Folie 1

1
Michael Berger (Center for Brain Research,
Medical University Vienna, Austria) Ligand/Recep
tor Interaction
L
http//cwx.prenhall.com/horton/medialib/media_port
folio/09.html
2

Wenn Du mit anderen ein Schiff bauen
willst, Antoine de Saint Exupery
3

Wenn Du mit anderen ein Schiff bauen
willst, beginne nicht, mit Ihnen Holz zu
sammeln, Antoine de Saint Exupery
4

Wenn Du mit anderen ein Schiff bauen
willst, beginne nicht, mit Ihnen Holz zu
sammeln, sondern wecke in Ihnen die
Sehnsucht nach dem großen weiten Meer. Antoine
de Saint Exupery
5
What is a receptor?
6
What is a receptor?
A physical target mediating the physiological
effect of a drug.
7
What is a receptor?
A physical target mediating the physiological
effect of a drug.
What is a ligand?
8
What is a receptor?
A physical target mediating the physiological
effect of a drug.
What is a ligand?
A substance that (strongly) binds to a tissue.
9
What is a receptor?
A physical target mediating the physiological
effect of a drug.
What is a ligand?
A substance that (strongly) binds to a tissue.
What is an agonist?
10
What is a receptor?
A physical target mediating the physiological
effect of a drug.
What is a ligand?
A substance that (strongly) binds to a tissue.
What is an agonist?
A substance that causes an effect, an active
change in the target tissue.
11
What is a receptor?
A physical target mediating the physiological
effect of a drug.
What is a ligand?
A substance that (strongly) binds to a tissue.
What is an agonist?
A substance that causes an effect, an active
change in the target tissue.
What is an antagonist?
12
What is a receptor?
A physical target mediating the physiological
effect of a drug.
What is a ligand?
A substance that (strongly) binds to a tissue.
What is an agonist?
A substance that causes an effect, an active
change in the target tissue.
What is an antagonist?
A substance that blocks the effect of an agonist
13
What is a receptor?
A physical target mediating the physiological
effect of a drug.
What is a ligand?
A substance that (strongly) binds to a tissue.
What is an agonist?
A substance that causes an effect, an active
change in the target tissue.
What is an antagonist?
A substance that blocks the effect of an agonist
What is a transmitter?
14
What is a receptor?
A physical target mediating the physiological
effect of a drug.
What is a ligand?
A substance that (strongly) binds to a tissue.
What is an agonist?
A substance that causes an effect, an active
change in the target tissue.
What is an antagonist?
A substance that blocks the effect of an agonist
What is a transmitter?
A natural agonist released by a cell and acting
on a neighboring cell.
15
Association
BL
B L BL
KA
B . L
16
Association
BL
B L BL
KA
B . L
KA association equilibrium constant
17
Association
BL
B L BL
KA
B . L
KA association equilibrium constant
Dissociation
B . L
BL B L
KD
BL
KD dissociation equilibrium constant
18
Association
BL
B L BL
KA
B . L
KA association equilibrium constant dimension
(concentration)-1
Dissociation
B . L
BL B L
KD
BL
KD dissociation equilibrium constant
19
Association
BL
B L BL
KA
B . L
KA association equilibrium constant dimension
(concentration)-1
Dissociation
B . L
BL B L
KD
BL
KD dissociation equilibrium constant dimension
concentration
20
Association
Dissociation
B L BL
BL B L
21
Association
Dissociation
B L BL
BL B L
Strong binding equilibrium is on right
side on left side
22
Association
Dissociation
B L BL
BL B L
Strong binding equilibrium is on right
side on left side
BL
B . L
KA
ltlt 1
KD
gtgt 1
BL
B . L
23
Association
Dissociation
B L BL
BL B L
Strong binding equilibrium is on right
side on left side
BL
B . L
KA
ltlt 1
KD
gtgt 1
BL
B . L
ln KD negativ
ln KA positiv
24
Association
Dissociation
B L BL
BL B L
Strong binding equilibrium is on right
side on left side
BL
B . L
KA
ltlt 1
KD
gtgt 1
BL
B . L
ln KD negativ
ln KA positiv
Van't Hoff ?Go - RT . ln KA RT . ln KD
?Go change in free enthalpy (Gibbs
energy) R universal gas constant, 1.987
cal/(Mol . K) or 8.314 J/(Mol . K) T absolute
temperature
25
The Vant Hoff equation allows the calculation of
the free enthalpy change of a reaction from the
reactions equilibrium constant
?Go (20 C) ?Go (20 C)
KA KD kcal/Mol kJ/Mol
107 M-1 10-7 M -9.4 -39.2
108 M-1 10-8 M -10.7 -44.8
109 M-1 10-9 M -12.0 -50.3
Van't Hoff ?Go - RT . ln KA RT . ln KD
?Go change in free enthalpy (Gibbs
energy) R universal gas constant, 1.987
cal/(Mol . K) or 8.314 J/(Mol . K) T absolute
temperature
26
Examples for the change in free enthalpy Go in
various reactions
?Go (kcal/Mol)
Glucose 6 O2 6 CO2 6 H2O -686 H2 ½
O2 H2O -46 ATP ADP Pi
-7.3
27
Examples for the change in free enthalpy Go in
various reactions
?Go (kcal/Mol)
Glucose 6 O2 6 CO2 6 H2O -686 H2 ½
O2 H2O -46 ATP ADP Pi
-7.3
In these reactions, Go is reduced (exergonic
processes)
28
Examples for the change in free enthalpy Go in
various reactions
?Go (kcal/Mol)
Glucose 6 O2 6 CO2 6 H2O -686 H2 ½
O2 H2O -46 ATP ADP Pi
-7.3
In these reactions, Go is reduced (exergonic
processes)
Bond dissociation energies
HO-H HO H 118 CH3CH2-H CH3CH2
H 101 CH3-CH3 CH3 CH3 90
29
The free enthalpy change ?Go of a reaction is
composed of 2 terms
Gibbs Helmholtz ?Go ?Ho T . ?So
30
The free enthalpy change ?Go of a reaction is
composed of 2 terms
Gibbs Helmholtz ?Go ?Ho T . ?So
change in enthalpy
31
The free enthalpy change ?Go of a reaction is
composed of 2 terms
Gibbs Helmholtz ?Go ?Ho T . ?So
change in enthalpy
change in entropy, multiplied by absolute
temperature
32
! attention e ? ? mathematics ? 8
v attention !
The free enthalpy change ?Go of a reaction is
composed of 2 terms
Gibbs Helmholtz ?Go ?Ho T . ?So
change in enthalpy
change in entropy, multiplied by absolute
temperature
ln KD ?Go / RT ?Ho / RT - ?So / R

• KD measured at various temperatures
• ln KD plotted against 1/T

! attention e ? ? mathematics ? 8
v attention !
33
! attention e ? ? mathematics ? 8
v attention !
?Ho lt 0 exotherm (reaction mixture warms)
lg KD
0 -
55 C 0 C
?So gt 0 (order is decreased)
-3 -
-6 -
?
?
?
?
-9 -




1/T
0.001 0.002 0.003 0.004
Most common case The warmer (the lower 1/T), the
weaker the affinity (the less negative lg KD).
ln KD ?Go / RT ?Ho / RT - ?So / R

• KD measured at various temperatures
• ln KD plotted against 1/T

lg KD 0.434 . ?Ho/R . 1/T 0.434 . ?So/R
0.434 1/ln10
! attention e ? ? mathematics ? 8
v attention !
34
! attention e ? ? mathematics ? 8
v attention !
?Ho lt 0 exotherm (reaction mixture warms)
lg KD
0 -
55 C 0 C
?So gt 0 (order is decreased)
-3 -
-6 -
?
?
?
?
-9 -




1/T
0.001 0.002 0.003 0.004
Most common case The warmer (the lower 1/T), the
weaker the affinity (the less negative lg KD).
Intersection with ordinate gives information
about ?So.
ln KD ?Go / RT ?Ho / RT - ?So / R

• KD measured at various temperatures
• ln KD plotted against 1/T

lg KD 0.434 . ?Ho/R . 1/T 0.434 . ?So/R
0.434 1/ln10
! attention e ? ? mathematics ? 8
v attention !
35
! attention e ? ? mathematics ? 8
v attention !
?Ho lt 0 exotherm (reaction mixture warms)
lg KD
0 -
55 C 0 C
?So gt 0 (order is decreased)
-3 -
-6 -
?
?
?
?
-9 -




1/T
0.001 0.002 0.003 0.004
Most common case The warmer (the lower 1/T), the
weaker the affinity (the less negative lg KD).
Intersection with ordinate gives information
about ?So.
ln KD ?Go / RT ?Ho / RT - ?So / R
Slope allows access to ?Ho.

• KD measured at various temperatures
• ln KD plotted against 1/T

lg KD 0.434 . ?Ho/R . 1/T 0.434 . ?So/R
0.434 1/ln10
! attention e ? ? mathematics ? 8
v attention !
36
! attention e ? ? mathematics ? 8
v attention !
?Ho lt 0 exotherm (reaction mixture warms)
lg KD
0 -
55 C 0 C
?So gt 0 (order is decreased) ?So lt 0 (order
is increased)
-3 -
-6 -
?
?
?
?
-9 -




1/T
0.001 0.002 0.003 0.004
lg KD
0 -
If order is increased, driving force is even more
sensitive to high temperatures
-3 -
-6 -
?
?
?
?
-9 -




1/T
0.001 0.002 0.003 0.004
ln KD ?Go / RT ?Ho / RT - ?So / R

• KD measured at various temperatures
• ln KD plotted against 1/T

lg KD 0.434 . ?Ho/R . 1/T 0.434 . ?So/R
0.434 1/ln10
! attention e ? ? mathematics ? 8
v attention !
37
! attention e ? ? mathematics ? 8
v attention !
?Ho lt 0 exotherm (reaction mixture warms)
lg KD
0 -
55 C 0 C
?So gt 0 (order is decreased) ?So lt 0 (order
is increased)
-3 -
-6 -
?
?
?
?
-9 -




1/T
0.001 0.002 0.003 0.004
lg KD
0 -
If order is increased, driving force is even more
sensitive to high temperatures
-3 -
-6 -
?
?
?
?
-9 -




1/T
0.001 0.002 0.003 0.004
It may be difficult to obtain solid data that
allow to decide, if ?So is gt or lt 0.
ln KD ?Go / RT ?Ho / RT - ?So / R

• KD measured at various temperatures
• ln KD plotted against 1/T

lg KD 0.434 . ?Ho/R . 1/T 0.434 . ?So/R
0.434 1/ln10
! attention e ? ? mathematics ? 8
v attention !
38
! attention e ? ? mathematics ? 8
v attention !
?Ho gt 0 endotherm (reaction mixture cools)
?Ho lt 0 exotherm (reaction mixture warms)
lg KD
lg KD
0 -
0 -
55 C 0 C
?So gt 0 (order is decreased) ?So lt 0 (order
is increased)
-3 -
-3 -
-6 -
-6 -
?
?
?
?
?
?
?
?
-9 -
-9 -








1/T
1/T
0.001 0.002 0.003 0.004
0.001 0.002 0.003 0.004
lg KD
0 -
Endotherm binding is driven by decrease in order
only here, driving force increases with
temperature.
-3 -
-6 -
?
?
?
?
-9 -




1/T
0.001 0.002 0.003 0.004
ln KD ?Go / RT ?Ho / RT - ?So / R

• KD measured at various temperatures
• ln KD plotted against 1/T

lg KD 0.434 . ?Ho/R . 1/T 0.434 . ?So/R
0.434 1/ln10
! attention e ? ? mathematics ? 8
v attention !
39
Mechanisms contributing to ligand/receptor
interaction

1. Ionic interaction
2. Hydrogen bonds
3. Hydrophobic interaction
4. Cation/p interaction
5. Van der Waals interaction

40
ionic interaction
e1 . e2
attraction between 2 charges depends on
D . r2
r ... distance D ... dielectric constant
41
ionic interaction
e1 . e2
attraction between 2 charges depends on
D . r2
r ... distance D ... dielectric constant
vacuum ... 1.0 hexane ... 1.9 H2O ... 78
42
ionic interaction
e1 . e2
attraction between 2 charges depends on
D . r2
r ... distance D ... dielectric constant
vacuum ... 1.0 hexane ... 1.9 H2O ... 78
In water, ionic interaction is hindered by shells
of water molecules surrounding each ion.
43
hydrogen bonds
B L B?L
Formation of a hydrogen bond is highly exergonic,
yields 3-7 kcal/mol
44
hydrogen bonds
B L B?L
Formation of a hydrogen bond is highly exergonic,
yields 3-7 kcal/mol
However, enthalpy balance is poor
B?H2O L?H2O B?L H2O?H2O
45
hydrogen bonds
B L B?L
Formation of a hydrogen bond is highly exergonic,
yields 3-7 kcal/mol
However, enthalpy balance is poor
B?H2O L?H2O B?L H2O?H2O
1. Break this bond.
46
hydrogen bonds
B L B?L
Formation of a hydrogen bond is highly exergonic,
yields 3-7 kcal/mol
However, enthalpy balance is poor
B?H2O L?H2O B?L H2O?H2O
2. Break this bond.
1. Break this bond.
47
hydrogen bonds
B L B?L
Formation of a hydrogen bond is highly exergonic,
yields 3-7 kcal/mol
However, enthalpy balance is poor
B?H2O L?H2O B?L H2O?H2O
2. Break this bond.
1. Break this bond.
3. Form this bond.
48
hydrogen bonds
B L B?L
Formation of a hydrogen bond is highly exergonic,
yields 3-7 kcal/mol
However, enthalpy balance is poor
B?H2O L?H2O B?L H2O?H2O
4. Form this bond.
2. Break this bond.
1. Break this bond.
3. Form this bond.
49
hydrogen bonds
B L B?L
Formation of a hydrogen bond is highly exergonic,
yields 3-7 kcal/mol
However, enthalpy balance is poor
B?H2O L?H2O B?L H2O?H2O
4. Form this bond.
2. Break this bond.
1. Break this bond.
3. Form this bond.
Hydrogen bond formation mainly driven by increase
in entropy, since the water molecules get more
freedom (2 kcal per mol of water).
50
hydrophobic interaction
Molecules or parts of molecules (residues)
without charge, that cannot form a hydrogen bond,
are called hydrophobic. They aggregate together
to reduce the contact with water to a minimum.
51
hydrophobic interaction
Molecules or parts of molecules (residues)
without charge, that cannot form a hydrogen bond,
are called hydrophobic. They aggregate together
to reduce the contact with water to a minimum.
52
hydrophobic interaction
Molecules or parts of molecules (residues)
without charge, that cannot form a hydrogen bond,
are called hydrophobic. They aggregate together
to reduce the contact with water to a minimum.
53
hydrophobic interaction

• This example is nice, but wrong.
• Hydrogene bonds are never left open.
• In contact with an inert partner, water molecules
  are highly ordered.
• Reduction of contact area leads to reduced order.

54
hydrophobic interaction

• This example is nice, but wrong.
• Hydrogene bonds are never left open.
• In contact with an inert partner, water molecules
  are highly ordered.
• Reduction of contact area leads to reduced order.
• Reduction of Go by hydrophobic interaction is
  always due to the entropy term T . ?S

Gibbs Helmholtz ?Go ?Ho T . ?So
55
hydrophobic interaction

• This example is nice, but wrong.
• Hydrogene bonds are never left open.
• In contact with an inert partner, water molecules
  are highly ordered.
• Reduction of contact area leads to reduced order.
• Reduction of Go by hydrophobic interaction is
  always due to the entropy term T . ?S
• Empirical rule ? Go -0.03 x area hidden from
  water (in ?2).

56
cation/p interaction
A molluscan acetylcholine (AcCh) binding protein,
with high sequence homology to the AcCh binding
site of the nicotinic receptor, has been
crystallized. The binding pocket is surrounded by
tyr and trp residues (Bejc et al. 2001, Nature
411 269)
57
Van der Waals interaction
Two atoms touching each other with their
electron shells redistribute their charges,
resulting in attraction.
http//www.columbia.edu/cu/biology/courses/c2005/l
ectures/lec02_06.html
58
Van der Waals interaction
range 3-4 ?, turns into repulsion at shorter
distances contribution to ?Go 0.5-1.0 kcal/Mol
(lower than hydrogen bond) A good ligand
undergoes 5-10 van der Waals contacts with his
receptor.
hydrogen bond
Van der Waals interaction
59
Van der Waals interaction
The ensemble of van der Waals interactions is
responsible for the key/lock nature of
ligand/receptor interaction.
?1998 Leif Saul
60
(No Transcript)
61
Example for the interaction of a hypothetical
ligand with its receptor
kcal/mol
formation of a hydrogen bond ... - 5.0 loss of
hydrogen bond with H2O... 5.0
62
Example for the interaction of a hypothetical
ligand with its receptor
kcal/mol
formation of a hydrogen bond ... - 5.0 loss of
hydrogen bond with H2O... 5.0
preliminary balance 0
63
Example for the interaction of a hypothetical
ligand with its receptor
kcal/mol
formation of a hydrogen bond ... - 5.0 loss of
hydrogen bond with H2O ... 5.0 2 H2O set free
- 4.0 Hydrophobic interaction - 2.0 8
van der Waals contacts - 4.7

64
Example for the interaction of a hypothetical
ligand with its receptor
kcal/mol
formation of a hydrogen bond ... - 5.0 loss of
hydrogen bond with H2O ... 5.0 2 H2O set free
- 4.0 Hydrophobic interaction - 2.0 8
van der Waals contacts - 4.7

balance -10.7
65
Example for the interaction of a hypothetical
ligand with its receptor
kcal/mol
formation of a hydrogen bond ... - 5.0 loss of
hydrogen bond with H2O ... 5.0 2 H2O set free
- 4.0 Hydrophobic interaction - 2.0 8
van der Waals contacts - 4.7

balance -10.7
?Go (20 C) ?Go (20 C)
KA KD kcal/Mol kJ/Mol
107 M-1 10-7 M -9.4 -39.2
108 M-1 10-8 M -10.7 -44.8
109 M-1 10-9 M -12.0 -50.3
66
How many receptors do we expect in a responsive
tissue?
Which analytical tools will be necessary to
detect them?
67
How many receptors do we expect in a responsive
tissue?

• Theoretical assumption the tissue consists of
  cubes 10 µm x 10 µm x 10 µm
• Then, 1 mg tissue would consist of 100 x 100 x
  100 106 cells

68
How many receptors do we expect in a responsive
tissue?
Josef Loschmidt (1821-1895)

• Theoretical assumption the tissue consists of
  cubes 10 µm x 10 µm x 10 µm
• Then, 1 mg tissue would consist of 100 x 100 x
  100 106 cells
• If each cell bears 1 binding site, this would
  result in 106 binding sites / mg tissue
• 1 fMol 6 x 1023-15 6 x 108 molecules
• 106 molecules 1/600 fMol

69
How many receptors do we expect in a responsive
tissue?

• The most common binding sites occur at densities
  of 10 to several 100 fMol/mg tissue.
• This is much more than 1/600 fMol/mg tissue.
• Thus, receptor-bearing cells have not only 1, but
  several thousands of binding sites.

Freeze-fracture analysis of AMPA receptors
labelled with immuno gold antibodies (5 nm) at
the postsynaptic site on cerebellar Purkinje
cells (climbing fiber input). Tanaka et al (2005)
J Neurosci 25799
70
Which analytical tools will be necessary to
detect them?
Labelling Replacement of one or more protons by
tritium (3H molecule practically unchanged)
Marie Pierre Curie
71
Which analytical tools will be necessary to
detect them?
Radioactivity measured in

• Curie (Ci, mCi, µCi)

(the radioactivity of 1 g radium)
Marie Pierre Curie
72
Which analytical tools will be necessary to
detect them?
Radioactivity measured in

• Curie (Ci, mCi, µCi)
• Becquerel (Bq, decays / s)
• dpm (decays / min)

1 Bq 60 dpm
Henry Becquerel
73
Which analytical tools will be necessary to
detect them?
Radioactivity measured in

• Curie (Ci, mCi, µCi)
• Becquerel (Bq, decays / s)
• dpm (decays / min)

1 µCi 2 220 000 dpm 1 nCi 2 220 dpm 1 pCi
2.22 dpm
Henry Becquerel
74
Which analytical tools will be necessary to
detect them?
Comparison of 3H with other nuclides (1
radioactive atom / molecule)
t ½ 1 10 100 1000 fMol
14C 5 730 y
3H 12.4 y
35S 87 d
131I 8 d
The shorter the half-life, the hotter the
radioligand.
75
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?
76
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?
A rule of thumb is a principle with broad
application that is not intended to be strictly
accurate or reliable for every situation.
(Wikipedia)
77
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?

• 1 fMol 10-15 x 6 . 1023 6 . 108 molecules

78
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?

• 1 fMol 10-15 x 6 . 1023 6 . 108 molecules
• t½ 12.3 y 4 500 d 108 000 h 6.48 . 106
  min

79
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?

• 1 fMol 10-15 x 6 . 1023 6 . 108 molecules
• t½ 12.3 y 4 500 d 108 000 h 6.48 . 106
  min

General idea Since we know that half of the
radioactive nuclei will decay in 6.48 million
minutes, we might obtain the number of nuclei
decaying in 1 minute simply by dividing half of
the number of nuclei by 6.48 millions.
80
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?

• 1 fMol 10-15 x 6 . 1023 6 . 108 molecules
• t½ 12.3 y 4 500 d 108 000 h 6.48 . 106
  min
• dpm 6 . 108 . 0.5 / 6.48 . 106 46

General idea Since we know that half of the
radioactive nuclei will decay in 6.48 million
minutes, we might obtain the number of nuclei
decaying in 1 minute simply by dividing half of
the number of nuclei by 6.48 millions.
81
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?

• 1 fMol 10-15 x 6 . 1023 6 . 108 molecules
• t½ 12.3 y 4 500 d 108 000 h 6.48 . 106
  min
• dpm 6 . 108 . 0.5 / 6.48 . 106 46

0.5 would be correct, if the decay rate would be
the same for the whole decay period.
82
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?

• 1 fMol 10-15 x 6 . 1023 6 . 108 molecules
• t½ 12.3 y 4 500 d 108 000 h 6.48 . 106
  min
• dpm 6 . 108 . ln2 / 6.48 . 106 64

0.5 would be correct, if the decay rate would be
the same for the whole decay period. However,
radioactive decay follows an exponential law
therefore, 0.5 must be replaced by ln2 0.69.
83
Why the natural logarithm of 2?
84
! attention e ? ? mathematics ? 8
v attention !
Which analytical tools will be necessary to
detect them?
A number of radioactive nuclei k decay
constant
dA/dt -k . A ?(1/A)dA -k . ?dt ln(A/Ao) -k
. ?t
! attention e ? ? mathematics ? 8
v attention !
85
! attention e ? ? mathematics ? 8
v attention !
Which analytical tools will be necessary to
detect them?
A number of radioactive nuclei k decay
constant
dA/dt -k . A ?(1/A)dA -k . ?dt ln(A/Ao) -k
. ?t
A Ao . e-k.?t
! attention e ? ? mathematics ? 8
v attention !
86
! attention e ? ? mathematics ? 8
v attention !
Which analytical tools will be necessary to
detect them?
A number of radioactive nuclei k decay
constant
dA/dt -k . A ?(1/A)dA -k . ?dt ln(A/Ao) -k
. ?t
A Ao . e-k.?t
k is related to t½ ln(½) -k . t½ k ln2 /
t½
! attention e ? ? mathematics ? 8
v attention !
87
! attention e ? ? mathematics ? 8
v attention !
Which analytical tools will be necessary to
detect them?
A number of radioactive nuclei k decay
constant
dA/dt -k . A ?(1/A)dA -k . ?dt ln(A/Ao) -k
. ?t
A Ao . e-k.?t
k is related to t½ ln(½) -k . t½ k ln2 /
t½
for 1 min (?t 1) -?A k . A . 1 ln2
/ t½ . A
! attention e ? ? mathematics ? 8
v attention !
88
Which analytical tools will be necessary to
detect them?
Therefore, it can be expected that 6 . 108
tritium nuclei (1 fMol) will emit
6 . 108 . ln2 / 6.48 . 106 64 electrons / min.
A molecule labeled with one single 3H has a
specific radioactivity (short specific activity)
of 64 dpm / fMol.
89
Which analytical tools will be necessary to
detect them?
Therefore, it can be expected that 6 . 108
tritium nuclei (1 fMol) will emit
6 . 108 . ln2 / 6.48 . 106 64 electrons / min.
A molecule labeled with one single 3H has a
specific radioactivity (short specific activity)
of 64 dpm / fMol.
1 µCi 2 220 000 dpm 1 nCi 2 220 dpm 1 pCi
2.22 dpm
Remember
64 dpm / fMol 28.8 pCi / fMol 28.8 Ci / mMol
90
Which analytical tools will be necessary to
detect them?
64 dpm / fMol 28.8 pCi / fMol 28.8 Ci / mMol
91
Which analytical tools will be necessary to
detect them?
64 dpm / fMol 28.8 pCi / fMol 28.8 Ci / mMol
92
Which analytical tools will be necessary to
detect them?
Comparison of 3H with other nuclides (1
radioactive atom / molecule)
t ½ 1 10 100 1000 fMol
14C 5 730 y 0.14 1.4 14 140
3H 12.4 y 64 640 6.4103 64103
35S 87 d 3.3103 33103 330103 3.3106
131I 8 d 36103 360103 3.6106 36106
dpm / mg tissue
93
Which analytical tools will be necessary to
detect them?
Comparison of 3H with other nuclides (1
radioactive atom / molecule)
t ½ 1 10 100 1000 fMol
14C 5 730 y 0.14 1.4 14 140
3H 12.4 y 64 640 6.4103 64103
35S 87 d 3.3103 33103 330103 3.3106
131I 8 d 36103 360103 3.6106 36106
dpm / mg tissue
most common experimental condition
94
Properties of 3H

• can replace 1H present in every organic molecule
• does not change the properties of the labeled
  molecule (no isotope effect)
• t½ 12.4 y
• b decay (emits electrons)
• radiation reaches in air 6 mm, in liquid and
  tissue 6 µm
• relatively safe to work with (no shielding
  required)
• the only risk is incorporation of gt 1 mCi
• only reliable method of counting

95
Properties of 3H

• can replace 1H present in every organic molecule
• does not change the properties of the labeled
  molecule (no isotope effect)
• t½ 12.4 y
• b decay (emits electrons)
• radiation reaches in air 6 mm, in liquid and
  tissue 6 µm
• relatively safe to work with (no shielding
  required)
• the only risk is incorporation of gt 1 mCi
• only reliable method of counting liquid
  scintillation

96
Analytical techniques
B L BL
equilibrium dialysis
non-equilibrium techniques for receptors in
solution

• gel filtration
• charcoal adsorption
• precipitation
• adsorption to glass fiber filters

non.-equilibrium techniques for particulate
receptors

• centrifugation
• filtration
• slice autoradiography

97
Analytical techniques
B L BL
equilibrium dialysis
non-equilibrium techniques for receptors in
solution

• gel filtration
• charcoal adsorption
• precipitation
• adsorption to glass fiber filters

non.-equilibrium techniques for particulate
receptors

• centrifugation
• filtration
• slice autoradiography

98
Analytical techniques
B L BL
equilibrium dialysis
non-equilibrium techniques for receptors in
solution

• gel filtration
• charcoal adsorption
• precipitation
• adsorption to glass fiber filters

non.-equilibrium techniques for particulate
receptors

• centrifugation
• filtration
• slice autoradiography

99
Analytical techniques
B L BL
equilibrium dialysis
non-equilibrium techniques for receptors in
solution

• gel filtration
• charcoal adsorption
• precipitation
• adsorption to glass fiber filters

non.-equilibrium techniques for particulate
receptors

• centrifugation
• filtration
• slice autoradiography

100
Analytical techniques
B L BL
equilibrium dialysis
non-equilibrium techniques for receptors in
solution

• gel filtration
• charcoal adsorption
• precipitation
• adsorption to glass fiber filters

polyethylene glycol n 6 000 8 000
non.-equilibrium techniques for particulate
receptors

• centrifugation
• filtration
• slice autoradiography

101
Analytical techniques
B L BL
equilibrium dialysis
non-equilibrium techniques for receptors in
solution

• gel filtration
• charcoal adsorption
• precipitation
• adsorption to glass fiber filters

non.-equilibrium techniques for particulate
receptors

• centrifugation
• filtration
• slice autoradiography

102
Analytical techniques
B L BL
equilibrium dialysis
non-equilibrium techniques for receptors in
solution

• gel filtration
• charcoal adsorption
• precipitation
• adsorption to glass fiber filters

non.-equilibrium techniques for particulate
receptors

• centrifugation
• filtration
• slice autoradiography

103
Analytical techniques
B L BL
equilibrium dialysis
non-equilibrium techniques for receptors in
solution

• gel filtration
• charcoal adsorption
• precipitation
• adsorption to glass fiber filters

non.-equilibrium techniques for particulate
receptors

• centrifugation
• filtration
• slice autoradiography

104
Analytical techniques
B L BL
equilibrium dialysis
non-equilibrium techniques for receptors in
solution

• gel filtration
• charcoal adsorption
• precipitation
• adsorption to glass fiber filters

non.-equilibrium techniques for particulate
receptors

• centrifugation
• filtration
• slice autoradiography

105
Saturation non-specific binding
Saturability a radioligand can only be displaced
if the target density is low.
Other examples for saturability Langmuir
isotherme (mono-molecular layer on a surface),
enzyme reaction rate (Michaelis-Menten).
http//www.steve.gb.com/science/membranes.html
106
Saturation non-specific binding
107
Saturation non-specific binding
At low nM concentrations, most of the
radioligand L is bound to saturable high affinity
sites.
108
Saturation non-specific binding
At high concentrations, the linearly
rising non-specific binding will dominate,
and specific binding can no longer be
detected.
At low nM concentrations, most of the
radioligand L is bound to saturable high affinity
sites.
109
Saturation non-specific binding
B . L
BL B L
KD
BL
KD dissociation equilibrium constant
With increasing L more binding sites are
occupied (BL) and free sites (B) are lost. The
sum BL B BM remains constant.
110
Saturation non-specific binding
B . L
KD
BL
replace B by BM BL
111
Saturation non-specific binding
B . L
KD
BL
replace B by BM BL
(BM - BL) . L
KD
BL
solve for BL
112
Saturation non-specific binding
B . L
KD
BL
replace B by BM BL
(BM - BL) . L
KD
BL
solve for BL
KD . BL BM . L BL . L
113
Saturation non-specific binding
B . L
KD
BL
replace B by BM BL
(BM - BL) . L
KD
BL
solve for BL
KD . BL BM . L BL . L
BL . (L KD) BM . L
114
Saturation non-specific binding
B . L
KD
BL
replace B by BM BL
(BM - BL) . L
KD
BL
solve for BL
KD . BL BM . L BL . L
BL . (L KD) BM . L
L
BM .
BL
Langmuir isotherm
L KD
115
Saturation non-specific binding
B . L
KD
BL
replace B by BM BL
(BM - BL) . L
KD
BL
solve for BL
KD . BL BM . L BL . L
BL . (L KD) BM . L
L
BM .
BL
Langmuir isotherm
Irving Langmuir 1881-1957 Nobel price 1932
L KD
116
Saturation non-specific binding
B . L
KD
BL
replace B by BM BL
(BM - BL) . L
wrong!
KD
BL
solve for BL
KD . BL BM . L BL . L
BL . (L KD) BM . L
L
BM .
BL
Langmuir isotherm
Irving Langmuir 1881-1957 Nobel price 1932
L KD
117
Saturation non-specific binding
B . L
KD
BL
replace B by BM BL
(BM - BL) . (Lo BL)
correct
KD
BL
118
! attention e ? ? mathematics ? 8
v attention !
Saturation non-specific binding
B . L
KD
BL
replace B by BM BL
(BM - BL) . (Lo BL)
KD
BL
solve for BL
KD . BL BM . Lo BM . BL BL . Lo
BL2
! attention e ? ? mathematics ? 8
v attention !
119
! attention e ? ? mathematics ? 8
v attention !
Saturation non-specific binding
B . L
KD
BL
replace B by BM BL
(BM - BL) . (Lo BL)
KD
BL
solve for BL
KD . BL BM . Lo BM . BL BL . Lo
BL2
BL2 BL . (BM Lo KD) BM . Lo 0
! attention e ? ? mathematics ? 8
v attention !
120
! attention e ? ? mathematics ? 8
v attention !
Saturation non-specific binding
Sweet memories
B . L
KD
BL
replace B by BM BL
(BM - BL) . (Lo BL)
KD
BL
solve for BL
KD . BL BM . Lo BM . BL BL . Lo
BL2
BL2 BL . (BM Lo KD) BM . Lo 0
! attention e ? ? mathematics ? 8
v attention !
121
! attention e ? ? mathematics ? 8
v attention !
Saturation non-specific binding
Sweet memories
B . L
KD
BL
replace B by BM BL
(BM - BL) . (Lo BL)
KD
BL
solve for BL
KD . BL BM . Lo BM . BL BL . Lo
BL2
BL2 BL . (BM Lo KD) BM . Lo 0
BL ½ . BM Lo KD - (BM Lo KD)2 4 .
BM . Lo½
! attention e ? ? mathematics ? 8
v attention !
122
! attention e ? ? mathematics ? 8
v attention !
Saturation non-specific binding
Sweet memories
B . L
KD
BL
replace B by BM BL
(BM - BL) . (Lo BL)
KD
BL
solve for BL
KD . BL BM . Lo BM . BL BL . Lo
BL2
BL2 BL . (BM Lo KD) BM . Lo 0
BL ½ . BM Lo KD - (BM Lo KD)2 4 .
BM . Lo½
In this case, quantities Lo and KD are not
entered as concentrations, but as moles in the
respective volume chosen, in the same units as BM.
! attention e ? ? mathematics ? 8
v attention !
123
! attention e ? ? mathematics ? 8
v attention !
Saturation non-specific binding
Sweet memories
B . L
KD
BL
replace B by BM BL
(BM - BL) . (Lo BL)
KD
BL
solve for BL
3 times more ligand than receptors at KD
concentration (8 loss)
KD . BL BM . Lo BM . BL BL . Lo
BL2
BL2 BL . (BM Lo KD) BM . Lo 0
BL ½ . BM Lo KD - (BM Lo KD)2 4 .
BM . Lo½
In this case, quantities Lo and KD are not
entered as concentrations, but as moles in the
respective volume chosen, in the same units as BM.
! attention e ? ? mathematics ? 8
v attention !
124
! attention e ? ? mathematics ? 8
v attention !
Saturation non-specific binding
Sweet memories
B . L
KD
BL
replace B by BM BL
(BM - BL) . (Lo BL)
KD
BL
solve for BL
3 times more receptor than ligand at KD
concentration (57 loss)
KD . BL BM . Lo BM . BL BL . Lo
BL2
BL2 BL . (BM Lo KD) BM . Lo 0
BL ½ . BM Lo KD - (BM Lo KD)2 4 .
BM . Lo½
In this case, quantities Lo and KD are not
entered as concentrations, but as moles in the
respective volume chosen, in the same units as BM.
! attention e ? ? mathematics ? 8
v attention !
125
Saturation non-specific binding
A realistic saturation function is a composite of
2 simultaneous processes
1 non-specific binding
It is sufficient to measure 2 points
extrapolation of L ? 0 results in the blank of
the measuring method ().
126
Saturation non-specific binding
A realistic saturation function is a composite of
2 simultaneous processes
1 non-specific binding
It is sufficient to measure 2 points
extrapolation of L ? 0 results in the blank of
the measuring method ().
2 specific binding
... Is sitting on the non-specific binding,
obtained as difference between total and
non-specific binding.
127
Saturation non-specific binding
A realistic saturation function is a composite of
2 simultaneous processes
1 non-specific binding
It is sufficient to measure 2 points
extrapolation of L ? 0 results in the blank of
the measuring method ().
2 specific binding
... Is sitting on the non-specific binding,
obtained as difference between total and
non-specific binding ().
128
Saturation non-specific binding
Mathematical combination of both processes
129
Saturation non-specific binding
Mathematical combination of both processes
1 non-specific binding
L
BU .
BL
L KU
130
Saturation non-specific binding
Mathematical combination of both processes
1 non-specific binding
L
BU .
BL
L KU
2 specific binding
L
BS .
BL
L KS
131
Saturation non-specific binding
Mathematical combination of both processes
1 non-specific binding
L
BU .
BL
L KU
2 specific binding
L
BS .
BL
L KS
KU (mM) gtgt Ks (nM)
132
Saturation non-specific binding
Mathematical combination of both processes
1 non-specific binding
L
BU .
BL
L KU
2 specific binding
L
BS .
BL
L KS
KU (mM) gtgt Ks (nM)
At reasonable ligand concentrations, L KU
KU and non-specific binding is a linear function
of L
L
BU
. L
BS .

BL
L KS
KU
133
Saturation non-specific binding
The most important value, the specific binding,
is not directly accessible. It must be calculated
by substracting the non-specific binding from
total binding.
134
Saturation non-specific binding
The most important value, the specific binding,
is not directly accessible. It must be calculated
by substracting the non-specific binding from
total binding.
The non-specific binding NB is measured as bound
ligand that is impossible to displace, even by
high concentrations of potent displacers.
135
Saturation non-specific binding
Strategies to keep non-specific binding low
136
Saturation non-specific binding
Strategies to keep non-specific binding low

• choose a biological source with a high density of
  high-affinity binding sites

137
Saturation non-specific binding
Strategies to keep non-specific binding low

• choose a biological source with a high density of
  high-affinity binding sites
• select a radioligand concentration around the
  expected KD ( a few hundred to a few thousand dpm
  will be sufficient as result)

138
Saturation non-specific binding
Strategies to keep non-specific binding low

• choose a biological source with a high density of
  high-affinity binding sites
• select a radioligand concentration around the
  expected KD ( a few hundred to a few thousand dpm
  will be sufficient as result)
• use a clean radioligand if necessary, any
  radioligand can be purified easily by thin layer
  chromatography

139
Saturation non-specific binding
Strategies to keep non-specific binding low

• choose a biological source with a high density of
  high-affinity binding sites
• select a radioligand concentration around the
  expected KD ( a few hundred to a few thousand dpm
  will be sufficient as result)
• use a clean radioligand if necessary, any
  radioligand can be purified easily by thin layer
  chromatography
• If you filter your samples and if you use a
  radioligand with an amino group, pre-treat the
  glass fiber filters with polyethylene imine

140
Saturation non-specific binding
Strategies to keep non-specific binding low

• choose a biological source with a high density of
  high-affinity binding sites
• select a radioligand concentration around the
  expected KD ( a few hundred to a few thousand dpm
  will be sufficient as result)
• use a clean radioligand if necessary, any
  radioligand can be purified easily by thin layer
  chromatography
• If you filter your samples and if you use a
  radioligand with an amino group, pre-treat the
  glass fiber filters with polyethylene imine
• optimise the rinsing procedure of pellets and
  filters, respectively

141
Radioligands for excitatory amino acid (EAA)
receptors
Classification of glutamate receptors
ionotropic receptors
metabotropic receptors
142
Radioligands for excitatory amino acid (EAA)
receptors
Classification of glutamate receptors
ionotropic receptors
metabotropic receptors
NMDA receptors
non-NMDA receptors
Group I Group II Group III
143
Radioligands for excitatory amino acid (EAA)
receptors
Classification of glutamate receptors
ionotropic receptors
metabotropic receptors
NMDA receptors
non-NMDA receptors
Group I Group II Group III
AMPA receptors
kainate receptors
144
Radioligands for excitatory amino acid (EAA)
receptors
Classification of glutamate receptors
ionotropic receptors
NMDA receptors
non-NMDA receptors
Schmid et al (2009) PNAS 10610320
AMPA receptors
kainate receptors
145
Radioligands for excitatory amino acid (EAA)
receptors
L-glutamic acid (S)-1-aminopropane-1,3-dicarboxyli
c acid
N-methyl-D-aspartic acid (NMDA)
D-Aminophosphonovaleric acid
CGP 39653 (E)-2-Amino-4-propyl-5-phosphono-3-pente
noic acid
146
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147
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148
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149
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150
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151
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152
Radioligands for excitatory amino acid (EAA)
receptors
Glycine
L-701.324 ( a phenyl quinolinone)
MK-801
MDL-105.519 (an indole carboxylic acid)
153
Radioligands for excitatory amino acid (EAA)
receptors
Glycine
L-701.324 ( a phenyl quinolinone)
MK-801
MDL-105.519 (an indole carboxylic acid)
3HGSK-931.145 radioligand for the glycine
transporter GlyT-1
(Herdon et al 2010 Neuropharmacol 59558)
154
Radioligands for excitatory amino acid (EAA)
receptors
kainic acid ( a pyrrolidine)
AMPA (a-Amino-3-hydroxy-5-methylisoxazol-4-propion
ic acid)
LY-354.740 (a bicyclo3.1.0hexan)
155
Radioligands for excitatory amino acid (EAA)
receptors
kainic acid ( a pyrrolidine)
AMPA (a-Amino-3-hydroxy-5-methylisoxazol-4-propion
ic acid)
LY-354.740 (a bicyclo3.1.0hexan)
Grant et al (2010) Neurotox Terat 32132
156
Radioligands for excitatory amino acid (EAA)
receptors
kainic acid ( a pyrrolidine)
AMPA (a-Amino-3-hydroxy-5-methylisoxazol-4-propion
ic acid)
Muscimol Ibotensäure
LY-354.740 (a bicyclo3.1.0hexan)
157
Radioligands for excitatory amino acid (EAA)
receptors
kainic acid ( a pyrrolidine)
AMPA (a-Amino-3-hydroxy-5-methylisoxazol-4-propion
ic acid)
LY-354.740 (a bicyclo3.1.0hexan)
LY-404.039 LY-379.268
158
The most important binding techniques
B L BL
... are all non-equilibrium techniques for
particulate receptor preparations

• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography

159
The most important binding techniques
B L BL
... are all non-equilibrium techniques for
particulate receptor preparations

• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography

... applied to weak ligands (KD gt 20 nM) ? you
need a high speed refrigerated certrifuge ?
plastic vials must support 40 000 x g ? after
centrifugation, pellet and inner wall needs
rinsing ? scintillation cocktail added directly
to the rinsed incubation vials.
160
The most important binding techniques
B L BL
... are all non-equilibrium techniques for
particulate receptor preparations

• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography

... Can only be applied to high affinity ligands
(KD lt 20 nM) ? you need a vacuum filter box
161
The most important binding techniques
B L BL
... are all non-equilibrium techniques for
particulate receptor preparations

• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography

... Can only be applied to high affinity ligands
(KD lt 20 nM) ? you need a vacuum filter box or
better a harvester ? for radioligands with amino
group, the glass fiber filter must be soaked in
0.3 polyethylenimine ?
162
The most important binding techniques
B L BL
... are all non-equilibrium techniques for
particulate receptor preparations

• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography

... Can only be applied to high affinity ligands
(KD lt 20 nM) ? you need a vacuum filter box or
better a harvester ? for radioligands with amino
group, the glass fiber filter must be soaked in
0.3 polyethylenimine ? for best results, filter
should be shaken in scintillation cocktail for 30
min.
163
The most important binding techniques
B L BL
... are all non-equilibrium techniques for
particulate receptor preparations

• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography

... applied to frozen slices prepared in a
cryostat / microtom (10-20 µm) ? tissue must be
shock-frozen (-40 C) in dry ice / isopentane ?
slices taken up to coated glass slides ? for
incubation, you can use..
164
The most important binding techniques
B L BL
... are all non-equilibrium techniques for
particulate receptor preparations

• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography

... applied to frozen slices prepared in a
cryostat / microtom (10-20 µm) ? tissue must be
shock-frozen (-40 C) in dry ice / isopentane ?
slices taken up to coated glass slides ? for
incubation, you can use a jar or...
165
The most important binding techniques
B L BL
... are all non-equilibrium techniques for
particulate receptor preparations

• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography

... applied to frozen slices prepared in a
cryostat / microtom (10-20 µm) ? tissue must be
shock-frozen (-40 C) in dry ice / isopentane ?
slices taken up to coated glass slides ? for
incubation, you can use a jar or simply a droplet
on the slide ? expose dried slices to film or
phosphoscreen ? evaluation by co-exposure of
stripes containing known amounts of radioactivity.