2 is low in plasma i.e. 10-4 M

•But bond to hemoglobin [O2] = 10-2 M or that of air

•Two alternative O2 transporters are

•Hemocyanin: a Cu containing protein

•Hemoerythrin: a non-heme containing protein

Myoglobin facilitates respiration in rapidly respiring muscle tissue.

The rate of O2 diffusion from capillaries to tissue is slow because of the solubility of oxygen

Myoglobin increases the solubility of oxygen

Myoglobin facilitates oxygen diffusion

Oxygen storage: myoglobin concentrations are 10-fold greater in whales and seals than in land mammals

Myoglobin Function

The Heme GroupEach subunit of hemoglobin or myoglobin contains a heme.•Binds one molecule of oxygen•Heterocyclic porphyrin derivative•Specifically, protoporphyrin IX

The iron must be in the Fe(II) form or reduced form (ferrous oxidation) state.

Myoglobin: Oxygen Binding

22 MbOO Mb

][MbO

][Mb][O K

2

2d

][OK

][O

][MbO [Mb]

][MbO Y

2d

2

2

2O2

Written backwards we can get the dissociation constant

Fractional Saturation solve for [MbO2] and plug in

Concentration of oxygen [O2] related to partial pressure of O2 or O2 tension (pO2). Therefore:

2d

2O pO K

pO Y

2

P50 = the partial oxygen pressure when YO2 = 0.50Note similarity to pKa and pH!

250

2O pO P

pO Y

2

What does the value of P50 tell you about the O2 binding affinity?

The shape of the curve of the plot of YO2 vs. pO2 is a rectangular hyperbola.

OR

P50 value for myoglobin is 2.8 torr

1 torr = 1 mm Hg = 0.133 kPa

760 torr = 1 atm of pressure

Mb gives up little O2 over normal physiological range of oxygen concentrations in the tissue

i.e., 100 torr in arterial blood

30 torr in venous blood

YO2 = 0.97 to YO2 = 0.91

What is the P50 value for Hb?

Should it be different than myoglobin?

Hemoglobin, Cooperativity, and The Hill Equation

E = enzyme, S = ligand, n= small number

ESn nS E Enzyme binding of 1 or more ligands

O2 is considered a ligand

ESn][

[E][S] K

n

1. [ESn] [E]n

n[ESn] Ys

2.

Fractional Saturation bound/total

As we did before combine 1. + 2. = 3.

KS][1

]E[

K[E][S]

Ys n

n

or n

n

[S]K

[S]Ys

Look familiar to Mb + O2 except for the n

3.

Continuing as before:

n50p K

n

2n

50

n2

OpOP

pO Y

2 4.

n = Hill Constant: Degree of Cooperativity among interacting ligand-binding sites or subunits

The bigger n the more cooperativity (positive value)

If n = 1, non-cooperative

n < 1, negative cooperativity

Hill PlotRearrange equation 4.

)nlog(p)pO(nlogY-1

Ylog 502

O

O

2

2

y = mx + b

n = slope and x intercept of -b/m

Hb subunits independently compete for O2 for the first oxygen molecule to bind

When the YO2 is close to 1 i.e. 3 subunits are occupied by O2 binding to the last site is independent of the other sites

However by extrapolating slopes: the 4th O2 binds to hemoglobin 100 fold greater than the first O2

Since: P50(1st O2) = 30; P50 (4th O2) = 0.3

When one molecule binds the rest bind and when one is released the rest are released.

Contrast Mb Binding to Hemoglobin

For Hemoglobin

YO2 = 0.95 at 100 torr

but

0.55 at 30 torr

a YO2 of 0.40

Hb gives up O2 easier than Mb and the binding is Cooperative!!

Remember, the YO2= .97 at 100 torr and .91 at 30 torr

Function of the Globin

Protoporphyrin binds oxygen to the sixth ligand of Fe(II) out of the plane of the heme. The fifth ligand is a Histidine, F8 on the side across the heme plane.

His F8 binds to the proximal side and the oxygen binds to the distal side.

The heme alone interacts with oxygen such that the Fe(II) becomes oxidized to Fe(III) and no longer binds oxygen.

Fe O O Fe

A heme dimer is formed which leads to the formation of Fe(III)

By introducing steric hindrance on one side of the heme plane interaction can be prevented and oxygen binding can occur.

The globin acts to:

•a. Modulate oxygen binding affinity

•b. Make reversible oxygen binding possible

The globin surrounds the heme like a hamburger is surrounded by a bun. Only the propionic acid side chains are exposed to the solvent.

Amino acid mutations in the heme pocket can cause autooxidation of hemoglobin to form methemoglobin.

The Bohr Effect (Are You Awake?)

Higher pH i.e. lower [H+] (more basic) promotes tighter binding of oxygen to hemoglobin

and

Lower pH i.e. higher [H+] (more acidic) permits the easier release of oxygen from hemoglobin

xH OHb O HOHb 1n22xn2

Where n = 0, 1, 2, 3 and x 0.6 A shift in the equilibrium will influence the amount of oxygen binding. Bohr protons

As the pH increases the P50 value decreases (i.e. the P in torr of O2 binding to Hb decreases), indicating the oxygen binding increases. The opposite effect occurs when the pH decreases.

At 20 torr 10% more oxygen is released when the pH drops from 7.4 to 7.2!!

As oxygen is consumed CO2 is released. Carbonic Anhydrase catalyzes this reaction in red blood cells.

-322 HCO H OH CO

About 0.8 mol of CO2 is made for each O2 consumed.

Without Carbonic Anhydrase, bubbles of CO2 would form.

The H+ generated from this reaction is taken up by the hemoglobin and causes it to release more oxygen. This proton uptake facilitates the transport of CO2 by stimulating bicarbonate formation.

R-NH2 + CO2 R-NH-COO- + H+

Carbamates are formed from the interaction of CO2 with the N-terminal amino groups of proteins.

Carbamate

About 5% of the CO2 binds to hemoglobin but this accounts for the 50% of the exchanged CO2 from the blood.

This is because only 10% of the total blood CO2 is lost through the lungs in each circulatory cycle.

As oxygen is bound in the lungs the CO2 comes off.

D-2,3-bisphosphoglycerate (BPG)

BPG binds to hemoglobin and decreases the oxygen affinity and keeps it in the deoxy form.

BPG binds 1:1 with a K=1x10-5 M to the deoxy form but weakly to the oxy form

The P50 value of stripped hemoglobin increases from 12 to 22 torr by 4.7 mM BPG

At 100 torr or arterial blood, hemoglobin is 95% saturated

At 30 torr or venous blood, hemoglobin is 55% saturated

Hemoglobin releases 40% of its oxygen. In the absence of BPG little oxygen is released. Between BPG, CO2, H+, and Cl-, all O2 binding is accounted for.

BPG and High-Altitude Adaptation

High Altitude = Less [O2] Binding (arterial),

Not as much change in venous binding?

BPG increases the release of O2 at high elevations between arterial and venous blood

My Thoughts on Simplifying These Observations

HbT + O2 HbR(O2) + 0.6 H+

Bohr Effect: Lower pH = Higher H+, Drives O2 Release

[H+]HbT + O2 HbR(O2) +

BPG Effect: BPG Stabilizes HbT, Drives O2 Release

HbT + O2 HbR(O2) + 0.6 H+

HbT(BPG)