Bark3304 lecture 11

Hemoglobin and Myoglobin
Because 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 Function
α2,β2 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 Function
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 10fold greater in whales and seals than in land
mammals

The Heme Group
Each 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

Mb + O 2 ↔

Written backwards we can
MbO 2 get the dissociation constant

[Mb][O 2 ]
Kd =
[MbO 2 ]
Fractional Saturation solve for [MbO2] and plug in

[MbO 2 ]
[O 2 ]
YO2 =
=
[Mb] + [MbO 2 ] K d + [O 2 ]

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

pO 2
YO 2 =
K d + pO 2

OR

pO 2
YO 2 =
P50 + pO 2

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

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

E + nS ↔ ESn

Enzyme binding of 1 or
more ligands

O2 is considered a ligand
n

[E][S]
1. K =
[ESn]

Fractional Saturation

n[ESn]
2. Ys =
n ( [E] + [ESn])
bound/total

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

[E][S]
K
Ys =
n
1 + [S]
[E]
K

(

)

3.
or

n

[S]
Ys =
n
K + [S]

Look familiar to Mb + O2 except for the n

Continuing as before:

K = ( p 50 )

( pO 2 )
=
n
n
( P50 ) + ( pO 2 )
n

n

4. YO 2

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 Plot
Rearrange equation 4.

 YO 2
log
 1 - YO
2

y


 = nlog(pO 2 ) − nlog(p50 )


=

mx

+

n = slope and x intercept of -b/m

b

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

Hb( O 2 ) n H x + O 2 ⇔ Hb( O 2 ) n +1 + xH

+

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!!
+

CO 2 + H 2 O ⇔ H + HCO 3

-

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

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+
Carbamate

Carbamates are formed from the interaction of CO2 with the Nterminal amino groups of proteins.

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

HbT + O2

HbR(O2) + [H+]

HbT + O2

HbR(O2) + 0.6 H+

HbT + O2
HbT(BPG)

HbR(O2) + 0.6 H+

BPG Effect: BPG Stabilizes HbT, Drives O2 Release

Bark3304 lecture 11

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Bark3304 lecture 11