1. Radioligand Binding Studies
A radioligand is a radioactively labeled drug that
can associate with a receptor, transporter, enzyme,
or any site of interest. Measuring the rate and
extent of binding provides information on the
number of binding sites, and their affinity and
accessibility for various drugs.
Radioligand binding can be used to:
(2)characterize receptors in their natural environment as well
as those transfected into cell lines;
(3)study receptor dynamics and localization;
(4)identify novel chemical structures that interact with receptors
(5)define ligand activity and selectivity in normal and diseased
tissues.

2. Radioligand Binding Studies
 Receptors exist in very small concentrations in
tissues.
 The most common method for detecting the
receptors in membrane preparations, tissue slices or
in the purified form is to use a radioactive drug which
has a high affinity and high degree of selectivity
 Incubate tissue with radioactive drug under the
appropriate experimental conditions, the radioactive
drug (D) will bind to the receptor (R) to form a drug-
receptor complex (RD).
The amount of drug-receptor complex (RD) can be
measured because it is now radioactive.

3. Law of mass action
At Equilibrium
kon Association rate constant or on-rate
constant
koff Dissociation rate constant or off-rate
constant
Kd Equilibrium dissociation constant

4. [Ligand] Fractional
Occupancy
0 0%
1.Kd 50%
4.Kd 80%
9.Kd 90%
99.Kd 99%

5. The model assumes:
• All receptors are equally accessible to ligands.
• Receptors are either free or bound to ligand. It doesn't
allow for more than one affinity state, or states of partial
binding.
• Binding does not alter the ligand or receptor.
• Binding is reversible.

6. Factors to consider in designing receptor
binding experiments.
1. Identify an appropriate radioactive ligand to
use for the experiment.
2. Tissue preparation to be used in the
experiment
3. Identify a method for separating bound from
free
4. Identify a method for distinguishing specific
from nonspecific binding

7. Criteria for selecting a radioactive ligand
a. The specific activity of the radioligand should be high
enough to detect the receptor in the tissue being
studied.
b. The radioligand should have a high affinity for the
receptor.
When using a filtration method to separate bound from
free, it usually takes 5 to 15 seconds to filter the sample
and rinse the filter. During this time less than 10% of the
radioligand should dissociate from the receptor.

8. c. The radioligand should have a high degree of
selectivity for the receptor being studied.
d. The radioligand should be chemically stable in the
assay media during the binding reaction.
e. The radioligand should be pure.

9. Radioligand binding could be :
3)Specific
The site that we want to study is referred to as
the SPECIFIC SITE.
2) Non-Specific
All other sites are called NONSPECIFIC SITES.

10. Examples of nonspecific binding sites
1) Other receptors from the same class:
[3H] rauwolscine binds to alpha-2A, alpha-2B, and
alpha-2C subtypes with similar affinities.
If all alpha-2 adrenoceptors are to be studied without
differentiating between subtypes then binding of [3H]
rauwolscine to all alpha-2 adrenoceptors would be
considered specific binding.
If only study alpha-2A subtype, then radioligand bound
to the alpha-2A subtype would be considered specific
binding and binding to the other subtypes would be
considered nonspecific binding.
• Other receptors from a different class: [3H]
rauwolscine binding to serotonin receptors as well as
to alpha-2 adrenoceptors.

11. 3)
•Binding to tissue protein: The radioligand may bind
to tissue protein or become trapped in the lipid membrane.
•Binding to test tube or glass fiber filters:
Many radioligands bind to test tubes and glass fiber filters
used to separate bound from free. This binding would be
considered nonspecific binding.
Binding to test tubes can sometimes be eliminated by
choosing the type of test tube (glass or plastic) used to do
the binding assay.
Binding to glass fiber filters can usually be eliminated to
some extent by trying different types of filters from
different manufactures or coating the filter with
polyethyleneamine which puts a negative charge on the
filter.

12. SATURABLE and NON-SATURABLE binding
SATURABLE binding sites are always present in
infinite amounts. Another way of thinking of this is that if you
add enough ligand all of the sites will be occupied with ligand.
NON-SATURABLE binding sites are sites that are present in
essentially infinite amounts. No matter how much ligand you
add, not all of the sites will be occupied with ligand. The site
is non-saturable.

13. Examples of saturable sites:
• Specific binding sites are always saturable
• All Receptors
• Test tubes and glass fiber filters may have saturable,
nonspecific binding sites as well as non-saturable binding
sites.
Examples of non-saturable sites:
• Low affinity tissue binding sites
• Binding to test tubes and glass fiber filters may be non-
saturable

14. Binding is measured in the presence of the highest concentration
of radioactive ligand used in a saturation curve. Under these
conditions all of the receptors would be occupied by radioactive ligand.
Binding is also measured in the presence of the same
concentration of radioactive ligand plus increasing concentrations
of unlabeled ligand. As the concentration of unlabeled ligand
increases the amount of radioactive ligand bound decreases until a
plateau is reached. At this point no matter how much unlabeled ligand
is added the amount of radioactive ligand bound remains constant.
This residual amount of radioligand bound represents binding to
nonspecific, non-saturable sites which can't be displaced by unlabeled
ligand.
The amount of inhibitor to use would be the concentration which
completely blocks the saturable binding sites.
In this example it would be between 10-9 to 10-8 M.

16. Guidelines -Appropriate assay conditions
Radioligand Selection
The selection of the radioligand is based on its stability,
specific activity, and pharmacological selectivity.
In general,
• Antagonists tend to bind to receptors with much higher
affinity than agonists.
• Additionally, agonists induce conformational changes in
receptor-effector complexes that can cause ligand-receptor
complexes to exist in multiple states with different binding
characteristics.
• Another advantage of antagonist radioligands is that they
do not activate the receptor which, in the case of binding with
metabolically active cells, can result in the desensitization,
and reduction in affinity, of the site.

17. Stability
Radioligand purity must be established periodically. In
some cases, the radioligand requires special storage
procedures, such as addition of antioxidants or protease
inhibitors in the case of a peptide radioligand, to slow or
prevent degradation.
Nonspecific binding
The physicochemical properties of a ligand determine its
level of nonspecific binding due to interactions with lipid
membranes and/or filter or scintillation bead material in
the assay.
Factors such as lipophilicity and aqueous solubility should
be taken into consideration when selecting a ligand to
radiolabel.

18. Buffers used
In most cases, a homogenization / assay buffer is selected that
yields the highest ratio of specific versus nonspecific binding.
Common buffers - Tris, Hepes, sodium phosphate and
glycylglycine, Krebs, Ringer, or Hanks’ balanced salt solution
(HBSS).
Buffers can affect the binding of radioligand to the receptor,
using the same buffer as for assays measuring response is best
so that the results can be compared.
Some binding assays require the presence of special ions. For
example, opioid receptor binding is modulated by sodium,
GABAA receptor binding by chloride, and the N-methyl-D-
aspartate (NMDA) glutamate receptor by magnesium.

19. pH of the assay buffer
The affinity of a ligand for a receptor generally varies with the
pH. Generally using a physiological pH, such as 7.4, is best so
that the results are comparable to what is seen in vivo.
Optically active radioligands
If a ligand contains a chiral center, use of the stereochemically
active form is preferable.
With few exceptions, most receptors differentiate between the
optical isomers of compounds,
with the pharmacologically-active isomer binding with higher
affinity than the less-active isomer.

20. Additional reagents
MgCl2 is used in many receptors binding assays. Initially this was
used to help precipitate the radioligand receptor complex during
centrifugation.
NaCl is often used in adrenergic receptor binding studies to convert
the receptor to a form that has a lower affinity for agonists.
GTP or its non-hydrolyzeable analog (Gpp(NH)p) are often used
when agonist binding is also going to be studied in subsequent
competition experiments. Gpp(NH)p converts the receptor into a
form that has a low affinity for agonist
Temperature of the binding reaction
Most binding assays can be conducted at room temperature.
Some assays work better at 4oC than at room temperature, and
sometimes the affinity of the radioligand for the receptor is higher at
4oC than at 25oC . It takes longer to reach steady-state when the
reaction is run at 4oC than at room temperature.

21. Major Types of Radio ligand Study
• Saturation binding experiments measure
equilibrium binding of various concentrations of the
radioligand. Analyze the relationship between binding and
ligand concentration to determine the number of sites,
Bmax, and the ligand affinity, Kd.
2. Competitive binding experiments measure
equilibrium binding of a single concentration of radioligand
at various concentrations of an unlabeled competitor.
Analyze these data to learn the affinity of the receptor for
the competitor.
3. Kinetics experiments measure binding at various
times to determine the rate constants for radioligand
association and dissociation.

22. 1. Saturation binding experiments
Experiment used to be analyzed with Scatchard plots
(more accurately attributed to Rosenthal), they are
sometimes called "Scatchard experiments".
The analyses depend on the assumption that you have
allowed the incubation to proceed to equilibrium (few
minutes to many hours, depending on the ligand, receptor,
temperature, and other experimental conditions.)
The lowest concentration of radioligand will take the longest
to equilibrate. When testing equilibration time, therefore, use
a low concentration of radioligand (perhaps 10-20% of
the KD).

23. Purpose of the Saturation experiment
1)To determine the affinity or Kd of a radioligand for a
receptor
Kd is the equilibrium dissociation constant. It is equal to the
concentration of radioactive ligand required to occupy 50 % of
the receptors.
2)The density (Bmax) of a specific receptor or receptor
subtype in a given tissue
Bmax is the total number of receptor sites in the tissue being
studied. It occurs when the all of the receptor molecules are
occupied by radioactive drug.

24. Basic characteristics of a saturation experiment
In a saturation experiment increasing concentrations of a
radioactive ligand are allowed to bind to the receptor until
steady-state conditions occur.
After reaching steady state, the bound ligand is separated
from the free ligand. The most widely used methods for
separation of bound ligand from free ligand are filtration and
centrifugation.
The amount of ligand bound to the filter or trapped in the pellet
is then measured. A radioactive ligand is used because the
radioactivity of low concentrations of ligand can be detected in
filters or membrane pellets.

26. Methods used to determine Kd and Bmax from a
saturation experiment
1. Saturation curve
2. Rosenthal plot (commonly
referred to as a Scatchard plot)
These experiments are called saturation
experiments because at the higher radioligand
concentrations all of the receptor molecules
are occupied (saturated) by radioactive ligand.

27. Results of the saturation experiment can be plotted with BOUND (the
amount of radioactive ligand that is bound to the receptor) on the Y
axis and FREE (the free concentration of radioactive ligand) on the X
axis.
The resulting graph is a hyperbola and is called a saturation curve.
Bmax is the density of the receptor in the tissue being studied.
Kd is the concentration of ligand required to occupy 50% of the
binding sites

29. where B is Bound
F is Free
By fitting the data to the equation for a saturation
curve.
Getting an accurate estimate of Kd and Bmax from this
graph by eye is difficult. The curve is usually analyzed
by nonlinear regression analysis

30. Rosenthal Plot
The data from the saturation experiment can be
plotted with Bound/Free on the Y axis and
Bound on the X axis.
Single site binding data can be analyzed by
linear regression to give a straight line.
The slope of the line is -1/Kd and the X-intercept
is Bmax. This is a Rosenthal Plot.
Most scientists call it a Scatchard Plot.

31. Rosenthal Plot

32. Advantages of Rosenthal Plot
It is easy to visually compare two sets of data on
a Rosenthal plot.
If the Kd for both sets of data are similar the
slopes will be similar.
If the Bmax changes then the X intercept will
change.
Two-site fit to the data can be visualized more
easily with a Rosenthal Plot than with a
saturation curve.

33. Experimental conditions that need to be
considered in doing saturation experiments
5. Concentration of radioligand used
2. Concentration of tissue used

34. 1. Concentration of radioligand used
 At least six radioligand concentrations equally spaced on
either side of the Kd should be chosen if the radioactive
ligand is bound to a single site.
 The lowest concentration should be approximately 1/10 of
the Kd.
 The highest concentration should be approximately 10
times the Kd value.
 Ninety-one percent of the receptors are occupied by
radioactive ligand concentrations which are 10 times the
Kd.
 Six serial 2:5 dilutions of the radioligand are then made
from the highest concentration. If the ligand is binding to
more than one site, using 15 to 25 points to clearly define
both binding sites may be necessary.

35. 2. The concentration of tissue used:
It is dependent on the number of binding sites per
mg of tissue. The tissue concentrations required
in the assay will vary for the same binding site in
different tissues and for different receptors in the
same tissue.

36. Factors deciding tissue (protein)
concentration
• It is helpful if the tissue concentration is high
enough so that at least 1000 cpm are bound when
all of the receptors are occupied with radioactive
ligand. This allows for reasonable accuracy in
counting the radioactivity bound to the tissue at the
lowest concentrations of radioligand.
• It is important that enough tissue be present so that
a measurable amount of the radioligand is bound at
the lowest radioligand concentration.

37. •However, the tissue concentration not be so high
that more than 10% of the radioligand is bound.
The equations used for generation of Kd and Bmax are
based on the assumption that the free concentration
of radioligand does not change.
It is generally assumed that these conditions are met
if less than 10% of the radioligand is bound to the
receptor.
One way to determine quickly whether 10% of the
radioligand is bound to the receptor is to look at the
position of Y-intercept in a Rosenthal plot.

38. Several factors can be determined from a Rosenthal plot
•The ratio of free to bound ligand that should be less than 10%.
Derivation of the equations for determination of Kd and Bmax assume
that the free ligand concentration is not changing during the binding
reaction. These conditions are generally assumed to be met if less
than 10% of the radioligand is bound to the receptor at steady
state conditions.
•Kd can be estimated as the inverse slope of the line.
•Bmax can be estimated from the X-intercept.

39. Calculating specific bound
by subtracting nonspecific bound from total bound.
Calculating free concentration of radioligand
The total concentration of radioligand is the amount added to
the assay tubes. This concentration is usually determined by
directly adding to scintillation vials, the same amount of the
radioligand as added to assay tubes . If a filter assay is used
to separate bound from free, then the filter is also placed in
the scintillation vial, and the radioligand is placed directly on
the filter.
When ligand is bound to the tissue, the free concentration of
radioligand decreases. The free concentration of ligand would
be the total amount of radioligand added to the assay tube
minus the amount bound.
Free = Total Added - Bound

40. If most of the nonspecific binding is to tissue and not to
filters used in a filter assay then bound would be total
bound. (Note that total bound represents the binding of the
radioligand to both specific and nonspecific sites.)
Free = Total Added - (Specific bound + Nonspecific bound)
or
Free = Total Added - Total bound
If the nonspecific binding comes predominately from the
binding of the radioligand to the filters used to separate
bound from free, then this nonspecific binding is not to the
tissue and occurs after the reaction is complete. In this
case, only specific binding should be subtracted from total
binding to give free.
Free = Total added - Specific bound

41. Nonlinear regression analysis of the
saturation curve
Saturation curves are best fit to the following equation
where B is bound and F is free.

42. Conditions that need to be met when doing
saturation experiments
•The measured amount of bound radioligand needs to reflect
the amount of radioligand bound under equilibrium conditions.
That is, equilibrium conditions need to be present when bound
is separated from free. The bound ligand should not dissociate
from the receptor during the course of separating free from
bound.
•A time course can be run at the lowest concentration of
radioligand under your experimental conditions to demonstrate
that equilibrium conditions are present when bound is separated
from free.

43. Less than 10% of the radioligand should dissociate from
the receptor in the process of separating bound from free.
Less than 10% of the radioligand should be depleted
from the reaction mixture.
The amount of bound radioligand needs to be
determined accurately
Specific binding must be correctly defined.
Ligand should not be degraded during the course of the
reaction.

45. Two-Site Saturation Experiments
Under the following conditions:
 When more than one subtype of the receptor is present.
 When the radioligand has a high affinity for more than
one type of receptor.
 When the radioactive ligand is an agonist.
For example: The G-protein receptors exist in at least two
conformations, one with a high affinity for agonist and one
with a low affinity for agonist. Unless a reagent, such as
Gpp (NH)p, is used to convert all of the receptor molecules
to the low affinity form, two binding sites may be detected in
the saturation experiments using a radiolabeled agonist

46. The equation for a two-site fit for a saturation plot is:
where
Bmax1 and Bmax2 are the binding site density
for sites 1 and 2
Kd1 and Kd2 are the Kd values for site 1 and
site 2

47. 2 Competition Experiments
Many ligands for receptors are not available in a radioactive
form. Since they are unlabeled there is no way to directly
measure their affinity for the receptor.
The affinity of the unlabeled ligand for the receptor can be
determined indirectly by measuring its ability to compete
with a radioactive ligand for the receptor.
In a competition experiment various concentrations of an
unlabeled ligand are allowed to compete with a fixed
concentration of a radiolabeled ligand for a receptor.
As the concentration of unlabeled ligand increases, the
amount of radioligand bound to the receptor decreases.

51. The competitive inhibitor can be either an agonist
or an antagonist.
It is called a competitive inhibitor because its
value is determined by measuring the ability of
the unlabeled drug to compete with a radiolabeled
drug for the receptor.
The Ki value for an unlabeled drug should be the
same as the Kd value obtained for the same drug
in radiolabeled form.

52. Analyses of the results of the competition experiment
are simpler and more accurate if the radiolabeled
ligand is only binding to one site.
Hormone and neurotransmitter receptors exits in
conformations which have either high affinity or low
affinity for the agonist.
A true antagonist has equal affinities for the high and
low affinity states of the receptor.
The analysis of the data is simpler if the radiolabeled
ligand is an antagonist

53. Purpose of Competition Experiments
 Competition experiments can be used to determine
the affinity of the unlabeled ligand for the receptor
 The affinity of an agonist for a receptor.
 The pharmacological characteristics of subtypes
of a particular receptor.
 The classification of receptor subtypes in a tissue

54. Experimental Conditions for Competition Studies
The radiolabeled ligand should have a high affinity for the
receptor being studied.
The concentration of radiolabeled ligand used for the
competition studies should be between 0.75 and 1.0 times
the Kd value for the ligand determined using the same
experimental conditions as is planned for use in the
competition studies.
It is helpful to have at least 10 concentrations of unlabeled
ligand for one-site competition studies. There should always
be a tube which does not contain any drug but contains the
same volume of diluents as used to deliver the unlabeled
drug. Increments of 1 and 3 are often used
Binding needs to reach steady-state conditions when doing
either saturation or competition experiments

55. The value of the EC50:
• The affinity of the receptor for the competing drug. If the
affinity is high, the EC50 will be low. The affinity is usually
quantified as the equilibrium dissociation constant, Ki.
The Ki is the concentration of the competing ligand that will bind to
half the binding sites at equilibrium, in the absence of radioligand
or other competitors. If the Ki is low, the affinity of the receptor for
the inhibitor is high.
• The concentration of the radioligand. If you choose to use a
higher concentration of radioligand, it will take a larger conc of
unlabeled drug to compete for half the radioligand binding sites.
• The affinity of the radioligand for the receptor (Kd). It takes
more unlabeled drug to compete for a tightly bound radioligand
(low Kd) than for a loosely bound radioligand (high Kd).

56. Two-Site Competition Experiments
Two site binding in a competition experiment under the
following conditions:
•If the unlabeled ligand is an agonist. Receptors have
both low and high affinity states for agonist. Unless steps are
taken to convert all the receptor molecules into either high or
low affinity states, agonist will bind to more than one affinity
state of a receptor in a competition experiment.
•If the unlabeled ligand binds to different subtypes of
the receptor with different affinities and there is more
than one subtype in the tissue you are studying.
•If the unlabeled ligand binds to more than one receptor
with different affinities. This assumes the radioligand is
also binding to more than one receptor.

57. The characteristics of a competition curve with
one-site binding are a sigmoid curve with:
• a slope of 1.0.
• there is a 81 fold difference in the concentration between
90% specific bound and 10% specific bound.

58. The characteristics of a competition curve with
two-site binding is a sigmoid curve with:
• a slope less than 1.0
• or evidence of two slopes separated by a plateau.

59. Determination of Receptor Subtype using
Competition Studies
Methods used to identify the pharmacological profile for a
specific subtype of a receptor
•To initial identify the pharmacological profile of a receptor
subtype it is important to first have a tissue which selectively
expresses only that receptor subtype.
•Competition studies are done to determine the affinity of a
large number of drugs for the receptor. It is important to use
drugs which have both a high and low affinity for the receptor.
The affinity of these drugs for the receptor define the
pharmacological profile for the receptor. This profile is used to
determine the identify of receptor subtypes in specific tissues

60. Methods used to identify the receptor subtypes in an
unknown tissue
• First, the pharmacological profiles for the known receptor
subtypes need to be determined. It is helpful to identify drugs
which have a high affinity for each of the subtypes of the
receptor.
• The pharmacological profile for the unknown tissue
needs to be determined. In doing this profile, choose
drugs which have high affinities for each of the known
subtypes. Drugs which have similar affinities for multiple
subtypes are not as effective.
• Compare the pharmacological profile for the unknown tissue
with the pharmacological profile of the known subtypes. So
for example, suppose a drug has a high affinity for subtype A
and low affinity for subtype B and C and it also has a high
affinity for the unknown receptor in the tissue you are
studying. This would suggest that the subtype in the unknown
tissue is subtype A.