Myoglobin and Hemoglobin Quaternary structure and allosteric properties of proteins Fe

Myoglobin

and

Hemoglobin Quaternary structure and allosteric properties of proteins

Fe

FeFe

FeFe

The importance of quaternary structure in proteins isbest illustrated by the binding of O2 to hemoglobin

Philosophy:

By comparing hemoglobin with myoglobin, (a singlesubunit protein), subunits can be seen to regulatethe strength of binding and give rise to a propertyof proteins called “cooperativity”

Cooperativity is the basis of another property of proteins called “allosterism” or “allostery”

Binding of O2 to Myoglobin

Mb + O2 MbO2

= MbO2

MbO2 + Mb

[MbO2 ]

[Mb][O2]K =

Filled Sites

Total Sites

= the fraction of filled sites = YO2

Define (YO2)

Derive an expression for as a function of O

We are asking: How do the sites filled vary with oxygenpressure?

[MbO2 ]

[Mb][O2]K =

MbO2 = K[Mb][O2]

= MbO2

Mb + MbO2

Mb + O2 MbO2

Sites Filled

Total Sites71% filled

10 out of 14

Substitute the value for MbO2 into the equation

=K[Mb][O2]

K[Mb][O2] [Mb] + MbMb1

= K[O2]

1 + K[O2]KK

K

1K = or Kd = O2 pressure that half fills sites

= P50

When 1/K = [O2], = 0.5 (also called Kd)Therefore:

(P50 distinguishes the specific O2-binding protein)

Redefining

O2 = pO2

Charles Law: Quantity of gas absorbed by a liquid is proportional to the partial pressure of thegas above the liquid.

= pO2

pO2P50 +

Note: Mb is left out of the final equation.

Note: Only connection to Mb is 1/K or P50

P50

100

50

pO2

BINDING PARAMETERSO2

(x 100)

When O2 binds tomyoglobin, the bindingincrease is hyperbolicwith pressure increase

Two points on the binding curve are apparent:(1) the point of half saturation(2) the point of full saturation (never attained)

Full saturation

Half saturation

P50

100

50

pO2

BINDING PARAMETERSO2

(x 100)

Weaker Binding

StrongBindingThe binding is considered weaker when it takes a greater oxygen pressure (larger P50)

to reach half-saturation

P50

= pO2

pO2P50 +

Assume P50 = 10

pO2

1 0.092 0.175 0.33

10 0.5020 0.67

100 0.91

= pO2

pO2P50 +

n

n

n = number of binding sites on the protein

What about Hemoglobin?

For Hb, n = 4

100

50

pO2

Hemoglobin

P50

Region of Cooperativity

Sigmoidal Curve

Why does Hemoglobin show Cooperative Binding?

Oxygen Transport

Inspired airAveolar air

Torrs158100

Arterial Blood 90Capillary 40Interstitial 30Cytosol 10

Binding

Release

Cooperativity is designed to RELEASE O2 at low Pressures

EXAM 1BICH 410 (all sections) 100 pts

Tuesday, Oct 4, 11:10 – 12:00, Rm 108 Biochemistry

81/2 x 11 scantron (Blue)

Calculator

Chapters 1-5: Basics of energy and acid-base, amino acids, peptides, proteins, myoglobin, hemoglobin, allostery

Protein-Ligand Interactions Revisited

P + L = PL

PL = P + L

Ka = [PL]

[P][L]

Kd = [P][L][PL]

Kd = 1/Ka = [L]0.5

orO = L+ L1/Ka

O = L+ LKd

Association

Disassociation

P + nL = PLn Hb + 4O2 = Hb(O2)4

Ka = [PLn]

[P][L]n Ka = [Hb(O2)4]

[Hb] [O2]4

O = Ln

+ LnKd

O = pO24

+1/Ka pO24

Any Ligand Hemoglobin n=4

O = Ln

+ Ln1/Ka

O = pO24

+ Kd pO24

Where Kd = 1/Ka = [L]n0.5

= Pn50

Where Kd = 1/Ka = [O2]40.5

= P450

Multi-Ligand Interactions

ALLOSTERIC BINDING

Rearranging the Equation

n log pO2 - nlog P50=

n = Hill Coefficient

pO2 1 –

=P50

n

n

log

1 –

O = pO2n

+ P50n pO2

n

log

1 - n log pO2 - nlog P50=

y = m x + b

nH = 1.0 No Cooperativity

nH = > 1.0 Positive Cooperativity

nH = < 1.0 Negative Cooperativity

nH = n Full Cooperativity

How is nH Determined

log

1 -

log pO2

Myoglobin

nH = 10

-3

+3

0 1.0-1.0

CooperativeTransition

nH = 3

HemoglobinLow Affinity

nH = 1

HemoglobinHigh Affinity nH = 1

Hemoglobin

R-State

T-State

How is nH Determined

log

1 -

log pO2

Myoglobin

nH = 10

-3

+3

0 1.0-1.0

nH = 3

HemoglobinLow AffinitynH = 1

HemoglobinHigh Affinity

The higher the O2 pressure, the stronger the binding, (binding mode)The lower the O2 pressure, the weaker the binding, (release mode)