2.

03 Receptor Theory and the Ligand–Macromolecule

Complex
J P V Heuvel, Penn State University, University Park, PA, USA
ª 2010 Elsevier Ltd. All rights reserved.

2.03.1 Introduction 28
2.03.2 Historical Perspective 28
2.03.2.1 Receptors and Chemical Selectivity 29
2.03.2.2 Law of Mass Action and Occupancy Theory 29
2.03.2.3 The Concept of Efficacy 30
2.03.2.4 The Concept of Spare Receptors 31
2.03.3 Quantitation of Receptor Occupancy 32
2.03.3.1 The Law of Mass Action 32
2.03.3.2 Assumptions Inherent to the Law of Mass Action 33
2.03.3.3 Assessment of Receptor Occupancy 33
2.03.3.3.1 Choice of label 33
2.03.3.3.2 The incubation 34
2.03.3.3.3 Separation of bound from free ligand 34
2.03.3.3.4 Nonspecific binding 36
2.03.3.4 Plotting Methods 37
2.03.3.4.1 Plotting saturation binding data 37
2.03.3.4.2 Scatchard plots 38
2.03.3.4.3 Double reciprocal plot 38
2.03.3.4.4 Hill plot 39
2.03.3.4.5 Linear regression and analysis error 40
2.03.3.4.6 Fitting a curve to determine Bmax and Kd 40
2.03.3.4.7 Evaluation of binding data: Have the assumptions been met? 40
2.03.3.5 Competitive Binding Experiments 41
2.03.3.5.1 Why use a competitive binding curve? 41
2.03.3.5.2 Performing the experiment 41
2.03.3.5.3 Analyzing competitive binding data 42
2.03.3.5.4 Calculating the KI from the IC50 43
2.03.3.6 Ligand Depletion 43
2.03.4 Quantification of Receptor Responses 43
2.03.4.1 Why Examine Receptors Based on Responses? 43
2.03.4.2 Assumptions for Determining Kd Values for Receptor–Ligand Interactions in Intact
Tissue Preparations 44
2.03.4.3 Full Agonists 44
2.03.4.4 Quantitation of Pharmacologic/Toxicologic Antagonism 46
2.03.4.4.1 Functional antagonism 47
2.03.4.4.2 Competitive antagonism 47
2.03.4.4.3 Noncompetitive antagonism 48
2.03.4.4.4 Irreversible and pseudoirreversible antagonists 49
2.03.4.4.5 Mixed antagonism 49
2.03.4.5 Partial Agonists 49
2.03.5 Conclusion 50
References 50

27
28 Basic Principles
Abbreviations
PEG polyethyleneglycol
2.03.1 Introduction
A key concept in biological sciences including phar-
macology and toxicology is that bioactive small
molecules such as drugs, hormones, and nutrients
must achieve adequate concentration at a target site
in order to elicit a biological response. For many
chemicals, the ultimate site of action is a cognate
protein or ‘receptor.’ The main criteria for the opera-
tional term ‘receptor’ are the functions of recognition
and transduction. By this definition, a receptor must
recognize a distinct chemical entity and translate
information from that entity into a form that the cell
can interpret, and alter its state accordingly. This
altered state may be a change in permeability, activa-
tion of a guanine nucleotide regulatory protein, or an
alteration in the transcription of DNA. To differenti-
ate a receptor from an enzyme, the recognition unit
should not chemically alter the small molecule and, to
differentiate from a binding protein, a receptor must
produce a biochemical change and transmit the signal.
Often the receptor is a protein and a single compo-
nent of a large complex of macromolecules that may
include other proteins, RNA, and DNA.
The quantification of the interaction between a
macromolecule and a xenobiotic is often called recep-
tor theory. As will be discussed later in the chapter, the
development of receptor theory predates many of the
modern techniques of molecular biology and coin-
cides with developments in analytical biochemistry,
basic enzymology, and pharmacology. The study of
structure–activity relationships and modifications of
chemicals to fit the active site of the macromolecule
have become standard in the pharmaceutical indus-
try and are an important part of modern
pharmacology and toxicology. Also, the basic tools
of quantification and characterization of a bimolecu-
lar interaction are applicable to many disciplines
including enzymology (Michaelis complex between
enzyme and substrate), immunology (formation of
antibody–antigen complex), pharmacology (drug–
receptor complex), and toxicology (toxicant–
receptor complex).
Much of the conceptual framework regarding
receptor theory evolved from pharmacology and
the investigation of drug action. Consequently, the
FP fluorescence polarization
historical account of the development of receptor
theory contains many references to drugs as opposed
to hormones, neurotransmitters, and xenobiotics.
The term ligand (L) is used interchangeably with
drug in this case, and simply denotes a chemical
that binds with affinity and specificity to a receptor.
Drugs and certain xenobiotics presumably bind to
receptors designed for interaction with endogenous
hormones and neurotransmitters. By way of defini-
tion, agonists are analogous to endogenous hormones
and neurotransmitters, in the sense that they elicit a
biological effect, although the effect elicited may be
stimulatory or inhibitory. In contrast, antagonists are
defined as agents that block receptor-mediated
effects elicited by hormones, neurotransmitters, or
agonist drugs by competing for receptor occupancy
or by interfering with agonist binding in other ways.
Antagonists may not have an endogenous, physiolo-
gically relevant counterpart in the strict sense of a
competitive inhibitor of receptor occupancy.
It is also worth noting that ligands, whether natu-
rally occurring, pharmaceutical, dietary, or
environmental, have the same basic mechanism of
interaction with receptors. The ligands as discussed
herein are small chemicals that associate with the
protein receptor, irrespective of the source of the
molecule. We will discuss how this bimolecular
interaction is studied, the pertinent end points and
approaches used to define the ligand–receptor com-
plex. The interpretation of this data will vary based
on whether the small molecule of interest is a drug or
a pollutant, for example, but the steps taken to derive
meaningful measurements of affinity are identical.
2.03.2 Historical Perspective
The general treatises of ‘receptor theory’ have a long
history that date back at least 100 years and for all
intents and purposes mark the advent of contempor-
ary pharmacology and toxicology. Modern scientists
often take it for granted that drugs elicit their effects
via interaction with specific receptors. However, this
concept was not always evident and evolved as a
result of the remarkable insights of early scientists
exploring a number of fundamental living processes.
 Receptor Theory and the Ligand–Macromolecule Complex
In particular, pioneers such as Claude Bernard
(1813–78), Paul Erhlich (1854–1915), John Langley
(1852–1926), and A. J. Clark (1885–1941) are respon-
sible for much of our understanding of quantifiable
receptor responses. For an extensive review of this
historical perspective, the reader is directed to a book
of great value – Cell Surface Receptors: A Short Course on
Theory and Methods by L. E. Limbird (1996).
2.03.2.1 Receptors and Chemical
Selectivity
Although Claude Bernard did not talk about recep-
tors per se, he demonstrated that the effects of a drug
or toxin (in his case curare) depended on its ability to
access a particular site of action, what he called a locus
of drug effect. Bernard concluded from a detailed set
of experiments with curare and frog gastrocnemius
muscle contractions that this chemical did not affect
the sensory nerves, but instead altered motor nerve
function. Through isolating particular nerves, apply-
ing curare followed by electrical stimulation, Bernard
was able to show a locus of effect whereby some sites
were resistant to curare’s blockade of nerve transmis-
sion. His experiments revealed the existence of a
neuromuscular junction prior to the demonstration
of the muscular endplate as a discrete anatomic struc-
ture. Also, these studies showed that an unknown
component of particular cells made them more or
less sensitive to chemical insult.
The receptor concept is generally attributed to
Paul Erhlich, who expanded the idea of chemical
‘selectivity.’ His most acclaimed studies, which
formed the basis of a Nobel Prize in Medicine in
1908, dealt with immunochemistry and antibody–
antigen interactions. Erhlich demonstrated that
antigen–antibody interactions could be described
as direct chemical encounters. He postulated that
mammalian cells possess entities or ‘side chains’
that can associate with certain chemical groups on
the antigen, and thus serve as the basis for attach-
ing the antigen to the cell. In addition to antigens,
he also felt that small molecules possessed distinct
domains for binding to the target cell. His own
studies established that the arsenoxide group of
organic arsenicals was essential for the lethal
effects of these agents. Target cells need to first
‘bind’ the arsenical, and cells that were unable to
recognize certain constituents were insensitive to
toxicity. Inherent in his ‘side chain theory’ was the
concept of specific cell surface receptors as the
basis for targeting bioactive agents to the
29
appropriate responsive cell. Erhlich conjectured
that the normal function of cellular side chains
was the binding of cell nutrients, and that the
affinity of toxic substances for these groups was
the fortuitous analogy between the structure of
the exogenous toxic substance and the endogenous
nutrient. All of his studies form the basis of quote
‘‘Corpora non agunt nisi fixata’’ (agents cannot act
unless they are bound).
J. N. Langley studied the chemical basis for
autonomic transmission and neuromuscular commu-
nication and extended the work of Bernard. Langley
demonstrated that nicotine could chemically stimu-
late the frog gastrocnemius muscle even following
severing and degeneration of its motor nerves, and
that this action could be blocked by curare. The
mutually antagonistic effects of curare and nicotine
led Langley to conclude that nicotine and curare act
on the same site within the nerve or muscle which he
called a receptive substance. He postulated that com-
plexes are formed between the receptive substance
and the drugs according to some law by which the
relative concentration of the drugs and their affinity
for each other are critical factors. Thus, Langley first
stated the concept of drug–receptor interactions
which predated the algebraic description of these
interactions, the mass action law, often attributed to
A. J. Clark (see Section 2.03.2.2).
2.03.2.2 Law of Mass Action
and Occupancy Theory
As described above, the concept of receptor-
mediated events dates back to the late 1800s and
helped explain, qualitatively, the selectivity and
saturability of drug action. However, it was not
until A. J. Clark (1885–1941) performed his research
that the quantitative nature of receptor theory
emerged. Based on studies of the antagonism
between acetylcholine and atropine, Clark postu-
lated that drugs combine with their receptors at a
rate dependent on the concentration of drug and
receptor. Similarly, the resulting drug–receptor com-
plex breaks down at a rate proportional to the
number of complexes. These statements implied
that drug–receptor interactions obey the law of mass
action that was used to describe the absorption of
gases onto metal surfaces and other physical–chemi-
cal binding isotherms. Based on these principles, a
mathematical expression can be provided to describe
drug–receptor interactions:
30 Basic Principles
Rate of combination ¼k1 ½L½R
Rate of dissociation ¼k – 1 ½LR
where k1¼rate constant for combination (also called
kon); k1¼rate constant for dissociation (also called k2
or koff); L¼concentration of drug; R¼concentration
of unoccupied receptor; and LR¼concentration of
drug–receptor complex. At equilibrium (or apparent
equilibrium, steady state), the rate of combination
equals rate of dissociation, or
k1 ½L½R ¼k – 1 ½LR
which can be arranged to
k – 1 ½L½R
¼ ¼ Kd ð1Þ
k1 ½LR
The term Kd is defined as the equilibrium dissociation
constant and is usually expressed as a concentration.
(The equilibrium association constant, Ka, is equivalent
to K1
d , but is of limited use.) Equation [1] relates the
concentration of drug applied, L, to the proportion of
receptors occupied. Fractional occupancy (Y) is the
proportion of all receptors that are bound to ligand at
equilibrium.
½LR ½LR
Fractional occupancy ¼ Y ¼ ¼ oversimplification of the biological response and
½RTotal ½R þ ½LR
The above equation after some algebra (multiply the
numerator and denominator by [L] and divide by
[LR]) can be described as follows.
½L
Y¼ ð2Þ
½L þ Kd
It is important to note that the algebraic relationship
describing fractional receptor occupancy as a func-
tion of drug concentration (eqn [2]) is analogous to
the quantitative relationships between enzyme and
substrate introduced by Michaelis and Menton.
Subsequently, Clark extended his hypothesis that
drug–receptor interactions obeyed mass action law
by postulating that the fraction of receptors occupied,
Y, was directly proportional to the response of the
tissue. Some early data of Clark and others describing
concentration–response relationships in various
model systems were consistent with this postulate.
However, certain data conflicted with this simple
relationship between occupancy and effect. First,
the slope of the concentration–response relationships
reported was often steeper than predicted from eqn
[2]. Second, a number of examples existed where the
application of extremely high concentrations of drug
did not elicit a maximal response. These latter find-
ings suggested that even maximal saturating
occupancy of a receptor does not necessarily trans-
late into a maximal physiological effect.
The lack of maximal response in the presence of
maximal occupancy proved to be troubling to early
researchers and required the addition of a new con-
cept. Ariëns introduced the term intrinsic activity to
describe the ability of a drug to elicit its pharmaco-
logical effect. He expressed the relationship between
the effect (EL) elicited by drug L and the concentra-
tion of drug–receptor complexes as follows.
EL ¼ ½LR
He defined  as the ‘intrinsic activity’ of the
particular drug. The intrinsic activity of a drug was
meant to be a constant that determines the effect
elicited per unit of ligand–receptor complex formed.
This early definition of intrinsic activity addressed
the observation that maximal receptor occupancy by
some agonists did not elicit a maximal response.
However, this conceptualization still could not
explain dose–response relationships that were stee-
per than predicted by mass action law. Obviously, the
law of mass action and fractional occupancy were an
would require refinement. An important concept
that developed from the work of Ariëns and Clark
was that of efficacy, as described subsequently.
2.03.2.3 The Concept of Efficacy
It was Stephenson (1954) who introduced a major
conceptual advance in the understanding of the
quantitative relationship between receptor occu-
pancy and receptor-elicited effects (1). Stephenson
argued that even A. J. Clark’s experimental findings
were not in accord with a linear relationship between
occupancy and effect. Although Stephenson con-
curred that eqn [2] is the probable relationship
between the concentration of drug introduced and
the concentration of drug–receptor complexes
formed, he felt that there was no experimental justi-
fication for extending this relationship to the
response of the tissue. He postulated three principles
governing receptor-mediated functions that could
explain the previously anomalous observation that
agonist–response curves were often steeper than the
dose–response relationships that would be predicted
by simple mass action law. In addition, his postulates
offered an explanation for the observed progressive
 Receptor Theory and the Ligand–Macromolecule Complex
variation in the agonistic properties of a homologous
series of drugs:
1. An agonist can produce a maximum effect while
occupying only a small proportion of the
receptors.
2. The response is a function of receptor occupancy,
although not linearly proportional.
3. Different drugs may have varying capacities to
initiate a response and consequently occupy dif-
ferent proportions of the receptors when
producing equal responses. This property was
referred to as the efficacy of the drug and is a
property of the L and the LR complex. A pure
antagonist would have an efficacy of zero.
Stephenson followed these postulates with the fol-
lowing derivations:
S¼stimulus to the tissue from the ligand, L.
S¼eY (the stimulus is proportional to fractional occu-
pancy), where e is efficacy as described above and Y is
fractional receptor occupancy.
e½L
S¼ ðfrom eqn ½2 forY Þ ð3Þ
½L þ Kd
R¼response of a tissue and R¼f(S).
To test the validity of his postulates regarding
various efficacies for different agonists, Stephenson
studied a series of alkyl-trimethylammonium salts on
contraction of the guinea pig ileum. He noted that the
short alkyl chain lengths (e.g., butyl-trimethylammo-
nium) behaved as an agonist like acetylcholine,
whereas the longer chain length homologues acted
like atropine, an antagonist. He interpreted this antag-
onism as a property expected for a drug with low
efficacy. Thus, the drug produces a response much
less than the maximum even when occupying all or
nearly all of the receptors. However, because a drug
with low efficacy can nonetheless occupy the recep-
tors, it decreases the response elicited by a drug with
high efficacy when added simultaneously. Stephenson
termed these low-efficacy drugs that possessed prop-
erties intermediate between agonists and antagonists
partial agonists.
The concept of efficacy may be best described by
examining a dose relationship for a hypothetical ser-
ies of drugs (see Figure 1) with efficacies ranging
from 1 to 1000. Of course, an efficacy of zero would
result in no stimulus, and hence no response. The
response from efficacies greater than one would be a
function of the stimulus; an increase in e results in an
31
2
e[L]
S=
[L] + Kd
Stimulus
e
1
e=0
0
1 10 100 1000
Log[L]
Figure 1 Dose–stimulus relationship with drugs of
increasing efficacy (e).
increase in the maximal stimulus without a major
change in the shape of the curve.
2.03.2.4 The Concept of Spare Receptors
The law of mass action implies that the formation of a
ligand–receptor complex is reversible. However, it
was noted that certain antagonists did not follow this
rule and were in fact irreversible, that is, they form
covalent bonds with a receptor. (The types of recep-
tor antagonism will be discussed in detail in Section
2.03.4.3.) This was shown when extensive washing of
a tissue preparation was unable to reduce the antago-
nist’s effect. A receptor that is occupied by an
irreversible antagonist is permanently disabled; this
property of irreversible antagonists was used to
examine a phenomenon known as spare receptors.
Research performed by Furchgott (1967) and others
showed that there was a receptor reserve and that 100%
occupancy was not required for maximal response.
Consider a simplified model as shown in Figure 2 in
which drugs A, B, and C stimulate the same receptor.
Although all three agonists can elicit a maximal
response, each requires a different percentage of
receptors to be occupied (consider each drug–recep-
tor complex to have a different intrinsic activity or
efficacy). Upon irreversible inactivation of a popula-
tion of the receptors, it becomes possible to
determine the fractional occupancy and rank the
efficacies for the three agonists. If 50% of the recep-
tors are inactivated, the maximal response of the
agonist A is decreased by 50%, whereas drugs B
and C are unaffected. If 75% of the receptors are
irreversibly inhibited, the response of A is decreased
by 75%, B’s effects are decreased by 50%, and C still
32 Basic Principles
Agonist
A B C
Response
A Occupancy
B
C
Log(Dose)
With irreversible antagonist
A B
Response
A
C spare receptors. However, the assessment of receptor
B
C occupation per se follows the law of mass action in
Log(Dose)
Figure 2 Hypothetical model for examining the existence
of spare receptors. Three agonists (A, B, C) were examined in
the absence (top) or presence (bottom) of an irreversible
antagonist. In the case of drug A, there is no spare receptor
and full occupancy is required for maximal induction. With
drugs B and C only 50% and 20% occupancy is required.
When one-half of the receptors are inhibited, only drug A’s
effects are reduced. Greater than 80% inhibition would be
required to affect the maximal induction elicited by agonist C.
elicits a maximal response. Using this model system,
Nickerson (1956) demonstrated that occupancy of
only 1% of the histamine receptor population of
guinea pig ileum was required to elicit maximal
contractile response, suggesting the existence of a
large receptor reserve for histamine receptors in
this tissue. However, it is worth noting that receptor
reserves were not always so dramatic. For example,
Furchgott (1967) noted that for epinephrine and the
muscarinic receptor there was only a shift of a half
log unit, if anything, before a decrease in the max-
imum response was observed following exposure to
the alkylating agent dibenamine.
Goldstein (1974) offered a tenable teleological
explanation for such a phenomenon, that is, that
sensitivity to low drug concentrations and low affi-
nity is achieved by the spare receptor capacity. In
circumstances where the response desired is to be
rapid in onset and in termination, as in neurotrans-
mission, a spare receptor capacity provides a
mechanism for obtaining a response at a very low
concentration of an agonist that nonetheless has a
relatively low affinity for the receptor. As we shall
discuss subsequently, the low affinity (i.e., high Kd) of
the drug assures its more rapid rate of dissociation,
since Kd¼k1/k1. If, alternatively, sensitivity to low
concentrations of agonist were achieved by a high
affinity of the drug for the receptor, then the rate of
reversal of the effect would necessarily be slow.
2.03.3 Quantitation of Receptor
2.03.3.1 The Law of Mass Action
Much of the discussion above showcases the diffi-
culty in extrapolating receptor occupancy to
responses elicited, especially in light of the existence
of partial agonists, steep dose–response curves, and
many instances. We will reexamine the law of mass
action as outlined by Clark and others and subse-
quently explain how these derivations may be used
experimentally. Most analyses of radioligand-bind-
ing experiments are based on this simple model:
k1
!
LþR  LR
k–1
where L is the ligand, R is the receptor, and LR is the
ligand–receptor complex.
The model is based on several simple ideas:
1. Binding occurs when the ligand and receptor
collide due to diffusion, and when the collision
has the correct orientation and sufficient energy.
The rate of association (number of binding
events per unit of time) equals [L][R]k1, where
k1 is the association rate constant in units of
mol1l min1 .
2. Once binding has occurred, the ligand and recep-
tor remain bound together for a certain amount of
time that is influenced by the affinity of the recep-
tor and ligand for one another. The rate of
dissociation (number of dissociation events per
unit time) equals [L]k1, where k1 is the disso-
ciation rate constant expressed in units of min1.
3. After dissociation, the ligand is the same as before
binding. As noted earlier, this is to differentiate a
receptor from an enzyme.
4. Equilibrium is reached when the rate at which
new LR complexes are formed equals the rate at
which these complexes dissociate. Equilibrium
may be more appropriately termed apparent equili-
brium or steady state, since the achievement of true
equilibrium is often not possible in pharmacologic
systems.
At equilibrium, LR complexes form at the same rate
that they dissociate:
½L½Rk1 ¼ ½LRk – 1
 Receptor Theory and the Ligand–Macromolecule Complex
which may be rearranged to define the equilibrium
dissociation constant Kd.
½L½R k – 1
¼ ¼ Kd
½LR k1
The Kd, expressed in units of moles per liter or molar,
is the concentration of ligand that occupies half of the
receptors at equilibrium (see below). A small Kd
means that the receptor has a high affinity for the
ligand whereas a large Kd means that the receptor has
a low affinity for the ligand. Often Kd, the equilibrium
dissociation constant, is confused with k1, the dis-
sociation rate constant. However, they are obviously
not the same, as denoted by the fact that their units
are different.
The law of mass action predicts the fractional
receptor occupancy at equilibrium as a function of
ligand concentration. Fractional occupancy (Y) is
defined as the fraction of all receptors that are
bound to ligand:
½LR ½LR ½L
Y ¼ ¼ ¼ ðsee eqn ½2Þ
½RTOT ½R þ ½LR ½L þ Kd
This equation predicts the following. When no ligand
is available, the occupancy equals zero. When the
concentration of ligand is very high (many times
Kd), the fractional occupancy approaches (but never
reaches) 100%. When [L]¼Kd, the fractional occu-
pancy is 50%. Equation [2] predicts that the
approach to saturation as ligand concentration
increases is quite slow. When the ligand concentra-
tion equals 4 times its Kd, it will only occupy 80% of
the receptors at equilibrium. The occupancy rises to
90% when the ligand concentration equals 9 times
the Kd. It takes a concentration equal to 99 times the
Kd to occupy 99% of the receptors at equilibrium.
2.03.3.2 Assumptions Inherent to the Law
of Mass Action
The law of mass action is a convenient and simple
model to describe a bimolecular interaction.
However, in order to coerce a complex biological
interaction into a simple model, several assumptions
or criteria must be met. Although termed a ‘law,’ the
law of mass action is simply a model based on the
following assumptions:
1. All receptor sites are equally accessible to ligand. In the
simplest model, all receptor sites are considered to
have equal affinity for ligand and to be indepen-
dent. That is, occupancy of some receptor sites
33
does not alter the binding to other unoccupied
sites (cooperative binding is not present).
2. The two reactants (receptor and ligand) are either free or
bound to ligand. The model ignores any states of
partial binding. Also, the measured products do
not include degraded, metabolized, or other una-
vailable forms of drug or receptor. Importantly,
nonspecific binding must be accounted for such
that the concentration of the free or bound ligand
is measured accurately. Nonspecific binding
includes any nonreceptor site for ligand binding
that would diminish the free concentration of the
chemical being examined.
3. Binding is reversible. The association between a
ligand and a receptor depends only on the inter-
action of ligand and receptor and dissociation only
on the breakdown of the ligand–receptor complex.
The discussions that follow regarding the incubation
plotting methods are dependent on the assumptions
above. If it is not possible to meet all of these criteria,
two choices exist. If the first assumption is not satisfied,
there are more complicated model systems to describe
ligand–receptor complexes that are cooperative in nat-
ure. The reader is directed to Limberg (1996), Cell
Surface Receptors, for description of these models as
they are beyond the scope of the present chapter. If
assumptions 2 and 3 are not met, the only option is to
analyze the data in the usual way, but to interpret the
results as an empirical description of the data without
attributing rigorous thermodynamic meaning to the Kd
values and rate constants. In other words, the data must
be considered as highly suspect.
2.03.3.3 Assessment of Receptor
Occupancy
2.03.3.3.1 Choice of label
In order to examine the binding of ligand and recep-
tor, one of these constituents must be easily
quantified. The most common means to quantify a
ligand is to add an isotope to form a radioligand (often
depicted as L). One of the most critical considera-
tions in the successful application of radioligand-
binding techniques is the choice of an appropriate
isotope for radiolabeling the ligand. Each common
isotope has certain characteristics to be taken into
consideration (see Table 1). For example, tritium is a
popular choice because of its long half-life. However,
tritium exchanges with water may require repurifica-
tion. Carbon-14 (14C) has benefits of ease of
incorporation into organic ligands, but suffers from
34 Basic Principles

Table 1 Considerations when choosing an isotope for labeling of drug in receptor binding assays

Specific activity
Isotope (Ci/mmol) Half-life Other considerations
3
H 29.4 12.3 years Bioactivity usually unchanged by tritiation; can introduce >1 mole 3H/mole
ligand to increase specific activity
125
I 2125 60.2 days Require tyrosine or unsaturated cyclic system in ligand structure to achieve
incorporation of 125I; high-specific radioactivity, especially useful when
receptor availability is limited
32
P 9760 14.2 days Short half-life makes 32P a difficult choice
35
S 4200 86.7 days Good sensitivity; used in metabolic labeling
14
C 0.064 5568 years Poor sensitivity

From Limbird, L. E. Cell Surface Receptors: A Short Course on Theory and Methods, 2nd ed.; Kluwer Academic Publishers: Boston, MA,
1996; pp 238.

low-specific activity. Iodine-125 (I125) is the most
common choice for radioligand products and affords
examination of low-affinity binding and low concen-
tration of receptor. However, the incorporation of
iodine into a drug may affect its affinity for a biolo-
gical receptor. Taken together, the choice of isotope
is dependent on sensitivity issues such as receptor
density, counting efficiency, and availability of tissue,
as well as ease of synthesis. Another option that is
gaining popularity is to examine a drug or chemical’s
fluorescence, either natural or added exogenously.
Whether this method can be performed depends on
the ligand under question, but when available repre-
sents a sensitive and rapid means to examine
receptor–ligand interactions.
2.03.3.3.2 The incubation
Critical components of the incubation include the
labeled ligand, a source of receptor (crude membrane
preparation, recombinant protein, etc.) and a suitable
buffer. Also, the incubation must proceed for a suffi-
cient amount of time, at an appropriate temperature,
until enough of the drug–receptor complex is formed
to be detectable. In general, increasing the concen-
tration of either or both of the reactants L and R can
increase the overall rate of association. These manip-
ulations will also increase the amount of drug–
receptor complex formed and thus lengthen the
time required to reach equilibrium. Optimal incuba-
tion conditions are determined empirically for each
system and are chosen to maximize complex forma-
tion while minimizing the degradation of L and R.
The source of receptor is dependent on the sys-
tem under study and may include crude extracts,
membrane preparations, cytosol, or nuclear extracts.
Typical concentrations of receptors in crude
preparations are 11012 to 1109mol l1
(femtomoles to picomoles per mg tissue). However,
in many instances the receptor is expressed in bac-
teria, yeast, or insect cells, if possible. This allows for
a much higher concentration of receptor than seen in
vivo, often at higher purity (>1mg ml1). However,
receptors that are multiprotein complexes or require
posttranslational modifications are difficult to exam-
ine using recombinant proteins.
2.03.3.3.3 Separation of bound from free
ligand
To determine the quantity of LR accumulated, one
must have a method for determining the incubation
that permits the resolution of L from LR. When
receptors are embedded within membranes, the free
and bound forms of drug may be easily separated by
filtration or centrifugation. The speed of separation is
an important consideration in determining which
method to use to separate L from LR. If the rate of
dissociation is slow in comparison to the rate of
separation, the amount of LR formed can be deter-
mined accurately. This is the case for high affinity
ligands (low Kd). However, if the rate of ligand dis-
sociation is high, the rate of separation may become
an important consideration for accurately measuring
the amount of drug–receptor complex.
Consider the following equation (for a derivation
of eqn [4], see Limbird (1996)).
½L½R k – 1 6:9310 – 1 mol l – 1 s
Kd ¼ ¼ ¼ ð4Þ
½LR k1 t1=2
where t1/2 is the half-life of the LR complex (amount
of time required for LR to decrease by 50%). If a
10% loss of LR is considered acceptable during the
separation process, then 0.15 times the half-life is
the allowable time required. (Note that these
 Receptor Theory and the Ligand–Macromolecule Complex
Table 2 Relationship between equilibrium dissociation
constant Kd and allowable separation time of DR
Kd Allowable separation time (0.15 t1/2)
1012 1.2 days
1011 2.9 h
1010 17 min
109 1.7 min
108 10 s
107 0.1s
106 0.01 s
From Limbird, L. E. Cell Surface Receptors: A Short Course on
Theory and Methods, 2nd ed.; Kluwer Academic Publishers:
Boston, MA, 1996; pp 238.
calculations are based on a k1 of 106mol1 l s1 and
that k1 is equivalent to ln2/t1/2.) As shown in
Table 2, with low affinity ligand–receptor interac-
tions, the separation of L from LR must proceed
exceedingly rapid. In the following section, the
basic separation techniques will be briefly discussed.
For a more detailed description of these methods, see
Levitzki (1984) or Limbird (1996).
2.03.3.3.3(i) Equilibrium dialysis The dialysis
chamber consists of two compartments separated by
a membrane to which the receptor preparation is
added to one side and the radiolabeled ligand to the
other. After the solution reaches equilibrium, samples
from both chambers are taken and the radioactivity
measured. The amount of drug–receptor complex
formed is determined based on the assumption that
the free drug is equal on the protein- and nonprotein-
containing compartments.
Protein compartment LR þ L
– Nonprotein compartment – L
LR ðthe concentration
Difference
of bound radioligandÞ
The advantages of equilibrium dialysis include
the fact that equilibrium is not disturbed when sam-
ples are taken and therefore represents an accurate
estimate of LR. Also, this method is useful for low-
affinity receptor–drug interactions. Equilibrium dia-
lysis is no longer a frequently utilized method in
receptor-binding assays, predominantly due to the
long time required to reach equilibrium and second-
ary increases in the risk of protein or drug breakdown
and decreased throughput.
2.03.3.3.3(ii)Centrifugation The following tech-
niques require that the drug–receptor complex be
35
formed and reach equilibrium with the free drug.
There are several variations of centrifugal separation
of drug–receptor complexes. If the receptor is mem-
brane bound, the drug–receptor complex may be
centrifuged and a tight pellet formed. The amount
of radioactivity retained in the pellet represents LR
and the free drug is found in the supernatant. One
minor variation is to layer the receptor-containing
solution on a high-density solution (sucrose or oil).
This alteration results in less free drug being trapped
in the pellet, but also makes the assay more cumber-
some and increases the risk of LR dissociating during
the centrifugation.
In the case of soluble receptors, either the receptor
or the drug may be precipitated by centrifugation. In
the former situation, an antibody specific to the
receptor may be coupled to a large molecule such as
sepharose. Upon centrifugation, the pellet will con-
tain LR and the supernatant L, as described above for
membrane-bound receptors. Alternatively, agents
such as polyethyleneglycol (PEG) or Sephadex
G-50 may be used to nonspecifically precipitate pro-
teins. Alternatively, the radioligand may be
precipitated with the aid of dextran-coated charcoal,
leaving the DR in the supernatant.
Typically, centrifugation can pellet the bulk of
the particulate matter within 5 s, and as shown
Table 2 is appropriate for receptors with a Kd of
10–8mol l1 and lower. This method is relatively
fast and many replicates may be examined in one
assay. However, nonspecific binding and trapping of
free drug is often a major limitation.
2.03.3.3.3(iii) Vacuum filtration The incubation is
terminated by pouring the mixture through a filter
under vacuum. The proteins (R and LR) are retained
by the filter and the free radioactivity flows through.
This procedure can handle a large number of samples
and the filtration devices are commercially available.
The filters are generally made from glass, nylon, or
nitrocellulose in order to provide high affinity and
capacity for protein binding. The amount of LR
formed is determined by counting the amount of
radioactivity present on the filter after washing with
a suitable buffer. Filtration usually takes 2–5s per
wash, which means that the minimum ligand affinity
of the receptor that can be identified by this techni-
que is 108 moll1. As with other methods, the
washing of the drug–receptor complex is performed
with cold buffer to slow down the dissociation rate of
the drug. Nonspecific binding and trapping of
36 Basic Principles
radioligand to the filter are potential problems asso-
ciated with vacuum filtration.
2.03.3.3.3(iv) Scintillation proximity assays A
newer type of assay is the scintillation proximity
assay, which does not require the free and the bound
drug to be separated. In this assay, the receptor is
attached to a synthetic bead which contains a scintillin
(a material that emits photons when it absorbs radio-
active emissions). In most instances the receptor is
bacterially expressed, purified, and chemically
attached to the bead, although it is possible to adhere
membrane-bound or crude receptor preparations.
When a radioactive ligand is added to the medium,
only the portion that is bound to the receptor will be
close enough in proximity to the scintillin and thereby
produce a photon emission. Therefore, the LR is
assessed as the number of light units produced and is
a true equilibrium-binding assay. Scintillation proxi-
mity assays have become quite popular because of their
high-throughput capabilities. However, the production
of the synthetic beads containing the receptor of inter-
est may be an arduous task for many laboratories.
2.03.3.3.3(v) Fluorescence polarization
assays Fluorescence polarization (FP) is a powerful
tool for studying molecular interactions by monitoring
changes in the apparent size of fluorescently labeled
ligand. Similar to scintillation proximity assays, FP
enables the researcher to view molecular-binding
events in solution, allowing true equilibrium analysis
into the low picomolar range. FP measurements do
not affect samples, so they can be treated and reana-
lyzed in order to ascertain the effect on binding by
such changes as pH, temperature, and salt concentra-
tion. In addition, because FP is a truly homogeneous
technique, it does not require the separation of bound
and free species. The theory of FP is based on the
observation that when a small fluorescent molecule is
excited with plane-polarized light, the emitted light is
largely depolarized because molecules tumble rapidly
in solution during its fluorescence lifetime. However,
if the tracer is bound by a larger molecule its effective
molecular volume is increased. The fluorecent
ligand’s rotation is slowed so that the emitted light is
in the same plane as the excitation energy. The bound
and free states of the ligand each have an intrinsic
polarization value: a high value for the bound state and
a low value for the free state. The measured polariza-
tion is a weighted average of the two values, thus
providing a direct measure of the fraction of tracer
bound to receptor.
2.03.3.3.4 Nonspecific binding
In addition to ligand binding to receptors of physio-
logical interest, ligands often bind to nonreceptor
sites. Any interaction with a nonreceptor site would
be considered nonspecific binding and is found at excess
over true, specific interactions with the receptor.
When performing radioligand-binding experiments,
a measure of both total and nonspecific binding is
performed, and specific (receptor) binding is calcu-
lated as the difference (see Figure 3). The binding of
radioligand is examined in the presence of excess
saturating amounts of unlabeled drug. This techni-
que assumes that a 100-fold excess of nonradioactive
ligand saturates the specific receptor binding but has
no effect on the nonspecific sites. A useful rule of
thumb is to use the unlabeled compound at a con-
centration equal to 100 times its Kd for the receptor, if
that is known from previous experiments or esti-
mated from preliminary analyses of the system. The
drug used in excess may be the compound used as the
radioligand, but unlabeled, or it may be a chemical
that is known to bind to the same receptor. Ideally,
the same results should be obtained when defining
nonspecific binding with a range of concentrations of
several drugs. Nonspecific binding is generally pro-
portional to the concentration of radioligand (within
the range used) and will not plateau at high concen-
trations. However, as shown in Figure 4 and
discussed subsequently in the chapter, the specific
binding is saturable and exhibits a sigmoidal shape
when examined on a semilogarithmic plot.
2.03.3.3.4(i) Additional criteria The binding of a
drug to a receptor is an in vitro experiment performed
[L][R ]tot
[LR] =
K d + [L]
100
at ∞, y = Rtot = B max
[LR], bound ligand
75
B max [R]tot
50 or
2 2
25
0
0 20 40 60 80 100 120
[Ligand]
Figure 3 The hyperbolic relationship of ligand binding
isotherms.

150
125
[LR], bound ligand
100
75
50
25
0
0 20
Figure 4 Hypothetical binding curves. The left figure shows total and nonspecific binding. The difference between total and
nonspecific binding is the specific binding and if this is plotted on a log-dose scale, it has a sigmoidal shape (right). If an
insufficient number of concentrations are used, often the plateau is not evident in such a representation.
under well-controlled situations. The desire is to
extend the data to a physiologically relevant situa-
tion, and therefore the following criteria may be used
to assess whether the data generated can be extra-
polated to a real-world (i.e., a real cell) situation. The
specific binding of L should be saturable, since a
finite number of receptors are expected in any
given biological preparation. The specificity of
agents in competing with L for binding to R should
parallel the specificity of physiological effect via the
putative receptor of interest. The use of competition
binding will be discussed subsequently. Further, the
kinetics and LR of binding should be consistent with
the time course of the biological effect elicited by L
and there should be consistency between Kd values
obtained using steady-state incubations and those
determined through kinetic experiments.
2.03.3.4 Plotting Methods
As shown in Figure 3, the relationship between
receptor occupancy ([LR]) and drug concentration
is hyperbolic when a saturable receptor population
binds to ligand in a freely reversible, bimolecular
interaction. Often the results are examined by linear
transformation of the experimental data. The advan-
tage of such transformations is that Kd and receptor
density (Bmax or Rtot) can be easily identified.
However, if the drug concentrations examined are
insufficient, as defined empirically, linear transfor-
mations may give a distorted view of the binding
phenomena. In this section, both nonlinear and linear
transformations will be discussed. All plotting
Receptor Theory and the Ligand–Macromolecule Complex 37
Total
Specific
Specific
Nonspecific
40 60 80 100 0.1 1 10 100
[Ligand] [Ligand]
methods must meet the following criteria: (1)
assumptions of the law of mass action, as described
above, are met; (2) the incubation and separation
techniques are appropriate; and (3) nonspecific bind-
ing has been adequately measured.
2.03.3.4.1 Plotting saturation binding
data
Saturation binding experiments measure specific
binding at equilibrium at various concentrations
(often 6–12) of the radioligand to determine receptor
number and affinity. Use of drug concentrations that
allow binding to approach saturation is crucial for
accurate examination of the interaction between the
drug and receptor. The next consideration is that of
nonspecific binding. As mentioned above, a good rule
of thumb is to use a concentration of unlabeled ligand
at 100 times the Kd. When examining binding in the
presence of 100Kd, the amount of [LR] detected is
considered nonspecific, and as shown in Figure 4 is
linear with respect to free ligand concentration. Total
binding is the amount of [LR] detected in parallel
samples without this competitor included in the
incubation. The difference (total – nonspecific) is
the specific binding, and is the data that should conform
to the law of mass action. It is this binding that we
will examine in more detail.
One of the criteria stated previously for a physio-
logically relevant receptor is that the binding sites are
saturable. To assess saturability, the characteristics of
binding as a function of increasing concentrations of
radioligand are determined. The saturation-binding
curve can be described by eqn [2]
38 Basic Principles
½LR ½L
Y ¼ ¼ ðsee eqn ½2Þ
½Rtot ½L þ Kd
which can be rearranged as follows
½L½Rtot
½LR ¼ ð5Þ
Kd þ ½L
Equation [5] is the equation of a rectangular hyper-
ax
bola y ¼ . An important consequence of this
bþx
equation representing a rectangular hyperbola is
that the horizontal asymptote is Rtot or Bmax. Thus,
Bmax can be obtained only at infinite concentrations
of L. This will be of importance when linear trans-
formations are discussed. Other important pieces of
information can be obtained from eqn [5]. The term
Kd is a measure of the affinity of the receptor for
ligand and is the concentration of ligand that occu-
pies one-half of the maximal binding sites. This can
be easily derived from eqn [5] if [LR] is replaced by
½Rtot
and solving for Kd (the result being Kd¼[L]).
2
The estimation of Bmax and Kd can be solved by either
linear transformation or by nonlinear regression,
which is described below.
2.03.3.4.2 Scatchard plots
The most common linear transformation of binding
data is the Scatchard plot. In this plot, the x-axis is
specific binding (usually labeled ‘bound’) and the
y-axis is the ratio of specific binding to concentration
of free radioligand (usually labeled ‘bound or free’).
Some of the terms used in Scatchard plots are as
follows:
B¼Bound (LR) Concentration of ligand in the incu-
bation that is specifically bound to
the receptor at equilibrium.
F¼Free (L) Concentration of free ligand present
in the incubation at equilibrium. As
discussed in Section 2.03.3.6, this is
often estimated by the concentration
of the drug added to the incubation.
Kd Equilibrium dissociation constant. In
the Scatchard plot, the slope of the
line is equal to Kd1. Kd is expressed
in the same units as the drug con-
centration (i.e., molarity).
Bmax (Rtot) Maximum number of binding sites
in the incubation at equilibrium, or
total receptor concentration. Bmax is
expressed in the same units as the
specific binding, usually as a
concentration relative to amount
of protein in the incubation (i.e.,
fmoles per mg protein).
The reason for plotting B/F as the y-axis and B as the
x-axis comes from rearranging eqn [5] (and substitut-
ing B for LR, F for L, and Bmax for Rtot):
ðF ÞðBmax Þ
B¼ becomes ðBÞðKd Þ þ ðBÞðF Þ ¼ ðF ÞðBmax Þ
Kd þ F
Dividing by F, and rearranging results in eqn [6] in
the form of y¼mxþb:
ðB=F ÞðKd Þ þ B ¼ Bmax
Bmax B
ðB=F Þ ¼ –
Kd Kd
Bmax B
ðB=F Þ ¼ –
Kd Kd
1 Bmax
ðB=F Þ ¼ – ðBÞ þ ð6Þ
Kd Kd
When making a Scatchard plot, you have to choose
units for the y-axis. One choice is to express both free
ligand and specific binding in counts per minute
(cpm), so the ratio of bound to free is a unitless
fraction. The advantage of this choice is that you
can interpret y-values as the fraction of radioligand
bound to receptors. If the highest y-value is large
(greater than 0.10), then the free concentration will
be substantially less than the added concentration of
radioligand, and the standard analyses are not appro-
priate. As such, one should either revise the
experimental protocol or use special analysis meth-
ods that deal with ligand depletion (see Section
2.03.3.6). The disadvantage is that the experimenter
cannot interpret the slope of the line without per-
forming unit conversions. An alternative is to express
the y-axis as fmol ligand bound per mg protein per
concentration (n mol l1). While these values are
hard to interpret, they simplify calculation of the Kd
which equals the reciprocal of the slope. The specific
binding units cancel when the slope is calculated.
The negative reciprocal of the slope is expressed in
units of concentration (n mol l1) which equals the Kd
(see Figure 5).
2.03.3.4.3 Double reciprocal plot
As the name implies, a double reciprocal plot is
simply the reciprocal of the amount of specifically
bound ligand versus the reciprocal of the free ligand
concentration. The y-intercept yields the reciprocal
of the value of maximal binding (1/Bmax) while the
 Receptor Theory and the Ligand–Macromolecule Complex 39

20 4
Slope = n
x-intercept = Kd when n = 1
3
15 n=2
n=1

Log(B/Bmax–B)
1 2
Slope = −Ka = −
B/F

10 Kd
1
n = 0.5
5
0

0 –1
0 20 40 60 80 100 120 0.1 1 10 100 1000
B Log[L]
x-intercept = Bmax
Figure 7 Hill plot. When the log (specific binding/
Figure 5 Scatchard analysis. The slope of B/F versus B is
(maximum specific binding–specific binding)) is plotted
equal to –1/Kd whereas the x-intercept estimates the total
versus the log [Ligand] a straight line with a slope equal to 1
binding sites (Bmax or LRmax).
is obtained, if the law of mass action is being followed. If
positive cooperativity is observed, the slope is >1; in the
case of negative cooperativity the slope is <1.
0.030

0.025 The difference between eqns [5] and [7] is the inclu-
sion of n or the Hill coefficient. The Hill coefficent is
0.020
the maximum number of ligand molecules bound to
each molecular receptor and is also a measure of
1/LR

0.015
cooperativity between binding sites. When a ligand
0.010
binds to a single class of noncooperative receptors,
1/Bmax
this binding is described by the law of mass action
0.005 and is n¼1. If n is greater than 1, evidence supports
–1/Kd positive cooperativity, while n less than 1 indicates
negative cooperativity or multiple classes of binding
–1.00 –0.75 –0.50 –0.25 0.00 0.25 0.50 0.75 1.00 sites.
1/L Equation [7] can be rearranged to give the follow-
Figure 6 Double reciprocal plot. When the inverse of ing linear equation:
specific binding (1/LR) is plotted versus the inverse of ligand
concentration (1/L), a straight line is obtained. The ½LR
y-intercept is 1/Bmax and the x-interecept in 1/Kd. Log ¼ nLog½L – nLogðKd Þ ð8Þ
½LRmax – ½LR

x-intercept represents the negative reciprocal of the Therefore, a plot of log[[LR]/([LR]max–[LR])] as a

equilibrium dissociation constant (1/Kd). Note that function of log[L] yields a line whose slope is n and
this is identical to the Lineweaver–Burke plot (see whose intercept on the ordinate is –nlogKD (see
Figure 6). Figure 7).
In addition to the Hill plot, the cooperativity
2.03.3.4.4 Hill plot between binding sites may be examined by
The Hill plot was originally used to describe the Scatchard analysis. In this case the plot is nonlinear
cooperativity of binding of oxygen to hemoglobin and concave when positively cooperative and con-
and has been adapted to examine ligand–receptor vex when negatively cooperative (see Figure 8).
interactions. The general equation from which the Both the Hill plot and the Scatchard may be used
derivation is based is as follows: to determine if the experiments are fulfilling the
criteria listed in Section 2.03.3.2, in particular if the
½Ln  ½LRmax binding sites are of a single population and inde-
½LR ¼ ð7Þ
Kd þ ½Ln pendent of each other.
40 Basic Principles
40
30 Positive cooperativity
Bound/free
20
10
Negative cooperativity
0 of free radioligand and is plotted on the x-axis. Bmax is
50 60 70 80 90
Bound
Figure 8 Nonlinear Scatchard plots. If the law of mass
action is being observed, a plot of B/F versus B will be a
straight line. However, in the case of cooperative binding,
the Scatchard plot is nonlinear.
2.03.3.4.5 Linear regression and analysis
error
While Scatchard, double reciprocal, and Hill plots
are useful plots for visualizing data, they may not
represent the most accurate way to analyze data.
Linear regression assumes that the data is normally
(Gaussian) distributed and that the standard devia-
tion is the same at every concentration of ligand. This
is not the case with data transformed in the Scatchard
plot. A second problem is that the value of X (bound)
is used to calculate Y (bound/free). Since the
assumptions of linear regression are violated, the
Bmax and Kd calculated by linear regression of
Scatchard transformed data are likely to be further
from their true values than the Bmax and Kd deter-
mined by nonlinear regression.
With many receptor-binding experiments, there
are insufficient data points throughout the binding
isotherm. This results in a clustering of data points
when the Scatchard analysis is performed. For
example, if there are few data points at the begin-
ning of the curve and many in the plateau region,
the Scatchard transformation will assign undo
weight to the data point collected at the lowest
concentration of radioligand (the upper left points
on the Scatchard plot). Although it is inappropriate
to analyze data by performing linear regression on a
Scatchard plot, it is often helpful to display data as a
Scatchard plot. As mentioned, visually interpreting
data with Scatchard plots can be of diagnostic value,
but it may not result in the most accurate values of
Kd and Bmax.
2.03.3.4.6 Fitting a curve to determine
B max and K d
Equilibrium-specific binding at a particular radioli-
gand concentration equals fractional occupancy
times the total receptor number (Bmax or Rtot):
½L  Bmax
Specific binding ¼ ½LR ¼ See Eqn ½5
Kd þ ½L
This equation describes a rectangular hyperbola or a
binding isotherm. As before, [L] is the concentration
100 the total number of receptors expressed in the same
units as the y-values (i.e., cpm, sites per cell, or fmol
per mg protein) and Kd is the equilibrium dissociation
constant (expressed in the same units as [L] usually
n mol l1). Typical values might be a Bmax of
10–1000fmol binding sites per milligram of protein
and a Kd between 10 p mol l–1 and 100 n mol l1.
To determine the Bmax and Kd, the most accurate
method is to fit the specific binding data to eqn [5]
using nonlinear regression. There are currently many
resources available for performing nonlinear regres-
sion and several statistics and plotting programs will
perform the analysis. An excellent source for a
detailed description of nonlinear regression may be
found at the GraphPad website.
2.03.3.4.7 Evaluation of binding data:
Have the assumptions been met?
In order for the binding data to result in physiologi-
cally relevant values of Kd and Bmax, the law of mass
action must be followed. Also, the choice of ligand,
separation procedures, and incubations must be
appropriate. When examining one’s own data, or
evaluating binding data within a manuscript, it is
advantageous to evaluate the quality of studies.
Here are some guidelines that may be used for eva-
luation of studies on receptor–ligand interactions.
Has the law of mass action been observed? In general,
there are benchmarks that can be used to assess
whether the law of mass action is being followed.
First, the Scatchard plot must be linear and the Hill
plot should have a slope equal to one. If a nonlinear
Scatchard is observed, more than one population of
receptor exists, cooperativity is an issue, and Kd is not
a constant. Second, ligand depletion should not occur
such that the amount of drug added to the incubation
is an appropriate approximation of free drug, or L.
The amount of binding (total binding) in the incuba-
tion should be less than 10% of the amount added.
See Section 2.03.3.6 for details on what to do if this
 Receptor Theory and the Ligand–Macromolecule Complex
value is exceeded. Third, the incubation must be at
steady state, or pseudo-equilibrium. To decrease the
time to steady state, one of the constituents should be
at considerably lower concentrations than the other.
Since the ligand is the constituent that is measured,
decreasing its concentration may affect sensitivity.
Therefore, the receptor concentration should be lim-
iting ([R]<<Kd; [R]<<[L]). Note, that this will also
minimize the risk of ligand depletion.
Is the binding physiologically relevant? The physiolo-
gical relevance often cannot be addressed without
conducting studies beyond receptor binding.
However, there are a few issues that can be exam-
ined. First, the formation of the ligand–receptor
complex must appear to be saturable. Note that
while 100% binding will never be achieved, com-
plete saturation should be approached. If a plateau is
not observed, either nonspecific binding has not been
properly accounted for, or a nonspecific interaction is
dominating the system. Second, the specificity of L
for R should parallel the physiological effects, if
known. That is, the Kd should be within an achievable
concentration at the receptor site. Examining a series
of chemicals and measuring their affinity for a parti-
cular receptor may also address physiological
relevance. Affinity and potency of effect should
have a similar rank order within this group of che-
micals. This may be easier competition binding
experiments, as described subsequently.
Have the experiments been properly designed and exe-
cuted? Several important issues regarding the design
and implementation of a ligand binding study was
discussed in Section 2.03.3.3. The following questions
should be asked when interpreting receptor charac-
terization studies.
1. Was the choice of radioligand and source of
receptor appropriate? In particular, the source of
receptor is often an issue. Bacterial and yeast
expression systems are used frequently to produce
soluble receptors. Often, these purification meth-
ods result in receptor preparations that
recapitulate the in vivo situation. However, if post-
transcriptional processing is required for receptor
function, these systems may not be appropriate.
2. Was the separation of bound and free drug per-
formed appropriately? In particular was the time
of separation adequate for the Kd of the receptor–
ligand interaction (see Table 1).
3. Was nonspecific binding measured correctly?
Remember the rule of thumb that the
41
concentration of competitor should be 100 times
the Kd of the drug–receptor complex.
2.03.3.5 Competitive Binding Experiments
2.03.3.5.1 Why use a competitive binding
curve?
Competitive binding experiments measure the bind-
ing of a single concentration of labeled ligand in the
presence of various concentrations of unlabeled
ligand. To quantify the potency of drugs in compet-
ing for the receptor, the IC50 (concentration that
inhibits 50% of the specific radioligand binding)
value is determined for each competitor.
Competitive binding experiments have several
advantages to direct binding assays and have been
used for the following types of experiments:
• Validate a direct binding assay. Competitive binding
experiments are an excellent way to examine the
physiological significance of a direct binding
assay. In this type of analysis the radioligand is
competed with drugs whose potencies are known
from functional experiments. Demonstrating that
these drugs bind with the expected potencies, or
at least the expected order of potency, helps
prove that the radioligand has identified the cor-
rect receptor.
• Determine whether a drug binds to the receptor.
Thousands of compounds can be screened to
identify drugs that bind to the receptor simply
by determining if they can effectively compete
with a known ligand. This can be faster and easier
than other screening methods. In fact, this is the
most common way the pharmaceutical companies
are identifying novel ligands for important recep-
tor systems.
• Investigate the interaction of low-affinity drugs with
receptors. Binding assays are only useful when the
radioligand has a high affinity (Kd<100 n mol l1).
A radioligand with low affinity generally has a fast
dissociation rate constant, and will not stay bound
to the receptor while you wash the filters or pellet
the complex.
2.03.3.5.2 Performing the experiment
The experiment is done with a single concentration
of radioligand, usually equal to the Kd concentration
of that ligand for the receptor as determined in pre-
vious studies. A higher concentration will increase
the sensitivity of the assay and decrease counting
42 Basic Principles

error but will also increase nonspecific binding and 120

time to equilibrium. 81-fold range
As with direct binding experiments, the incuba- 100
tion should reach equilibrium. However, the

Specific binding
80
reaction is more complicated in the presence of
an inhibitor, often at high concentrations. To 60
ensure that a steady state has been reached, the
incubation proceeds for 4–5 times the half-life of 40
the radioligand for receptor dissociation as deter-
20
mined in an off-rate experiment. In order to have a
complete profile of the competition, typically 0
12–24 concentrations of unlabeled compound span- 0.1 1 10 100 1000
ning about six orders of magnitude are examined. Log(Competitor)
Figure 10 Analyzing assumptions of the law of mass
2.03.3.5.3 Analyzing competitive binding action. The law of mass action predicts that the
data concentration of competitor that results in 90% and 10%
Visual inspection of competition of radioligand bind- inhibition cover a 81-fold (roughly two orders of magnitude)
range.
ing reveals a hyperbolic curve, with the most potent
ligands inhibiting specific binding at lower concen- binding curve is determined by the law of mass
tration than less potent chemicals (Figure 9). The action. As shown in Figure 10, the curve descends
plateau at the top of the curve, the radioligand bind- from 90% specific binding to 10% specific binding
ing in the absence of the competing unlabeled drug, with an 81-fold increase in the concentration of the
represents total binding. Note that total binding is unlabeled drug. More simply, nearly the entire curve
not the same as Bmax since the receptor is not fully will cover two log units (100-fold change in concen-
saturated (if using a Kd concentration of radioligand). tration). This generalization can be determined in
The bottom of the curve is a plateau equal to non- terms of [L], Kd, and the fractional saturation, Y.
specific binding (NS) with the difference between the
top and bottom plateaus being specific binding. The ½L
Y¼
IC50 is the concentration of unlabeled drug that Kd þ ½L
blocks half the specific binding.
If the labeled and unlabeled ligands compete for a The fractional occupancy at 90% and 10% satura-
single binding site, the steepness of the competitive tion will be
120 S0:9 S0:1
0:9 ¼ and 0:1 ¼
Kd þ S0:9 Kd þ S0:1
100 Total
Solving these equations simultaneously, and introdu-
Specific binding

80
cing the term cooperativity index (Rs), or the change
60 in drug concentration from 90% to 10%:
40 0:9
S0:9 0:1
Rs ¼ ¼ 0:1K ¼ 81
20 S0:1 0:9Kd
Nonspecific d
0
0.1 1 10 100 1000 When the interaction between competitor and
Log(Competitor) receptor does not proceed via the law of mass
Figure 9 Competition binding experiments. Total binding action, the value of Rs will not equal 81 (positive
of a single concentration radioligand is examined following cooperativity Rs<81; negative cooperativity
incubation with graded concentrations of competitor. Rs >81).
Shown are four competitors with different affinity for the
receptor. The plateau at the top of the curve represents total
Competitive binding curves are described by this
binding whereas the bottom plateau is nonspecific binding. equation:
The data may be analyzed using nonlinear regression to
Total – NS
Total – NS
calculate IC50 values. ½LRI ¼ NS þ . ½LRI ¼ NS þ ð9Þ
1 þ 10log½I – log IC50 1 þ 10log½I – log IC50
 Receptor Theory and the Ligand–Macromolecule Complex
where [LR]I (y-axis) is the amount of binding mea-
sured at each [I] (x-axis). Nonlinear regression to fit
your competitive binding curve to determine the
log(IC50). In order to determine the best-fit value of
IC50, the nonlinear regression problem must deter-
mine the 100% (total) and 0% (nonspecific) plateaus.
Alternatively, values of IC50 can also be ascer-
tained by logit-log plot (or pseudo-Hill).
½LRI
log ¼ n log½I þ n log IC50 ð10Þ
½LR – ½LRI
where [LR] is the amount of binding in the absence
of inhibitor and n is Hill coefficient. Plotting the log
([LR]I/[LR]–[LR]I) versus [I] will yield a straight
line with slope equal to n and an x-intercept equal to
IC50.
2.03.3.5.4 Calculating the K I from the IC 50
The IC50 value is not equivalent to Kd for the com-
petitor (or Ki) and is dependent on the amount of
radioligand in the incubation. Therefore, the IC50
value can vary between studies whereas Ki is a con-
stant. The Ki can be calculated from IC50 using the
Cheng and Prusoff method:
IC50
Ki ¼ ½L 
ð11Þ
1þ Kd
where Ki is the equilibrium dissociation constant for
the inhibitor and Kd is the equilibrium dissociation
constant for the radioligand L. Several assumptions
were made in the derivation of the Cheng and
Prusoff equation that must be met.
1. The radioligand and the inhibitor must interact
with the receptor according to the law of mass
action. That is, the binding of both chemicals
should be reversible and directed at a single popu-
lation of R. Whether the law of mass action has
been met can be determined from an indirect Hill
plot where the slope equals –1.
2. The concentration of L added should equal the
amount free. There should not be ligand depletion
([LR]<10%) and the concentration of receptor is
much less than Kd.
3. The incubation must have reached equilibrium
for radioligand and all concentrations of the
competitor.
2.03.3.6 Ligand Depletion
The equations that describe the law of mass action
include the variable [L] which is the free concentration
43
of ligand. All the analyses presented so far are based on
the assumption that a very small fraction of the ligand
binds to receptors (or to nonspecific sites), so you can
assume that the free concentration of ligand is approxi-
mately equal to the concentration added. In some
experimental situations, the receptors are present in
high concentration and have a high affinity for the
ligand, so that assumption is not valid. A large fraction
of the radioligand binds to receptors so the free con-
centration of radioligand is considerably lower than the
concentration added. If radioligand depletion occurs,
alternative analysis is required. For example, the free
concentration of ligand can be measured in every tube.
Alternatively, the free concentration in each tube is
calculated by subtracting the number of cpm of total
binding from the cpm of added ligand. One problem
with this approach is that experimental error in deter-
mining specific binding also affects the calculated value
of free ligand concentration.
2.03.4 Quantification of Receptor
Responses
2.03.4.1 Why Examine Receptors Based
on Responses?
The interaction between the receptor and ligand is
the first step in the elicitation of a biological response.
In fact, there are three components of a receptor
system: the ligand, the receptor, and the effector (E)
(see pathway below). The effector may be an
enzyme, an ion, or a transcription factor and it trans-
mits the biophysical interaction of ligand and
receptor into a biochemical or molecular signal.
Physiological receptors are linked to the signal trans-
duction apparatus of the cell. Ultimately, the dose–
response of a cell to a ligand is more complicated
than predicted by direct binding assays. While the
ultimate response is proportional to the amount of
ligand–receptor complex formed, direct proportion-
ality may not be observed. Therefore, it is often
difficult to determine Kd values for a ligand–receptor
interaction based solely on the response of that cell.
k1 k2
k3
! !
LþR  LR þ E  LRE! Effect
k–1 k–2
Beyond adding complexity, the effector system ampli-
fies the response. Therefore, detection of a receptor-
mediated event is often easier examined than direct
binding per se. This is the major reason why receptors
are often characterized by their responses prior to the
44 Basic Principles
implementation of direct binding assays. Sensitive
assays for detecting influx or efflux of Naþ, Kþ,
Ca2þ, or Cl, activation of adenylate cyclase, phos-
phorylation of a receptor or effectors, and alterations
in mRNA expression have been developed. In addi-
tion, binding of a chemical to a receptor says nothing
about the response elicited by the ligand. That is,
antagonists and agonists may bind with the same affi-
nity to a receptor (in fact antagonists are often more
avidly associated) but are coupled to the effector
molecular in a different manner. Therefore, receptor
responses are of more physiological relevance than
direct binding assays in isolation.
Prior to the advent of modern molecular biology,
which has allowed for cloning of receptors without
knowledge of their function or ligands, the existence
of a cognate receptor for a particular chemical was
based on cellular responses. Some of the criteria that
led investigators to believe that a given effect was in
fact receptor-mediated included:
1. Specificity of response. It is often the specificity of a
drug, hormone, or xenobiotic that persuades an
investigator that the effects are receptor-mediated.
Rank order of potency for effect of a series of
agonists should be characteristic of a particular
receptor.
2. Existence of antagonists. Antagonists can selectively
block receptor-mediated events. The order of
potency of antagonists should be characteristic of
a particular receptor.
3. Protection against irreversible receptor blockade. In the
presence of a ligand, the irreversible antagonist
cannot reach its site of action.
4. Cross-desensitization. If you can downregulate the
receptor, ligands will have less of an effect.
Transgenic knockouts can be viewed as the ulti-
mate desensitizer.
Examination of receptor responses offers a mechan-
ism by which the physiological relevance of a ligand–
receptor interaction can be confirmed. If the four
criteria listed above can be fulfilled, the receptor
system is considered to be properly characterized
and the chemical(s) under question can be purported
to elicit effects via a cognate protein.
2.03.4.2 Assumptions for Determining Kd
Values for Receptor–Ligand Interactions in
Intact Tissue Preparations
As mentioned above in the context of IC50 values
versus Ki, Kd values are more useful than are EC50
values. Both EC50 and IC50 are dependent on the
nuances of the incubation conditions whereas Kd is
only dependent on the receptor and its ligand.
However, there are many assumptions that must be
made in order to determine a Kd from a cellular
response.
1. The response of the tissue or cell should be due solely to the
interaction of an agonist with one type of receptor. As
such, the response on which Kd values are based
should not be a composite on effects of two or
more distinct receptor populations nor should it
be the result of nonspecific or nonreceptor-
mediated effects of the agonist.
2. The altered sensitivity to an agonist observed in the
presence of an antagonist should be due to competition
for a shared recognition site. This holds true for
pharmacological and toxicological antagonism of
the competitive and irreversible variety (see
Section 2.03.4.4).
3. The response obtained following addition of agonist
should be measured at a time when the maximal response
is elicited. Biological preparations especially suited
for determination of Kd values for receptor–ligand
interactions are those that maintain a maximal
response over a reasonable length of time.
4. When agonists or competitive antagonists are added to
the tissue incubation, the concentration of ligand free in
solution should be maintained at a known level. Tissue
uptake, degradation, and other losses must be pre-
vented or overcome by continual re-addition of
ligand.
5. The experimental design should contain proper controls
to permit measurement of, or correction for, any changes
in tissue sensitivity over time. Internalization of a
membrane-bound receptor, or proteolysis of solu-
ble receptors within the time frame of the
experiments may alter the sensitivity of the cell
to the ligand.
2.03.4.3 Full Agonists
The determination of a Kd for a full antagonist based
on responses is not as straightforward as Clark’s the-
ory of occupancy. Complicating factors such as the
existence of spare receptors and efficacy issues will
impact the calculations. Clark’s theory of occupancy
may also be used for examining responses, if examin-
ing a full agonist (efficacy e is 100%) and the receptor
requires full occupancy for maximal response. The
key feature of this formulation is that response is
proportional to the number of receptors occupied.
 Receptor Theory and the Ligand–Macromolecule Complex 45

k1 efficacy (e) of the drug–receptor complex. There

! ke
LþR  LR! Effect
k–1
are several ways to irreversibly inactivate a receptor,
and for many receptors these agents have been
Thus, if ke (the rate constant for coupling, or of the described. If an irreversible antagonist has not been
effector system) is known, for any given [LR] the characterized, chemical cross-linking may be uti-
extent of the response can be predicted. lized. In this instance, a highly reactive side chain
  such as an –azido group may be added to the ligand.
½L½LRmax
Response ð
Þ ¼ f ð½LRÞ ¼ f Upon binding to the receptor and activation with
Kd þ ½L
ultraviolet light, the ligand will covalently attach to
The maximal response
max will occur at [LR]max the protein. With peptide ligands, bifunctional

LR ½L reagents such as disuccinimidyl suberimidate may
¼ ¼ ð12Þ result in irreversible blockade. Nonspecific means

max LRmax Kd þ ½L
such as alkylation or proteolysis have been
As with receptor occupancy, the half-maximal employed. All means of irreversible inhibition must
response will occur at [L] equal to Kd. If it is assumed affect the receptor complex and not affect the effec-
that occupancy and response are proportional, the tor signaling pathways.
same graphical means to examine Kd used in receptor Substituting eqn [13] into that of [12] results in
binding may be employed. That is, a plot of fractional the following relationship:
response (
/
max) versus log[agonist] will result in  
a hyperbolic curve whereas log (
/(
max–
)) ver-
½L ½L
¼ fe ¼ f " LRmax  ð14Þ
sus log[agonist] represents a straight line.
max Kd þ ½L Kd þ ½L
However, as mentioned in Section 2.03.2, this If the receptor preparation is treated with an irrever-
simple relationship between occupancy and effect sible antagonist, the concentration of total active
does not adequately predict many biological receptors is reduced to a fraction (q) of the original
responses. In particular, dose–response relationships LRmax. Since efficacy is equal to "LRmax, in the pre-
to drugs and other chemicals tend to have a steeper sence of irreversible blockade, e¼q"LRmax, and the
slope than predicted and the existence of spare following equation is derived:
receptors shows that maximal responses do not  
require full occupancy.
9 ½L9 ½L9
¼ fe ¼ f " qLRmax  ð15Þ
Stephenson and Furchgott formalized a nonlinear
9max Kd þ ½L9 Kd þ ½L9
relationship between receptor occupancy and biolo- where
9,
9max, [L9] represent values after irrever-
gical response: sible blockade of the receptor. When comparing
LR equal responses before and after inhibition, for exam-
Response ¼ f ðSÞ ¼ fe
LRmax ple EC50 values, it is assumed that the stimulus
Intrinsic efficacy ð"Þ : e ¼ "LRmax ð13Þ applied to the tissue is the same (S¼S9).
LR ½L ½L9
Response ¼ f ð"LRmax x Þ ¼ f ð"½LRÞ "LRmax ¼ "qLRmax
LRmax Kd þ ½L Kd þ ½L9
eliminating "LRmax ð16Þ
where e is efficacy, " is intrinsic efficacy, and S is the
½L ½L9
stimulus applied to the tissue or cell. The introduc- ¼q
Kd þ ½L Kd þ ½L9
tion of the term " effectively divided efficacy (e) into
two components: a ligand-dependent (e) and a tissue- The advantage of comparing equal responses is that
dependent (LRmax) term. the unknown LRmax is eliminated and the function f
In order to use this relationship to examine Kd for relating occupancy to response is no longer required.
a ligand–receptor interaction, the experiments are Equation [16] can be rearranged to the linear
similar to those used to determine the spare receptor equation:
reserve (discussed in Section 2.03.2.4). Dose–
½L9 1 ½L9ð1 – qÞ
response data for a particular agonist before and ¼ þ ð17Þ
½L q Kd  q
after irreversible receptor blockade is obtained. In
effect, what these experiments are doing is changing A plot of [L9]/[L] versus [L9] results in a straight
LRmax in a preparation, and hence altering the line with a y-intercept equal to 1/q and a slope
46 Basic Principles
1.0
0.8 Control
0.6
Δ/Δmax
0.4
0.2
0.0
1 10 100
L1 L2 L1⬘ L3 L2⬘
[Agonist]
Figure 11 Determination of Kd for receptor–agonist interactions using irreversible receptor blockade.
ð1 – qÞ
equal to . The Kd value can be calculated as
Kd q
(y-intercept – 1)/slope (see Figure 11).
2.03.4.4 Quantitation of Pharmacologic/
Toxicologic Antagonism
The use of antagonists to block receptor responses
represents a classical approach in pharmacology and
a major mechanism by which chemicals cause toxi-
city. There are five basic types of antagonism:
1. Functional antagonism. This type of inhibition often
results from stimulation of two pathways with
opposite function (i.e., sympathetic versus para-
sympathetic nervous systems). Functional
antagonism results from action via two distinct
receptors and cannot be examined by the methods
described herein.
2. Competitive antagonism. A competitive antagonist is
binding to the receptor in roughly the same phy-
sical space as the agonist, precluding the latter
from interacting with the receptor to elicit a bio-
logical response. A competitive antagonist is
reversible, with a rate of dissociation that is rele-
vant to the time frame of the experiments.
3. Irreversible antagonism. As with the competitive
antagonist, the irreversible antagonist binds to
the same region of the receptor as the agonist.
However, in this instance, the rate of dissociation
is extremely slow or practically nonexistent.
4. Noncompetitive antagonism. Receptors are compli-
cated macromolecules that may interact with
small molecules in a variety of ways. A noncom-
petitive antagonist is defined as a chemical that
12
10
I1
8
I2
1−q
L/L⬘
slope =
6 Kd •q
I3 y − intercept
Kd =
4 slope
I4 1
2 y − intercept =
q
1000 0 200 400 600 800 1000
L3⬘
[L⬘]
binds to the receptor at a site other than the
agonist-binding pocket, yet binding precludes
the binding of the agonist. This is often caused
by the antagonist producing a conformational
change in the binding packet.
5. Mixed antagonism A combination of any of the
types of antagonism listed above.
As stated above, antagonism is an important mechan-
ism by which drugs and other chemicals exert
beneficial responses and how certain toxicants produce
their hazardous effects. Therefore, it is appropriate to
use the methods described below to characterize the
chemical and determine which type of inhibition is
occurring. Knowing the mechanism of action may aid
in designing more potent drugs or in ameliorating
toxicity. However, antagonists are useful tools to char-
acterize the receptor as well. This will be the focus of
the present section. To characterize a receptor using
antagonists, such interaction must fulfill certain
requirements:
1. Blockade must be selective for the family of ago-
nists in this receptor type.
2. Blockade of responses in different tissues by a
series of antagonists should result in a similar
rank order of potency.
3. Blockade should be sufficiently rapid to allow for
precise quantitative measurement.
4. An identical value for Ki in antagonizing different
agonists that act at the same receptor should be
obtained.
5. Values for the dissociation constants Ki derived from
studies assessing tissue response should be identical
to antagonist dissociation constants obtained in stu-
dies of binding to a particular type of receptor.
 Receptor Theory and the Ligand–Macromolecule Complex 47

2.03.4.4.1 Functional antagonism ½L

LR ½LR Kd
Functional antagonism is defined as antagonism of ¼ ¼ ¼ ð18Þ

max LRmax ½R þ ½LR þ ½IR 1 þ ½L þ ½I
tissue response that is unrelated to blockade at Kd KI

drug receptors but instead represents blockade of
tissue response at a site distal to receptors. This
precludes its precise quantitation using the meth-
ods described. It may affect second messengers or
depress cellular excitability and/or energy status.
Alternatively, it may produce an opposing action
through a different receptor.
2.03.4.4.2 Competitive antagonism
Competitive antagonists are ligands that compete
with agonists, usually for a common binding site in
a receptor. The interaction of agonist (L), competi-
tive antagonist (I) with receptor (R), is described
using the following scheme.
I with the same shape dose–response curve, only
þ K!
d Ke
Lþ  LR! Effect
R
Ki "#
IR
Note that the inhibitor–receptor complex is ineffec-
tual, and cannot couple occupancy with a biological
response. The formation of the ligand–receptor and
the inhibitor–receptor complex may be described by
the Clark equations and have equilibrium dissocia-
tion constants of Kd and Ki respectively. As a result of
IR formation, there are fewer receptors available for
LR formation.
This can be rearranged (divide by [L]/Kd):

1
¼   ð19Þ

max 1 þ Kd 1 þ ½I
L Ki
As you can see, the extent of antagonism depends on
the agonist and antagonist concentration, as well as
their dissociation constants, Kd and Ki. In the presence
of competitive inhibitor I, fractional occupation
[LR]/[LR]max decreases as a consequence of an
increase in the apparent equilibrium dissociation
constant of the agonist L. This decrease is from a
value of Kd in the absence of inhibitor to a value of KD
(1þ[I]/Ki) in the presence of I. The increase in
apparent dissociation constant yields an equation
shifted to the right with no effect on maximal
response (see Figure 12).
Since the competitive antagonist acts by excluding
the agonist from binding, it does not change the rela-
tionship between occupation and response. At equal
fractional occupancy, equal responses are observed.
When
/
max in the absence of competitor equals

9/
max of the presence of competitor I, then
½L ½L9
¼   ð20Þ
½L þ Kd ½L9 þ Kd 1 þ ½I
K i
Where L and L9 are the concentration required to
generate equivalent responses
/
max and

(a) (b)
Raw data Schild plot
1.0 10
No inhibitor
I = 50
I = 500
0.8 I = 1000
8
Log(x−1)
Δ/Δmax

0.6 6

0.4 4

0.2 2
−LogKI

0.0 0
0.1 1 L 10 L⬘ 100 1000 10000 0 2 4 6 8 10
Dose agonist −Log inhibitor

Figure 12 Determination of the equilibrium dissociation constant (KI) for a competitive antagonist. (a) The fractional response
was calculated using eqn [18] with a Kd of 10, KI of 10 and varying the inhibitor concentration from 0 to 1000. (b) Schild plot
with log(dose ratio1) versus concentration of inhibitor. The KI may be calculated from either the x- or y-intercept.
48 Basic Principles

9/
max in the absence and presence of I. This 2.03.4.4.3 Noncompetitive antagonism

equation can be simplified: A noncompetitive antagonist interacts with the
receptor, but this interaction takes place at a site
½L9 ½I
–1 ¼ different than that of the agonist. Upon binding to
½L Ki
the noncompetitive antagonist, the affinity of the
And by taking the logarithms of this equation and receptor for the agonist is altered, possible the result
substituting x for [L9]/[L], the Schild equation is of a conformational change in the protein structure.
derived: The interaction of agonist (L), noncompetitive
antagonist (I) with receptor (R), is described using
logðx – 1Þ ¼ log½I – log Ki ð21Þ
the following scheme.
Note that the Kd is removed from the equation and
I I
hence is not required to calculate Ki. The term dose þ Kd þ Ke
ratio (x) is applied to the expression [L]/[L9]. Dose L þ R $ LR ! Effect
ratios depend on the [I] and Ki. (This approach Ki "# Ki "#
assumes that the dose ratio is independent of the IR $ LIR
agonist used and that a single receptor type is being An antagonist of this sort produces quantitative
examined.) The determination of dose-ratios is iden- changes in the agonist dose–response curves: the max-
tical to that described above for full agonists (Section imal response is decreased while the EC50 of the
2.03.4.3), in which concentrations in the presence and
agonist does not change (Figure 14). The antagonist
absence of inhibitor result in equivalent responses.
does not alter the concentration of bound agonist
The Schild equation does not assume there is a linear
(LRþLRI) but the LRI complex is nonfunctional.
relationship between occupancy and effect. However,
The response of the agonist may be derived as follows:
the Lineweaver–Burke plot does. Therefore it is not
appropriate to calculate a KI, but is of diagnostic value.
ke ½LR ke ½LR
¼ ¼
As shown in Figure 13, double reciprocal plots in the
max ½RTotal ½R þ ½LR þ ½IR þ ½ILR
presence of increasing amounts of competitive inhibi- Which can be rearranged to : ð22Þ
tor intersect at 1/Bmax. Non-competitive inhibitors

ke ½L
will result in a plot with the intersection occurring at ¼  

max ð½L þ Kd Þ 1 þ ½I
–1/Kd, as discussed below. KI

Competitive Noncompetitive

100

80

60
1/Δ/Δmax

40

20

−0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 −0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75
1/[Ligand] 1/[Ligand]
Figure 13 Comparison of double reciprocal plots for competitive and noncompetitive antagonists.
 Receptor Theory and the Ligand–Macromolecule Complex 49

1.0 I=0
I=1
0.8

I=5
Δ/Δmax

0.6
KI = IC50
I = 10
0.4

0.2
I = 50
I = 100
0.0
0.1 1 10 100 1000 0.1 1 10 100 1000 10000
Log agonist Log antagonist
Figure 14 Determination of the equilibrium dissociation constant (KI) for a noncompetitive antagonist.

The response is decreased by a factor whose magni- 2.03.4.5 Partial Agonists

tude is increased by [I] and low values of Ki (i.e., high
A partial agonist has affinity, but less than maximal
affinity). For noncompetitive antagonists Ki¼IC50,
efficacy. The only way to determine the Kd for a
the concentration of antagonist that inhibits the
partial agonist (designated Kdp) is to compare it to
response by 50%.
a full agonist (Figure 15). However, many assump-
tions will be made to simplify the comparisons.
2.03.4.4.4 Irreversible and First, it is assumed that the concentration of full
pseudoirreversible antagonists agonist (A) resulting in a response is small relative
Some types of antagonists produce irreversible to Kda. We can therefore simplify eqn [2], for a full
antagonism of agonist-mediated responses. This is agonist to
most commonly observed when an antagonist pro-
duces a covalent modification in a receptor. ½A
YA ¼
Irreversible antagonists will yield curves that are Kda
identical to noncompetitive antagonist if the
response is proportional to the number of receptors For a partial agonist, eqn [2] cannot be simplified and
occupied. (However, remember spare receptors.) remains
Note that if the k1 is sufficiently slow, competitive ½P
antagonists may appear to be irreversible. Yp ¼
½P þ Kdp
Noncompetitive, irreversible, and pseudoirreversible
antagonism share a common property in that they are
nonsurmountable antagonists, that is, increased ago- To determine the Kdp of a partial agonist, a dose–
nist does not cause maximal response. response curve for the full agonist A and the partial
agonist P is obtained. As is the case in the previous
examples, comparisons of [A] and [P] that elicit equal
2.03.4.4.5 Mixed antagonism responses are used.
Mixed antagonism is observed when antagonists have
multiple types of interaction with receptor systems. S ¼ eA Ya
For example, the antagonist may initially block S ¼ eP Y p
receptors competitively but over time may become
At equal response
irreversible. Two other possibilities exist if the LRI
complex is able to produce a response or if the KI for ½A ½P
eA ¼ eP
R does not equal that of LR. Kda ½P þ Kdp
50 Basic Principles

1.0 12

0.8 Full agonist 10

slope
Kdp =
y − intercept

1/[Full agonist]
8
0.6
Δ/Δmax

Partial agonist
6
0.4

4
0.2
2
0.0
1 10 100 1000 0 200 400 600 800 1000
A1 A2 P3 P2
[Agonist] 1/[Partial agonist]
Figure 15 Determination of the equilibrium dissociation constant (Kdp) for a partial agonist.

which can be arranged to the following linear
equation
 
1 eA Kdp 1 eA
¼ þ
½A eP Kda ½P eP Kda
Thus, a plot of 1/[A] versus 1/[P] should be linear
eA Kdp
with the slope equal to and a y-intercept of
eP Kda
eA
. The Kdp can be determined by dividing the
eP Kda
slope by the y-intercept. It is important to stress that
an assumption was made regarding the Kda versus the
concentration of a full agonist that results in a
response. If this is not the case, the mathematics
become much more difficult (see Limbird 1996).
2.03.5 Conclusion
The quantification of the interaction between a
macromolecule (receptor) and a xenobiotic (ligand)
is called receptor theory and is often overlooked in
modern molecular toxicology. The proper use of
receptor theory can be used to examine structure–
activity relationships and modifications of chemicals
to fit the active site of the macromolecule. Also, the
basic tools of quantification and characterization of a
bimolecular interaction are applicable to many
disciplines including enzymology (Michaelis com-
plex between enzyme and substrate), immunology
(formation of antibody–antigen complex), pharma-
ð23Þ cology (drug–receptor complex), and toxicology
(xenobiotic–receptor complex). The shape of the
dose–response curve, the most important relation-
ship examined in toxicology, is dictated by receptor
theory. Therefore, by applying the equations
described herein, taking into account all assumptions
and limitations, the estimation of risk posed by che-
mical exposure may be more accurately determined.
References
Limbird, L. E. Cell Surface Receptors: A Short Course on Theory
and Methods, 2nd ed.; Kluwer Academic Publishers:
Boston, MA, 1996; pp 238.
Levitzki, A. Receptors: A Quantitative Approach, Benjamin/
Cummings Pub. Co.: Menlo Park, CA, 1984; pp xv, 142.
Taylor, P.; Insel, P. A. In Principles of Drug Action: The Basis of
Pharmacology; Pratt, W. B., Taylor, P., Goldstein, A., Ed.,
Churchill Livingstone: New York, 1990; pp 103–200.
Relevant Websites
http://www.graphpad.com – Introduction to nonlinear
regression, Copyright 1999 by GraphPad Software, Inc.