Biomolecular Ligand-Receptor Binding Studies: Theory, Practice, and Analysis

Charles R. Sanders,
Dept. of Biochemistry, Vanderbilt University
Table of Contents
Introduction
The simplest case: 1:1 stoichiometry
A short introduction to binding kinetics
The variables of binding studies
Relationship between thermodynamics and kinetics of binding
The attractiveness of study binding using pure ligand(s) and receptor
The model for 1:1 binding
Fitting a model to data
A little more on 1:1 binding
Alternatives to direct and Scatchard plots
Measuring concentrations in binding studies
What if the receptor/ligand system is more complicated than 1:1?
Multiple sites for a single ligand, Kd some for all and constant
When 1 ligand binds to multiple receptors
Multiple equivalent sites that are homocooperative
Heterocooperativity
Two ligands compete for the same binding site
Survey of methods used to monitor binding
Equilibrium dialysis
Spectroscopic methods
UV spectrophotometry
Fluorescence
NMR
Circular dichroism
Binding/Dissociation is slow on spectroscopic time scale
Binding/Dissociation is fast on spectroscopic time scale
Enzyme kinetics and dose/response pharmacology
Calorimetry
Chromatographic methods
Filter-based methods
Gel mobility shift assays
Ligand competition assays
Checklist of system variable to consider when planning binding studies
Checklist of experimental variables to consider when planning binding studies
Acid/Base equilibria
Strong acids and bases
Weak acids and bases
Buffers
Choice of a buffer
Buffer table
Buffers: a summary
Chelating agents
Chelating agent table
Concentration units when working with membrane proteins and/or
membrane associated ligands.

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One of the ways biological chemistry distinguishes itself from traditional chemistry is by the
degree to which biochemistry is based on NON-COVALENT and REVERSIBLE binding. This
is vital to life. For example, enzymes are the machines that catalyze the chemistry of
physiological processes. Enzymes reversibly and non-covalently bind their substrates as part
1

of the catalytic cycle. The same principle extends to the regulation of biochemical processes.
If it is said that a certain ligand directly "regulates" an ion channel, it is generally implied that
the molecule reversibly binds to the ion channel, causing it to shift towards open or closed
states. The entire immune system is based upon the body being able to produce "custom
non-covalent binders" for just about any molecule it wants to.
Binding can be also be exploited by humankind The exploitation of natural binding processes
lies at the heart of a number of basic techniques in molecular biology and biotechnology. For
example, various immunoassays, such as Western blots, are based on antibody binding to
signature molecules associated with various biological cells and processes. Endonucleases
are exquisite in their ability to distinguish particular nucleotide sequences from all other
sequences, leading to specific binding followed by cleavage, and are thereby standard tools
of molecular biology.
Drugs are typically molecules that are bound by biomolecules (usually proteins or nucleic
acids) with a high degree of affinity and specificity, leading to a medically relevant
physiological response. Most drugs interfere with or modulate a target protein through noncovalent binding.
In common lab practice, "affinity chromatography", such as metal ion affinity chromatography,
is based on attaching protein or ligand molecules to a gel matrix and then passing a mixture
through the column: only the molecules that have a strong binding affinity for the matrixbound molecule will stick to the column- everything else passes right on through. The now
pure molecule on the column is then displaced by passing a solution through the column that
competes with the matrix for the specially-bound molecule being purified.
It may be helpful to review some vocabulary with respect to biomolecular
association/dissociation (binding):
Ligand and Receptor: For any two non-identical molecules that associate it is possible refer
to one as the "Ligand" and to one as the "Receptor". Typically, the receptor will be a protein
and the ligand will be a smaller molecule, but not always. "Receptor" for the purposes of this
discussion is being used generically does not imply a biological function as a receptor (for
example, enzymes and transport proteins will be called "receptors" in this discourse). In fact,
one persons ligand may be another's receptor. Consider protein-DNA association. Which
is the ligand and which is the receptor? It really doesn't matter, the terms are basically
interchangeable. Usually, the receptor will be the larger of the two molecules. The reason
for adopting this terminology lies in the fact that in most binding studies, the "receptor"
concentration is usually held constant (or nearly so) and the ligand concentration is varied.
Molecular Recognition: Humans recognize objects and people using the 5 senses. In fact,
humans have extraordinary abilities to discern. Molecules are somewhat less sophisticated.
Molecules "recognize" one another when they come close enough to "feel" the presence of
each other- either by physically bumping into each other or through the interactions of the
fields (electrostatic) between each other. Usually we use "molecular recognition" in a positive
sense, meaning that molecules that are energetically compatible will associate.
Specificity: The "specificity" of a ligand for a receptor (or vice versa) is a description of how
favorable the binding of the ligand for the receptor is compared with its possible binding to
2

other types of receptors that may also be present. For a receptor, specificity describes how
much the receptor favors a particular ligand relative to the other ligands that may also be
present. In real biological systems the specificity of either ligands or receptors is rarely
100%- this is one of the reasons why drugs tend to have side effects. For example, it is well
known that most proteins that bind a given nucleotide (like ATP) are not completely specific
for ATP, but can bind a variety of ATP analogs like thio-ATP, AMPPCP, or even GTP.
Affinity: "Affinity" simply refers to how strong the binding is (as judged by Kassociation or Kdissociation and
Go). "High affinity" refers to very strong binding (large negative Go and a very small Kd). The
association or dissociation constant is often referred to as the "affinity or binding constant.
Stoichiometry: "Stoichiometry" refers to how many molecules of ligand can bind to a single receptor.
Cooperativity: "Cooperativity" (sometimes called synergism) refers to situations where the binding of
one (or more) molecules to the receptor enhances (or weakens) the binding of additional molecules to
that same receptor. Cooperative binding effects are also known as "allosteric effects".
Reversible vs. Irreversible Binding: All non-covalent binding processes are reversible, meaning that
the ligand can both bind to and dissociate from the receptor. Equilibrium is reached when the time
following mixing is long compared to the t1/2 binding and dissociation. However, sometimes noncovalent binding is so tight that the ligand does not dissociate for a very long period of time
(sometimes days). In such cases, the association is effectively irreversible and does not reach
equilibrium within the relevant time frame.
Kinetics: "Kinetics" is a rather generic term used to describe both the rates at which processes occurs
and the field associated with the study of rates. Binding and dissociation processes will be
characterized not only by the equilibrium constants, but also by how fast association/dissociation
occur.

The Simplest Case: 1:1 Stoichiometry

R + L
RL
R: receptor: could be enzyme, transporter, carrier protein, receptor, etc.
L: ligand: could be substrate, inhibitor, drug, metabolite,
hormone, DNA/RNA, another protein, etc.
It is equally valid to write the equilibrium constant in either of two ways:
[RL]

Kassociation = Ka =

[R]

Kdissociation = Kd =

[R]

[L]

Where the concentrations of

the free R, free L and the complex
are the concentrations at equilibrium

[L]

[RL]
Ka = 1/Kd

It is also easy to show that Goassociation = -Godissociation

A Short Introduction to Binding Kinetics

Before proceeding to a more detailed consideration of binding theory and analysis, it is
important to first understand basic kinetics, a short review of which is presented here.
Binding and other equilibrium constants are fundamentally related to the rates of interchange
between the states involved in the equilibrium process. "Rate" is, of course, a description of
how frequently something happens.
A unimolecular rate is how fast one molecule does something and will have units of "per
second" ( = sec-1 = Hz) or "per minute" ( = min-1). Unimolecular rates are sometimes referred
to as zero order rate constants where "zero" means that the rate is independent of any
concentration. One example of a zero order rate constant is the radioactive decay of a single
isotope (which is determined completely by the type of isotope, not by chemical
concentrations or compositions). Another example is the enzyme turnover number: kcat. This
rate constant tells the maximum rate that a single enzyme molecule can execute a chemical
reaction under conditions where it is saturated with substrate.
A "first order" reaction rate is a rate that describes a process that is dependent upon the
concentration of a single species. It will have units of
[concentration]/time

(e.g. mM product produced/minute) or quantity/time (e.g. micromoles product

produced/second). An example of a first order reaction would be the production of an
enzyme-product complex from an enzyme-substrate complex:
ES

EP

In this case the rate of EP production will be dependent upon two things:
i. How much ES is present (its concentration)
ii. A factor that describes how often ES will get
converted into EP under standard conditions
The factor mentioned above is the first order rate constant, "k". The rate will be defined:
rate of EP production = [EP]/t = k . [ES]
From this, it can be seen that the units of the first order reaction rate will be time-1. Thus, if
we define the rate as mMolar of EP produced per minute, the units of k will be min-1. In the
above example, if the assumption can be made that ES cannot be converted into anything
except for EP, it also would have been possible to define the rate of EP production as the
[ES]/time.
Another example of a first order process is the dissociation of a 1:1 receptor-ligand complex
to form free ligand and free receptor. While two separate species are produced, the rate at
which they are produced will be dependent upon a single concentration: that of the complex.
A "second order reaction" is a reaction whose rate is dependent upon the concentrations of 2
species. For example in the reaction
A

the reaction rate could be defined as either [C]/time or [D]/time, but the rate will be
dependent upon both [A] and [B]:
rate = [C or D]/time = k . [A] . [B]
where k is the second order rate constant. k is a factor that describes how often A and B
react under standard conditions. It can be seen that the units for a second order rate
constant must be concentration-1 . time-1. For example: per molar per second (M-1 . sec-).
An example of a second order process is the binding of a ligand (such as a hormone) to a
receptor (such as a GPCR) to form a 1:1 ligand-receptor complex. In this case, the rate is
dependent upon both concentrations: L and R can associate only if they bump into each
other and the probability that they will bump into each other is determined by their
concentrations.
The Variables of Binding Studies
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As we shall see, there is diversity in the classes of binding processes- a tremendous range of
possible stoichiometries, variable affinities, cooperativity, etc. There is also a remarkable
wealth of techniques for examining binding experimentally. We will therefore selectively
survey binding theory and techniques. Towards this end, a logical starting point is overview
the possible variables relevant to studies of biomolecular association.
Consider the simple case of the formation of a 1:1 complex:
R
where:

RL

[R] . [L]
Kd = 1/Ka = ------------------[RL]

(1)

From this equation it can be seen that Kdissociation and Kassociation for a given system can be
determined any time the concentrations of [R], [L], and [RL] are measured under equilibrium
conditions. This is the basis for one entire class of experimental methods to study binding.
The free energy in favor of binding (negative is favorable) is:
Go = R . T . lnKdissociation = -R . T . lnKassociation

(2)

Note that in addition to being useful as an equilibrium constant, Kd gives the free ligand
concentration at which the total populations of free and complexed receptors will be equal
(half maximal binding). This is an important fact to know.
Relationship Between Thermodynamics and Kinetics of Binding
The strength of binding is related to the "kinetics" of ligand-receptor association-dissociation:
how fast the ligand binds and how fast it dissociates.
As described in the previous section the rate of bimolecular processes is dependent upon the
concentrations of the species involved and a rate constant. For a generic 1:1 ligand/receptor
binding system:
L

LR

The forward rate is kon . [R] . [L] , while the reverse rate is koff . [RL]. kon is a second order rate
constant while koff is a first order rate constant. By definition, at equilibrium the rate of the
forward process equals that of the reverse process, meaning:
kon . [L] . [R] = koff . [LR]

(3)

where the concentrations are equilibrium concentrations. This expression can be rearranged:

koff

[L] . [R]
6

-------- =
kon

---------------[LR]

(4)

which is (of course) equal to the dissociation constant, Kd. From this derivation, it can be
inferred that another class of experimental binding study methods is based upon making
kinetic measurements (because if the on and off rate constants are determined, then Kd is
determined).
The rates are related to the time that the ligand spends in the free and bound environments.
For example, the "half-life" describing the average amount of time a ligand will spend as part
of a complex is:
t1/2 = 0.693/koff

(5)

This actually can tell us something important. The on/off times will influence the choice of
binding methods to study binding in a particular system (see below).
What determines kon? It is usually determined primarily by how fast (how often) the receptor
and ligand bump into each other. This is determined mostly by the rate of diffusion of the
ligand. For typical sized ligands, kon will fall roughly in the range of 106-108 per molar
receptor per second. This is very fast compared to the off rate under normal conditions.
What will the range of off-rates and "complex" dissociation half lives be? It is Kd-dependent
because Kd = koff/kon. So, if kon falls in the range of 106-108 M-.sec-, we can roughly estimate
the following:
Kd (M)

koff range (sec-)

t1/2 range_____

10-11

10-5 to 10-3

hours to days

10-9

10-3 to 0.1

seconds to hours

10-7

0.1 to 10

0.1 to 10 seconds

10-6

1 to 100

msec to 1 second

10-3

103 to 105

10 sec to 1 msec

The take-home message of this table is two-fold. First, when binding is very tight, complexes
can persist for a long time (hours). This has implications for design of binding experiments
(see below). Secondly, when a ligand and a receptor are mixed, it will take at least 5 times
t1/2 for equilibrium to be approached. In cases where binding is very strong, this means
waiting a very long time! If one can't wait long enough, then for all practical purposes the
binding is irreversible.
One caveat that should be mentioned is that there are certain classes of ligand and receptors
for which kon is lower than the diffusion rate limit. This is known as "slow binding" behavior
and is not rare. For a given Kd, a much slower on rate also means a much slower off rate
compared to the values given in the table. Slow binding behavior arises from factors that
7

cause a lowering of the probability of successful association every time the two molecules
bump into each other. Lowering the probability of productive contact means a lower
association rate. For example, if a protein can exists in two major conformations, one that
binds and one that doesn't, every time its ligand bumps into its inactive form, they will fail to
form a complex.
From this section, it can be seen that a measurement of Kd requires at least the
measurement of the concentrations of the free and bound species at equilibrium or
determination of the on and off rate constants.
The Attractiveness of Studying Binding Using Pure Ligand(s) and Receptor
One fact that should be apparent is that binding studies become much more straightforward if
one is working with pure ligand and receptor and under well-defined experimental conditions.
Whenever a system is studied in vivo (or in a biological extract containing numerous types of
molecules), the number of experimental variables becomes very high, to the point that it may
be difficult to obtain unambiguous results (see following sections).
The Model for 1:1 Binding
In the very simplest type of binding study involving a simple 1:1 association of ligand and
receptor to form a complex, a primary goal might be to determine the Kd.
The standard definition of Kd can be algebraically manipulated to yield the following equation:
[L]free
[RL]/[R]total = fraction of sites occupied = fR = --------------------Kd + [L]free
Ka . [L]free
= ---------------------1 + Ka . [L]free

(6)

(7)

where [L]free is the free ligand concentration. From this equation it is observed that if one
could measure the fraction of sites occupied as a function of [L]free, the data would map out a
curve that could be fit to yield a value for Kd.
Equations 6 and 7 predict hyperbolic (fractionR) vs [L]free plots (see below). Such plots are
sometimes referred to as "isotherms" (for relatively obscure thermodynamic reasons).

The binding isotherm equation for 1:1 binding can be plotted:

saturation

1.0

0.5

fR

half maximal saturation

free ligand concentration required

to achieve fR = 0.5 is equal to Kd
0
0

[L] (free ligand concentration)

Maximum % change in fR per unit [L] is in the 0-0.5 range

Important implications and considerations for 1:1 binding isotherm:
Remember, it is free [L] that is being plotted. However, since total ligand is often much
higher than total receptor, this means that the % ligand that forms a complex with R is often
going to be small. In this (very common) case [L]free is effectively equal to [L]total. This is
fortunate, because the total ligand concentration is often easily determined, but not the free
ligand concentration. THE ASSUMPTION THAT Lfree IS EFFECTIVELY EQUAL TO Ltotal
CAN OFTEN BE SAFELY MADE, BUT NOT ALWAYS. THIS ASSUMPTION SHOULD
ALWAYS BE SCRUTINIZED BEFORE BEING MADE.
To approach 100% saturation requires that [L] be many times higher than Kd. If [L] is a drug
there may be negative tradeoffs involved in going to very high drug concentrations in order to
achieve maximum efficacy.
When collecting data it is important to choose [L] concentrations both below Kd, at Kd and >>
Kd in order to get data from each part of the binding curve only with thorough
representation of all parts of the curve will it be possible to get a reliable fit of the model to the
data (that yields a reliable value of Kd) and to verify the applicability of the 1:1 model.
At [L] concentrations up through 1-2 Kd, fR is very sensitive to ligand concentration.
9

The isotherm equation (eq. 6) becomes equal to the Michaelis-Menten equation for enzyme
kinetics if fR is replaced with v/Vmax and Kd with Km. Km is sometimes equal to the true Kd, but
not always, depending on the enzyme/substrate.
Often, a dose-response may be quantitated in terms of the concentration of ligand required to
give 50% maximum effect. Such values are then reported as Ki, LD50, I50 or Kapparent. These
values MAY reflect a true Kd, but often do not because a 1:1 binding model may not be valid
or because measurements are not being made under equilibrium conditions.

Fitting a Model to Data

If binding is involves 1:1 model, then data should be fit by a hyperbola.
1.0

fR

[L] (free ligand concentration)

If model is not appropriate, then the model will not be well fit to the data.
1.0

fR

[L] (free ligand concentration)

Here, the data is clearly reflecting a sigmoidal shape. A 1:1 binding model does
not describe to the data adequately. A different model would be more appropriate.
10

A Little More On 1:1 Binding

The obvious problem of actually using eqs. (6) and (7) is the question of how to
experimentally determine the fraction of sites occupied, [RL]/[R]t at a given composition. This
parameter (fraction of sites filled, fR) is what most experimental binding study methods are
designed to yield (see below). For now, let us assume we can find the fraction. Then, how
can [L]free be determined? Sometimes it is measured directly (see following sections).
However, if [L]total is known in advance (it usually is), [L]free can be determined as follows:
[L]t = [L]free + [RL]
thus:

(8)

[L]free = [L]t - [RL]

and: since fraction of R complexed = fR = [RL]/[R]total and [RL] = [R]t . fR, then
[L]free = [L]t - fR . [R]total

(9)

Accordingly, [L]free can be determined if we know the fraction of sites occupied, [L]t, and [R]t.
What if [R]t is not known, as might be the case in studies involving a crude biological extract?
If the assumption can be made that [RL] << [L]t, then we can make the approximation that [L]
= [L]t. This is often the case in real systems. If not, then we may have to use equation (9)
but treat [R]t as an additional unknown constant that must determined during the analysis.
Based on the above, it can be seen that binding studies can generally be reduced to
measuring the fraction bound as a function of the free ligand concentration and possibly the
total receptor concentration.
It should be emphasized that there are other ways of rewriting equations 6 and 7. The
Scatchard equation is an example of a linear form:
1
fR
fR
---------------- = ------- - ------------[Ligand]
Kd
Kd

(10)

A Scatchard plot for data that satisfies the 1:1 binding model is shown below.

intercept = 1/Kd
slope = -1/Kd

fR/[L]

1.0
11

A variation of the Scatchard plot/equation given above is the closely related equation that
leads to a plot where [RL]/[L]free is the y variable and [RL] is the x variable.
If we wished, we could rewrite equations 6-7 so that the function is dependent upon the
fraction of total ligand that is complexed rather that the fraction of receptor:
fL = fraction of L complexed =
so:

[RL] = fL . [L]t

[RL]
----------[L]t

which can be substituted for [RL] in equation 6 so that:

fL . [L]t
-------------[R]t

[L]free
= ---------------------Kd + [L]free

which is easily simplified using algebra to:

Rt . [L]free
fL = --------------------------([L]t . (Kd + [L]free)
which could be rearranged into a number of linear forms (including Scatchard). From this
equation, it can be seen that Kd could also be determined by monitoring the fraction of [L] that
is complexed as a function of [L]free and [R]t.

Alternatives to "Direct" and Scatchard Plots

In addition to determining parameters using the direct (eqs. 6-7) and Scatchard-type plots
(equation 12) there are a number of other types of plot that are often encountered. For the
case a single ligand and a single receptor site here are some examples:
"Bjerrum" Plot:
-log[L]free = -logKd + log[(1-fR)/fR]

12

1.0

fR

0.5

log[L]

-logKd

free

As shown, the predicted curve is sigmoidal, with the point of inflection giving a simple read
out of -logKd. Such plots are often used for acid-base titrations because for such titrations logKd = pKa.
"Double Reciprocal" or "Benesi-Hildebrand" Equation/Plot::
1
----- =
fR

Kd
-------------[L]free

That this equation is similar to the Lineweaver-Burke plot, where fR corresponds to the
observed enzyme reaction rate, Kd corresponds to Km, [L]free corresponds to the substrate
concentration, and "Vmax" corresponds to the "maximum fraction bound" which is, of course,
simply 1.0 (for 1:1 stoichiometry).
slope = Kd

1/fR
y intercept = 1

1/[L]
Measuring Concentrations in Binding Studies
Ligand. This is often straightforward. The ligand is usually titrated into a solution containing
the receptor. The concentration of the stock solution is known either because a precise
amount of ligand was weighed out and dissolved in a known quantity of water or because of
its specific radioactivity, its UV absorbance, or some other method. Thus, based on the
13

known stock solution and the amount added to a known volume of receptor solution, the total
ligand concentration is easily calculated (it's just a dilution problem).
Receptor. In some, but not all cases, there are straightforward ways to determine the
concentration of a macromolecule:
Depending on the method for determining the fraction of receptor sites filled by ligand, it is
sometimes possible to determine the receptor concentration in the course of the actual
binding study. For example, if it is possible to measure the fraction of ligand that forms a
complex, it is easy to determine [L]free, such that the fraction ligand complexed (fL) versus
[L]free data can be fit to yield both Kd and [R]t.
Occasionally, the "receptor" can be weighed out (assuming it is pure and available in mg
quantities).
If R is pure and is a protein a couple of common protein assays (in cases where it is not
available in dried form or when there is not enough to directly weigh out) are:
1. Colorimetric assay (such as the Lowry and Bradford assays): a dye is added to the protein
solution that changes colors (its light absorbance properties) upon binding to the protein. The
amount of color change (as determined by UV spectrophotometry) is directly proportional to
the protein concentration.
2. UV absorbance: many proteins absorb light strongly at 280 nanometers, due to Tyr and
Trp. For proteins of known sequence, the extinction coefficient (how much light at a give
wavelength gets absorbed from a protein solution of a given concentration) can be calculated.
Based on this, one can simply measure A280 and then calculate the protein concentration
using Beer's law. UV absorbance at 259 nm is a standard way to measure the concentration
of nucleic acids (RNA/DNA).
If the receptor is not pure (or anywhere close), it can be difficult to directly determine the
concentration. A couple of less direct ways sometimes used are:
1. Measure the "activity" of the protein. For a pure enzyme that, when pure, catalyzes a
reaction with a specific activity of 1000 units/(min-mg), if a cell extract shows an activity of 1
unit/ml, then it can be estimated that there is 1 microgram of enzyme per ml of extract.
Another "activity" that might be used in quantitation would be (in the case of a cellular
membrane receptor) a measurable physiological outcome whose magnitude is directly
proportional to the amount of the receptor (does/response).
2. Affinity-based assay. A tagged antibody (or some other molecule) that specifically binds
only the receptor of interest is mixed with the receptor solution followed by quantitating how
much complex forms. For example, if the receptor is found on a cell surface it may be able to
take a gold-labeled antibody solution and incubate it with the cells. The cells can then be
washed to remove free labeled antibody and electron microscopy can be carried actually
visually count the number of gold "dots" appearing on the cell surface.
What if the Receptor/Ligand System is More Complicated than 1:1 Binding?
14

Many systems (e. g. proteins involved in regulation, signal transduction, etc.) involve
receptors that have more than one of binding site for one or more of ligands. Even when all
sites are for the same ligand, they may not all have identical affinities and their affinities may
vary depending on whether the other sites are filled or not. These complications of greaterthan-1:1 stoichiometry (for a single ligand), binding of more than one type of ligand, nonidentical sites, and/or cooperative sites make experiments harder to design and correctly
interpret. Let's try to sort out the adjustments that have to be made in such situations.

Multiple Sites for a Single Ligand, Kd is Same for All Sites Regardless of the Current Number
of L bound.
RL0 to n-1

RL1 to n

where n is the number of equal affinity binding sites on R for L.

In this case:
fractionR

[RL] + 2.[RL2] + 3.[RL3] +... n.[RLn]

= ---------------------------------------------------------[R]tot
n . [L]free
= ----------------------Kd + [L]free

(11)

(12)

which leads to a linear Scatchard equation:

fraction
---------- = n/Kd - fraction/Kd
[L]free

(13)

Note that as defined in eq. 13, the maximum "fraction" is not longer equal to 1.0, but is equal
to n. The shape of the direct fraction vs. [L] curve will be exactly the same as for a 1:1
complex (a hyperbola).

The Case Where There are Multiple Independent Sites for the Same Ligand, but they Have
Differing Affinities.
In this case the following equation holds:
fractionR

[RL] + 2.[RL2] + 3.[RL3] +... n.[RLn]

= --------------------------------------------------------[R]tot

15

[L]free
-------------------------[L]free + Kd,n

(14)

In the more general case where there are n types of sites (each with its own Kd) and m of
each type, the more general equation is:

m . n . [L]free
----------------------[L]free + Kd,n

(15)

A fraction vs. [L] binding curve will be the sum of hyperbola. By the naked eye, it may be
hard to distinguish the observed curve from a true hyperbola.
This equation can be converted into a Scatchard-type equation:
fraction
---------- =
[L]free
n

m/Kd,n
----------------------1 + [L]free/Kd,n
=

(16)

(m/Kd,n - fraction/Kd,n)

n
Now, however, the plot will no longer be linear but will exhibit a curve.
The Case where 1 Ligand Binds to Multiple Receptors
In the above case, it was implied that while there were multiple sites present, they were all on
the same receptor (and will have the same concentrations, assuming equivalent
stoichiometries).
What about the more general case where there is more than one receptor? In such a case,
the fraction of receptor sites bound will not usually be measured (since there will be multiple
receptors). Instead one would usually measure the total fraction of ligand bound (to all
receptors). In this case, the relevant equation is:
mi . [L]free
fraction of L bound = ------------------------n
Kd,i + [L]free

(16)

where n is the number of receptors and m represents the number of identical sites on the "ith"
(where i = 1, 2 ... n) receptor.
The Case Where there are m Equivalent Binding Sites That are Homocooperative
R+L

RL + L

RL2 + L

RL3 ....... RLn

16

Kd,1

Kd,2

Kd,3

Sometimes (quite frequently in biology) a receptor will have multiple binding sites for a ligand
that are initially equivalent. However, when a ligand binds to one site it causes a change in
the receptor (typically a conformational change) that causes the affinity of the remaining sites
for the ligand to either increase ("positive" cooperativity) or decrease ("negative
cooperativity"). Binding of a second molecule of the same ligand may induce yet another
perturbation and so forth. The exact model for such a situation is:
K1.Lf + 2.K1.K2.L2 + ... n.K1.K2.....Kn.Ln
fR = ------------------------------------------------------1 + K1.Lf + K1.K2.L2 + ... + K1.K2.....Kn.Ln

(18)

where the K above are association constants (remember, these = 1/Kd) and n is the total
number of cooperative sites.
Positively cooperative systems are easy to recognize in plots of fR vs. [L] because of the
characteristic sigmoidal shape of the curves. Consider the case of 1 ligand that binds to a
receptor having two positively cooperative sites, where K are the association constants.

1 ligand, 2 sites, homocooperative:

R+L

RL
K1
fR =

RLL
K2

K1[L] + K1K2[L]2
1 + K1[L] + K1K2[L]2

17

fR

[L] (free ligand concentration)

(Where fR varies from 0 to 1.0) The relatively simple two site case is the simplest route to a
sigmoidal binding curve, but there is an abundance of more complicated cooperative systems
that can also lead to sigmoidal isotherms.
Cooperativity also leads to curved Scatchard plots (see below; recall that an alternate form of
the Scatchard is to plot [L]bound/[L]free vs [L]bound).

18

postively
cooperative

Fraction
[L]free

negatively
cooperative

fraction of sites filled

We see that even for the very simplest case of homocooperativity, we get a very complicated
equation (above). In practice, such equations are rarely used (think how many unknowns
there will be to fit for!). What is sometime instead used is an all-purpose set of equations
based on the Hill equation, an empirical equation.
For a generalized cooperative process going on involving n different sites, the following
equation holds:
[L]free
fraction = --------------------Kd,app + [L]

(19)

In this case the maximum fraction of sites filled is always set to 1. This equation is known as
the Hill equation. The Kd of this equation is not the true Kd, but is instead apparent (which
makes sense, since in a cooperative process there will be multiple true Kd).
"" is the Hill coefficient and is the "index of cooperativity". When is determined to be equal
to the number of binding sites, then it means the receptor is "maximally cooperative"
(positive), meaning that once a single ligand binds, the affinity of the other sites for L become
very high and they will be immediately filled. If is 1 then there is no cooperativity. An
between 1 and its maximal value means that the degree of positive cooperativity is
somewhere in between. Negative cooperativity shows up as < 1 with 1/(number of binding
sites) being the lowest possible value (fully negatively cooperative).
19

The Hill equation can be rearranged into a linar equation:

fraction
log (------------------------) = . log[L] - logKd,app
1 - fraction

(20)

This equation leads to the "Hill Plot", which provides a convenient way of determining .
log{fR/(1-fR)} is plotted vs. log[L]. The slope is the Hill coefficient and the y intercept =
-LogKd,app.
The Case of Heterocooperativity
What if there two different types of ligands (L1 and L2), both of which bind to different
sites on the receptor, each with its own dissociation constant? If the sites are independent
then Kd,1 and Kd,2 will be constant regardless of whether the site for the other ligand is
occupied or not. However, what if the binding of one ligand to its own site perturbs the
subsequent binding of the other ligand to its own site?:
R

L1

KL1

RL1

L2

L2

KL2
RL2

KL2
+

L1

KL1

RL1L2

In this case, one would likely study the fraction of binding for one site as a function of the
concentration of ligand for that site and the concentration of the other ligand:
fraction of L1 sites filled = function([L1],[L2])
What is the appropriate equation? If there is 1 site each for L1 and L2 and the fraction of L1
sites complexed is monitored:
fR,L1 = [L1] / {[L1]+(KL1(1+[L2]/KL2)/(1+[L2](1-)/KL2)} (needs checked)

(21)

where = (1-KL1)/KL2, (all dissociation constants). The 4th dissociation constant KL1 does not
appear in the above equation because it can be expressed as a function of the other three
dissociation constants. The above equation is complicated!

20

The Case where Two Ligands Bind to the Same Binding Site (Competitively)
Even for the simplest case of a single receptor with a single binding site, there are several
variables that must be considered:
1. Are the two ligands structurally identical, except that one is radiolabeled and one is not?
2. Will the experiment be run such that one can determine the fraction of receptor sites filled
by each ligands, or only the total fractions of sites filled by both?
3. Will the fraction of L1 and/or L2 complexed be monitored (instead of the fraction of receptor
sites filled)?
4. Is Kd,1 or Kd,2 known ahead of time?
If the two ligands are structurally non-equivalent the fraction of R that is complexed with L1 is
given by:
fR = [L1] / ([L1]+KL1(1+[L2]/KL2))
(22)
where the K are dissociation constants and the concentrations are the free ligand
concentrations.
When one of the competing ligands is the "natural" ligand for the binding site, the other is
thought of as an "inhibitor", the "IC50" gives the inhibitor concentration at which the binding of
the "natural ligand" is inhibited by 50%. Unlike Kd, the "IC50" has no standard state, but
depends both on the Kds of both ligands, and also upon the actual solution conditions in
which it is measured. Hence IC50 tends to be rather subjective since it will be different (for a
given pair of ligands) under differing conditions. Nevertheless, IC50 is frequently encountered
in the literature. If Kd for the "inhibitor" is dubbed "Ki" and Kd for the "natural ligand" is simply
"Kd", the following relationship holds:
IC50 . Kd
Ki = ----------------[L] + Kd

(23)

If the two ligands are chemically the same, but one of them (L') is radiolabeled the fraction of
radiolabeled ligand that is bound will typically be measured as follows:
[L'R]/[R]total

[L']
= -------------------------Kd + [L] + [L']

(24)

where Kd is the dissociation constant (which is, of course, identical for both labeled and
unlabeled ligand).
(For more detailed theory of competition assays, see Lepre et al., Chem.
Rev. 104, 3641-3675 (2004)

21

A Survey of Methods Used to Determine the Fraction of Sites Filled or the Fraction of
Ligand Complexed.

Equilibrium Dialysis.
Equilibrium dialysis involves putting the receptor (if it is a big molecule) into a dialysis tube or
chamber that is separated from the surrounding reservoir solution by a semi-permeable
membrane that will not allow the receptor to escape, but that will allow the free ligand (which
must be much smaller) to freely diffuse into and out of the tube/chamber. The ligand is
added to the outside solution and the system is allowed to equilibrate. When equilibrium has
been reached (usually at least several hours) the total concentrations of ligand and (usually)
the receptor inside of the bag/chamber are measured. Also the total concentration of the
ligand in the outside reservoir solution is determined. This yields the following data:
[R]total, outside = 0
[L]total, outside = [L]free
[R]total, inside = [R]free + [RL]
[L]total, inside = [L]free + [RL]
[RL] is easily calculated from this data as [RL] = [L]inside-[L]outside and [R]free is determined:
[R]free = [R]inside - [RL].

chamber 1

chamber 2

Dialysis Membrane: Allows Ligand,

But Not Receptor to Freely Equilibrate
Between the Two Chambers

We see from this that it is possible to determine Kd from a single point since:
22

Kd =

[R] . [L]
------------[RL]

However, a single point determination cannot confirm that a simple 1:1 binding model actually
applies and also has a lot of error compared to a result obtained from many measurements.
Thus, one would normally vary the ligand concentration and make several measurements
and fit the data either to eq. 6 or a closely related equation (such as the Scatchard equation).
What if [L]in and [L]out can be measured but not [R]in? In this case a series of [L]free vs. fL data
points would have to be collected and the data fit by the appropriate model to yield both [R]t
and Kd.
The simplest type of equilibrium dialysis simply involves a dialysis bag in a beaker of solution.
However, many companies now sell special equilibrium dialysis systems where there are two
small chambers separated by a single layer of tubing.

Spectroscopic Methods
Often the ligand and/or receptor interacts with electromagnetic radiation (such as light) so
that they either perturb the radiation in a measurable manner or respond to the input radiation
by emitting their own characteristic signals. Examples of this include:
Ultraviolet Spectrophotometry: Light is passed through a solution and it is determined how
much gets absorbed at one or more specific wavelengths.
Fluorescence Spectrophotometry: Shine light of a certain wavelength through the molecule
and monitor, at one or more other wavelengths, light emitted by the molecule. If a range of
wavelengths is scanned for emission, the plot of intensity vs. emission wavelength is known
as an emission spectrum. Alternately, it is possible to vary the wavelength of the excitation
light and measure intensity at a single emission wavelength. The plot of intensity versus
excitation wavelength is known as an excitation plot.
Nuclear Magnetic Resonance Spectroscopy: In a strong magnetic field, the molecule is
pulsed with broad frequency radio waves and the amount of radiation absorbed as a function
of frequency is recorded. Each NMR-active atom will absorb to produce a signal at a discrete
frequency.
Circular Dichroism: Polarized light of a particular wavelength passes through solution
followed by detection the difference in absorbencies of the right and left-handed light (the
ellipticity, ). The ellipticity is plotted as a function of wavelength.
For each type of spectroscopy the general idea is that a given free ligand and/or
uncomplexed receptor has a characteristic "signal" or "signature" However, when a complex
is formed, the spectroscopic properties of the molecule(s) as part of the complex may be very
different than when free. In such cases, the changes in spectroscopic signals can be used to
determine the relative concentrations of free and bound ligand and/or receptor: data that can
be used to extract Kd.
23

For the moment let us assume that only the ligand (in free and complexed form) gives off a
signal while the receptor is "spectroscopically silent". In this case, when there is a mixture of
complexed and uncomplexed ligand, the observed spectra will fall into one of the following
categories:
Binding/Dissociation is Slow on the Spectroscopic Time Scale:
Signals from both free and bound receptors and/or ligands are directly observed (sometimes
getting stacked on top of each other). When the spectroscopic technique has a "shutter
speed" is rapid relative to the rate of exchange, the technique will be able to resolve
spectroscopic "pictures" of both the bound and free species, not a time average. In this case,
the relative intensities (integrals) of each signal will be proportional to the relative
concentrations (or "populations") of the molecule and the complex.
Signal from
R-L complex

Signal from
Free R

[L] = 0

Frequency or Wavelength

In this case, the relative intensity of the signal from the free ligand will be proportional to the
free ligand concentration. The relative intensity of the signal from the complexed ligand will
be proportional to the concentration of the bound ligand.
UV, fluorescence and CD will usually fall in this "rapid spectroscopy" or "fast shutter speed"
category. Obviously, the use of these techniques in binding studies requires that the spectra
from the free and complexed molecules not be identical.

Receptor-Ligand Association-Dissociation is Fast on the Spectroscopic Time Scale:

Some spectroscopic techniques have much slower "shutter speeds" so that they cannot
distinguish between free and bound species. In these cases the observed signal is the
24

population-weighted average of the two species (only a single average signal is observed).
This is often (but by no means only) the case for NMR spectra.
Signal from
R-L complex

Signal from
Free R

[L] = 0

Frequency or Wavelength
It is easy to see how the fraction of ligand or protein complexed can be determined from such
a series of spectra. The changes observed in the spectra are proportional to the amount
complexed vs. the amount free, which is easily converted to the fraction of ligand or receptor
complexed)
It must be re-emphasized that these spectroscopic methods only work if the "intrinsic" signals
from the free and complexed molecule are significantly different. This is not always the case.
Enzyme Kinetics and Dose-Response Pharmacology
We saw above that there is often a spectroscopic response to complex formation and
that such responses can be used to quantitate molecular association. Often, binding of a
ligand to a receptor brings about some physiologically-relevant response.
L

LR

physiological response directly

proportional to [LR] concentration

25

For example, substrate binding to an enzyme leads to a chemical reaction. By measuring the
ligand concentration dependence of the reaction rate we can sometimes determine the
binding constant. Consider the Michaelis-Menten equation:
Vmax . [S]
v = -----------------------Km + [S]

(25)

where is v the rate (the observed "response"), Vmax (the maximum possible response) is
observed only when all sites on the enzyme are filled with substrate, [S] is the free substrate
(ligand) concentration, and Km is the Michaelis constant (which is either equivalent or closely
related to Kd). It is easy to rearrange this equation to yield:
v
-----Vmax

[S]
-----------------------Km + [S]

This equation is very similar to equation 6, with v/Vmax being equal to the fraction complexed.
This make sense: v = Vmax only when the enzyme is fully saturated with substrate. "Fully
saturated" means all sites filled, which means fR,max = 1.0.
If the "response" to binding is not a chemical reaction, but a physiological process (such as a
change in blood pressure or death rate in a population) a similar equation holds (assuming
many assumptions are true):
degree of response
----------------------------max possible response

[L]free
---------------------------------Kd + [L]free

(26)

(Keep in mind that max possible response is observed only when all sites are filled).
Because this type of assay is frequently used to test potential drug molecules, such binding
studies are often called "dose-response" studies.
It should be noted that these equations only hold true in cases where the on/off rates are
rapid compared to the "response" rate. This is known as the "rapid equilibrium" assumption.
If this assumption is not valid (as is sometimes the case), then the above equations still hold,
except that Kd becomes an apparent equilibrium constant- not the true dissociation constant.
This apparent equilibrium constant is often referred to as the "IC50": the "inhibitor"
concentration at which 50% of maximal response is elicited.

Calorimetry
When a ligand binds to a receptor, heat will be generated (or absorbed). In calorimetry, a
receptor is titrated with a ligand in a calorimeter and heat production is measured at each
point in the titration. The heat generated is proportional to the amount of additional binding
that takes place at that titration point. Binding isotherms are generated by plotting heat
production vs. free [L] concentration. It is important to also account for heat that is produced
during titration by secondary processes that have nothing to do with the binding event of
26

interest, such as heat generated by mixing stock ligand solution with the receptor solution
and any non-specific binding of the ligand to the receptor or to other molecules/assemblies.

Chromatographic Binding Methods

There are at least two classes of such methods:
1. Gel chromatography-based methods. If one equilibrates a size exclusion chromatography
column with an eluting buffer containing a particular [L]t concentration, a constant
concentration of ligand will be flowing through and eluting from the column. Now, if a small
volume of large molecule receptor-containing solution is applied to the column, followed by a
resumption of elution with a constant [L] solution, the "pulse of receptor in the column will
immediately bind the ligand out of the nearby elution solution. Because the receptor (being a
large molecule) passes through the column more quickly than the small molecules in the
eluting buffer, it takes the bound ligand through more quickly, resulting in a surge of total
ligand concentration coming off of the column ( = bound + free ligand concentration) followed
by a droop in the ligand concentration (because of depletion by the faster moving receptor),
and then by a resumption of the normal ligand concentration. It is possible to determine the
binding constant from a plot of [L] eluted from the column vs. time, if total [R] is known.

[L] = [L]free

[R]

=0

total

ElutionTime After Adding R to Column

2. Affinity chromatography-based methods. In these methods a column is used with a
chromatography column resin coated with tightly bound ligand or receptor. A solution of the
receptor or ligand is applied to the column so that it gets stuck to the molecules on the resin.
Then, elution is carried out by applying a low to high concentration gradient of a second
molecule, whose affinity for the ligand or receptor is known. The concentration at which the
molecule of interest displaces the receptor or ligand from the column is directly related to the
dissociation constant. Note that this method requires that ligand/receptor complexes have
27

very long lifetimes: long on the time scale that it takes to run the column. Otherwise the
bound receptor or ligand would be washed off of the column even the absence of a
competitor. From the table above, it can be seen that Kd in the range of 100 nanomolar or
tighter are required for the lifetimes of the complexes to be long enough for this approach to
work.
Filter-Based Binding Assays
These methods are commonly used in pharmacology to detect binding of radioisotopically
labeled ligand to membrane receptors. Usually, isolated cell membranes (that have both the
receptor and many other membrane proteins), whole cells, or lysed cells are used. In other
words, these experiments typically involve looking at interactions between a small molecule
and a receptor that is part of a gigantic (filterable) superstructure.
The cells/membranes containing the receptor are mixed with the ligand. The mixture is then
filtered so that the cells/membrane containing the receptor and bound ligand are stuck to the
filter. The filter is then washed to remove any residual unbound ligand and then subjected to
scintillation counting to detect and quantitate the amount of bound ligand. An important
control experiment is to repeat measurement on cells/membranes that are identical except
that they have no receptor. By repeating measurements under conditions where a cold (nonlabeled) ligand is used to compete off the hot ligand, it is possible to get binding curves.
This is another example of a method that works well only if the complex between the receptor
and the ligand is long-lived relative to the time it takes to carry out filtration and then wash off
excess unbound ligand.
1. Load receptor
onto column
containing
resin-bound
ligand.

2. Run gradient
of free ligand
into column to
compete with
bound ligand
for receptor.

3. Record free
ligand concentration
at which receptor is
eluted from the
resin.

28

Gel Mobility-Shift Binding Assay

In polyacrylamide gel electrophoresis a polymer matrix is used, which is a network of
interconnected pores of fairly uniform size. The size of the pores is determined by the %
cross-linking of the polymer-- higher cross-linking means smaller pores. To a well in one edge
of a slab of such a polymer is loaded protein and/or DNA. An electric field is then applied
across the plane of the slab. The molecules in the sample well are then electrically dragged
into the gel by the current and will migrate through the gel. Other factors being equivalent,
large molecules tend to migrate slower because it is harder for them to get through the pores.
What if both DNA/RNA and a protein that binds to the nucleic acid is loaded into the sample
loading well of the gel? Once equilibrium has been reached, there will be both free and
complexed protein/nucleic acid populations in the well. When the current is turned on
everything is quickly pushed into the gel where the free nucleic acid or protein (whichever is
lightest) is quickly separated from the complex (because of very different molecular weights).
As the gel runs, very little of the complex dissociates because it is confined in the pores of the
gel.
Thus, after running the gel, the exact amount of free nucleic acid or protein in the original
sample (and sometimes the concentrations of the complexes) can be easily determined by
autoradiography, densitometry, or scintillation counting. By running several lanes in which
the amount of ligand is varied (while the receptor is held constant), it is possible to obtain the
ususal fractionR as a function of [L]free data, from which a dissociation constant can be
determined. Typically, the oligonucleotide serves as the "receptor" in these studies and is
radioactively labeled, while the concentration of the unlabeled protein ligand is varied from
lane to lane.

Binding of tRNAfMet to Trbp111. (A) Gel-motility

shift assay of Trbp111 binding to radioactively
labeled tRNAfMet. Trbp111 was incubated at
increasing concentrations with 12 nM [5'-32P]
Met
tRNAf and electrophoresed on a native
acrylamide gel at 4C. The top band denotes
formation of a complex. (B) Binding profile
obtained after determination of fraction of
Met
tRNAf bound to Trbp111 (squares) at various
concentrations. Data were simulated using the
curve corresponding to a simple-binding
equilibrium (solid line). The apparent
dissociation constant for tRNAfMet binding was
determined as 32 nM (dimer concentration).
(From: Morales et al., The EMBO Journal (1999)
18, 34753483.)

29

One limiting factor for these experiments seems to be the "dead time" it takes the DNA and
protein to enter at the beginning after turning the current on. If all of the sample does not
enter the gel very rapidly, changes in the concentrations in the loading wells can cause
problems. Since the time it takes all sample to enter into gel is on the order of seconds, the
technique may tend to be limited to fairly tight (or slow binding) where the t1/2,off is also on the
order of seconds or longer. This condition that is frequently met in the case of protein-nucleic
acid interactions, for which binding is often avid.
Ligand Competition Assays
Frequently, more than one type of ligand can compete for the same binding site. In
such cases the binding of "Ligand 1" to the receptor can be studied as a function of the
amount of "Ligand 2" bound to the receptor.
L1-R

L1 + R + L2

L2-R

We already covered the theory behind this class of experiments. There are two forms of
these experiments to be especially aware of are:
1. L1 is exactly the same as L2, except that one of them is radiolabeled. A typical study
involves forming a complex of labeled ligand with the receptor and then adding aliquots of
unlabeled ligand and watching the degree of displacement of the labeled ligand from the
complex.
2. The case where the dissociation constant of Ligand 1 is known in advance. This makes
determination of Kd for L2 very easy.
A Checklist of System Variables to Consider When Planning Binding Studies
Is the receptor pure? If it is not pure, are there other receptors or sites to which the ligand
could also bind? If so, then the total ligand concentration will be:
[L]t = [L]free + [L]bound to receptor + [L]bound to other molecules
This may be a serious complication if the third term is similar in size to either the free or
receptor-associated ligand concentrations. If the receptor is not pure, it may be difficult to
quantitate exactly how much of it is there- this can complicate analysis.
Is the ligand pure? If not, there will similar complications as for impure receptor. Can
"impurities" also bind to the receptor and compete for the same binding sites as the ligand of
interest? Do impurities make it hard to determine the ligand concentration?
Are the receptors and ligands stable during the time it takes to do a binding study? If not,
how does one deal with the fact that the total concentrations of ligand and/or receptor are
degrading during the course of an experiment?
Is receptor or ligand associated with membranes? micelles? If the receptor is membrane
bound then one may have to be concerned that the ligand may bind to the membrane as well
30

as directly to the receptor site. Is this taken into account by the model? If "reconstituted"
model membranes like liposomes or micelles are used, does the reconstitution procedure
perturb the binding properties of the receptor from its properties in the original native
membranes? If it is inserted into liposomes, is the "business end" of the receptor located on
the outer side of the bilayer? on the inside? on both sides?
If in vivo studies are being carried out, can the ligand physically make it to the receptor? Lots
of things can happen in a real physiological system to a ligand. For example, it may be
chemically degraded or altered by an enzyme. It may not be able to reach the target receptor
of interest because it cannot get into the cell or because, once in the cell, a transport protein
may kick it out again.
If the receptor is an enzyme, is the ligand a substrate? If so then it may be possible be able
to study binding using enzyme reaction kinetic methods, but not using equilibrium methods
(unless some enzyme cofactors or co-substrates are missing), because the ligand will be
converted into product.
Will the ligand bind metal ions or other small molecules present in the system? If so, how
tight is the binding? Will multiple types of metal-ligand complexes be present? Which forms
of these complexes will bind to the receptor and how does one sort out all of the possibilities?
If the ligand has ionizable moieties, what will be the ionic form under the conditions of the
experiments? Will multiple ionic forms be present? Which ionic forms of the ligand will bind
to the receptor and how will the possibilities be sorted out?
If one is studying a binding process in vitro, can results be extrapolated to the natural in vivo
process?
If binding is being monitored as a physiological response to the ligand, is it certain that the
response is the direct result of ligand-receptor association, or could it be an indirect
connection? Maybe the ligand activates a protein that stimulates a protein kinase that
phosphorylates a receptor that is then activated to bring about the physiological response of
interest. If so, is the response really proportional to the original extent of the association of
the ligand with receptor?
A Checklist of Experimental Variables to Consider in Planning Binding Studies
How tight is binding likely to be and will the chosen technique be well matched to determine
binding constants in the expected range? For example, NMR is good at measuring relatively
weak binding constants (Kd > 100 M), but not effective for tighter binding. Conversely,
techniques that rely on physically separating free ligand from bound ligand often will only
work if binding if very tight (long-lived complexes).
What is the time resolution of the chosen technique? In other words, does the chosen
method for quantitating the concentrations of free ligand/receptor and/or the complex
measure the time average concentrations of a system that is at equilibrium during the time it
takes to make a measurement, or can the chosen method be used in a time resolved manner
to follow the approach of a mixture of ligand and receptor to reach equilibrium. If
31

spectroscopy is being used to monitor binding, does the technique allow one to directly
observe and quantitate both free and complexed species, or does the technique give a signal
that reflects a population-weighted average between free and bound species?
Will binding be monitored under equilibrium conditions? If not, binding may best be
characterized using kinetic methods (measuring kon and koff), which may require very different
models for data interpretation.
Will the extent of binding be monitored directly or will it be monitored indirectly through a
process such as enzyme catalysis or a physiological event that is thought to be coupled to
the binding of the ligand and receptor of interest? In these cases, be aware of all of the
assumptions required by such methods if the interpretation of the data in terms of binding is
to be valid.
How direct is the relationship between the measured parameter in a binding study and the
degree of binding? (See below)
Can the total, free, and complexed concentrations of all of the relevant ligands and receptors
present in the system be directly quantitated? As noted earlier, if the answer is no, it may
complicate analysis.
Will the total ligand concentration be large (at all points in the experiment) compared to the
concentrations of the Ligand-receptor complex? If so, then data interpretation may be
simplified.
Can control experiments be designed that will eliminate all possible ambiguity in results?
Is the system stable enough and available in sufficient quanitity to take multiple points?
What is known about the system in advance? Is the stoichiometry known? Is it know
whether there is any cooperative binding? etc.
There are a lot of things to think through before (and after) embarking upon a binding study!
Acid-Base Equilibria
A "Bronsted acid" is a molecule that can give up a H+, while a "Bronsted base" can accept
one. In biochemistry this usually means giving up or accepting protons in aqueous (water)
solutions:
AH

H2O

A-

H3O+

When we refer to H+ as acid, we really mean H3O+. For the common definition of pH: pH =
log(1/[H+]), it is more accurgate to define: pH = log(1/[H3O+]). In this section H+ and H3O+ will
be used interchangeably.
In the above equilibrium, we there is a great fundamental paradox: "A" exists in two forms- in
one form it is clearly an acid and in the other form it is a base! This is true of most acids and
32

bases and for this reason molecular pairs represented by AH and A- are said to be related as
the "conjugate acid" and "conjugate base" of one another.
Water is kind of unique because when it dissociates it forms both acid and base:
H3O+

2 H2O

OH-

This is like any other equilibrium. For pure water:

[H3O+] [OH-]
[1 X 10-7] [1 X 10-7]
Keq = -------------------- = --------------------------------- = 3.3 X 10-18
[H2O]2
[55]2
Since [H2O] will always be 55 M, it is traditional to drop the bottom of this equation and
redefine Keq as "Kw", where:
Kw = [H3O+][OH-] = 1 X 10-14
From the above, we see that the concentrations of free H+ and OH- in neutral water are very
low. Water is a very "weak" acid and base.
This leads to a definition. The pH of an aqueous solution is defined as:
pH = -log[H+] = log(1/[H+])
while the pOH is defined as
pOH = -log[OH-] = log(1/[OH-]).
Thus, for pure water pH = pOH = 7.0.
pH and pOH are related:
pH + pOH = 14

(always)

Strong Acids and Bases

HCl in water dissociates in water, effectively to infinity:
HCl

H2O

H3O+

Cl-

From this equation, it is seen that Cl- is the "conjugate base" of HCl. However, in aqueous
solution, this equilibrium lies very far to the right because Cl- has virtually no tendency to pick
up a proton (if it did, NaCl would be a base!). Hence, while Cl- may technically be a
conjugate base, in practice it isn't. For this reason, HCl and certain other acids such as
H2SO4 and HNO3 are referred to as "strong" acids, meaning they can be assumed to totally
dissociate when dissolved in aqueous solution.
33

Acids and bases are usually supplied as highly concentrated aqueous solutions. The actual
molar concentrations can be obtained from various sources (such as a table that appears in
the Merck Index). Concentrated HCl is 11.6 M in HCl (it is prepared by bubbling HCl gas
through water until the water is saturated with the acid).
Just as there are strong acids, there are strong bases as well- most notably the alkali metal
hydroxides (NaOH, KOH). Be aware that while we think of ammonia (NH4OH) as a strong
base from a household standpoint, it is not a strong base from a chemical standpoint: it does
not fully dissociate into NH4+ and OH- in water.
When a certain amount of a strong acid or a strong base is added to water, it is very easy to
calculate the pH. Since the equilibrium constant is effectively infinity in favor of H+ or OHformation, the moles of strong acid or strong base added is equal to the moles of H+ or OHthat form in solution. For example, what if 1 ml of concentrated HCl is added to 1 liter of
water?
final [H+] = moles HCl added/liter final solution + initial conc. of H+ in water
= (0.001 liters conc. HCl . 11.6 moles HCl/liter conc. HCl
.
1/(1 liter final solution) + 1 X 10-7 M =
0.01160001 M = [H+]
So the pH is log(0.0116)-1 = 1.9

Weak Acids and Bases

From the above we saw that in the case of a strong acid or a strong base, the assumption is
made that dissociation in water is infinite. This is obviously not the case for water, nor for
other weak acids and bases. For example, a little acetic acid is added to water, the pH goes
down, but not nearly as much as for an equimolar amount of a strong acid: this is because
some protons remain attached to the parent acid. The amount by which the pH goes down is
determined by the degree of dissociation:
HOAc + H2O

H3O+ +

OAc-

The position of this equilibrium for HOAc dissolved in pure water is determined (big surprise)
by the dissociation constant. However, by convention, for acids and bases the H2O
concentration is not included in acid base equilibrium constants. Thus:
[H+] [OAc-]
Kd = -------------------[HOAc]
This dissociation constant is typically referred to as the acid dissociation constant (Ka, not to
be confused with the association constant). Similarly, if a conjugate base form of acetic acid,
sodium acetate, is dissolved in water, the acetate ion could accept a H+ from water:
34

OAc- + H2O

HOAc +

OH-

and the "base dissociation constant': could be determined:

[HOAc] [OH-]
Kb = ------------------[OAc-]
Kacid and Kbase for all conjugate acid/base pairs are related:
Ka . Kb = 1 . 10-14
So, acids with relatively large Kas are relatively strong acids (they tend to release their
protons into solution) and a similar relationship hold for Kbs and bases.
What all does Ka (or Kb) tell us? As we learned earlier, for any binding process the
dissociation constant provides (among other things) the ligand concentration at which the
"receptor" is half saturated by a "ligand" (half of the sites are filled). In this case H+ can be
thought of as "the ligand" and the conjugate base that gets protonated can be thought of as
"the receptor". Thus, if we know the Ka, we know the [H+] at which the "receptor" is 50%
protonated (half of the molecules will be in acid form, half in base form).
Secondly, if Ka (or Kb) is known for an acid and we throw some into water, the [H+]
concentration (and hence the pH of the solution) can easily be calculated.
Just as we don't talk about [H+] in a real lab situation (we use "pH"), we don't talk much about
Ka and Kb. Instead, we usually talk about pKa (equal to -logKa) or pKb (equal to
-logKb). This is useful, because the pKa will tell us the pH at which the molecule will be 50%
acid form, 50% base form. The significance of this becomes apparent when we talk about
buffers below. It should go without saying that the pKa and pKb for a conjugate acid/base pair
are related:
pKa + pKb = 14
What determines the pKa of a substance? One factor is the nature of the chemical moiety
and its covalent environment. For example, amines are usually bases- becoming protonated
in the pH 8-12 range, carboxylic acids are usually acidic having pKas in the pH 2-5 range.
Secondly, the local environment of the molecule can influence pKa. High salt concentrations
typically reduce the pKa (because the salt cations compete with protons for the lone pairs).
Low polarity environments can shift a pKa in either direction, favoring either the acid or base
form depending upon which is neutral.

35

Buffers
In the above section we learned that weak acids/bases exist in both conjugate forms in
aqueous solutions. Let's think about this a bit more. Acetic acid has a pKa of 4.8 (hence a Ka
of 1.4 X 10-5). What will the pH be if 10 ml of glacial acetic acid (17.4 M) are dissolved into a
liter of water?
pH = log(1/[H+])
[H+] = 1 X 10-7 (from neutral H2O) + the [H+] from acetic acid dissociation
[H+]from acetic acid = ???
[Acetic acid total] = 0.010 liters . 17.4 moles/l . 1 liter = 0.17 moles/liter = [acid form] +
[acetate ion]
So...
-5

Ka = 1.4 X 10 =

[x] . [x]
------------------[0.17 - x]

where [x] = [OAc-] = [H+]

This can be solved for x using the quadratic equation leading to [H+] = 0.0015 M and pH =
2.8. As would be expected if only the acid form is dissolved in water, the pH ends up below
the pKa for the acid.
For this 0.17 M acetic acid solution (where pH = 2.8), what happens if we now titrate in pure
OH- (in the form of a strong base like NaOH)? What is initially observed is that the pH
changes only slowly with the amount of added base, until at some point it shoots up:

What is going on here? Remember, the acid is part of an equilibrium:

36

A-

AH

H+

with the "position" of the equilibrium being determined by Ka. Now, when OH- is added (as
NaOH or KOH), the situation becomes more complex:
AH

A- +

H+

+ OH-

A- + H2O

(In the above, it is presumed that OH- does not directly steal AH's proton, but only combines
directly with H+. This is really not true, but for the purposes of this discussion it is an
acceptable working approximation.) As shown above, the second equilibrium lies almost
infinitely in favor of OH- combining with H+ to form H2O. Thus, after adding OH- overall
equilibrium is restored only when all of the added OH- has been neutralized by H+. What is
the effect of OH- addition upon the first of the two equilibria? Well since the original
equilibrium concentration of H+ gets soaked up (in part) by the added OH- more AH
dissociates to restore equilibrium. Thus, even though OH- has been added the ability of AH
to dissociate dictates that the equilibrium H+ concentration (and therefore pH) doesn't change
all that much. In this manner AH acts as a buffer of the pH and can continue to do so (as
more OH- is added) until the added OH- has depleted all of available protons (AH is all gone,
only A- remains) and the pH shoots up when there are no more AH left to donate a proton to
pair with the added OH-.
The above process illustrates how a weak acid or a weak base can act as a pH buffer. We
saw that as long as there was enough AH around, the addition of base really didn't change
the pH very much. This same thing would hold true if a strong acid is added to a solution of
A-. A- would "soak up" the added acid until it was completely converted to AH, at which point
further addition of H+ would cause the pH to plummet.
From this, we can see that weak acids and weak bases can be used to buffer solutions pHwise against dramatic variations in pH when small amounts of strong base or acid are added.
What if one needs to maintain a solution at a certain pH in a situation in which some strong
acid or strong base might be added? In this case, it would not be appropriate to have a
situation where the buffer was almost exclusively in either its acid or base forms. This is
because each will only buffer against added base or acid, respectively. However, what if the
solution contains a buffer that is already 50% acid/50% conjugate base at the pH one wishes
to work at? In this case, the buffer could soak up both strong acid and strong base.
How does one find a buffer that will be the desired 50/50 conjugate acid/base at a given pH?
This is easy. Find an acid/base whose pKa is very near to the working pH: when pH = pKa
the buffer molecule population will be 50% in acid form and 50% in base form.
Where can lists of buffer/pKas be found? Tables. (See Table below).
It should be noted that a number of compounds have more than 1 pKa because they can lose
more than 1 proton. For example, phosphoric acid:
H3PO4

Ka,1

H2PO41- + H+

Ka,2

HPO42- + H+

Ka,3

PO43- + H+
37

Choice of a Buffer
Calbiochem will send a nice booklet on buffers on request- see their catalog. Segel has an
exhaustive listing of buffers and pKas in its appendices.
The rule of thumb is that a buffer needs to have a pKa that is within 1 unit of the working pH if
the buffer is to be effective.
One should anticipate how much acid or base may be produced in the course of an
experiment and make sure the buffer is suitable to maintain the pH. On the other hand, it is
undesirable to use too high of a concentration (rarely > 0.1 M) because this can lead to
various artifacts. In calculating the expected pH change during a process, the "HendersonHasselbach" equation can be very helpful:
pH = pKa + log([A-]/[HA])
Zwitterionic buffers are preferred to anionic or cationic buffers. A zwitterion is a molecule that
has both positive and negative charges, but in which the net charge is zero. There are a
whole series of buffers known as the "Good" buffers that will be zwitterionic in at least one of
their conjugate forms. These are listed in the Sigma catalog and include HEPES, PIPES,
MOPS, etc.
HEPES (pKa = 7.55)
acid form (zwitterionic):
+
HO

base form:

H
HO

SO3

SO3
pH << 7.4

12 >> pH >> 7.4

The advantage of zwitterionic buffers over ionic buffers is that they are very non-reactive and
are less likely to produce experimental artifacts. For example, phosphate (negatively
charged) will bind to metal ions that can cause troubles in studies of the effect of metal ions
on biologically relevant processes. Zwitterions, with their net 0 charges have less affinity for
metal ions. The only problem with zwitterionic buffers is that they tend to be expensive.
Thus, ionic buffers such as Tris (positively charged) and phosphate remain popular,
especially for high volume procedures.
Tris buffers are not very compatible with standard Ag/AgCl pH electrodes (I think because
Tris has an unusual avidity for silver). Thus, calomel (mercury-based) electrodes are usually
used.
The pKa of buffers and the pH of their solutions is temperature dependent (see Table, as high
as 0.03 pH or pKa units per degree of deviation from the 25 values). This should be kept in
mind when performing studies on ice or in a warm water bath.
38

Borate buffers are not suitable for working with nucleic acids because it reacts with the diol of
the ribose moieties to form insoluble complexes.
Very high salt concentrations can make the pH meter readings unreliable- in such cases one
must dilute the solution by a factor of 10 or so and then measure the pH. Assuming the
solution is buffered, the pH will change little due to dilution.
Some buffers will specifically interact with the biomolecules of interest. For example, one
would not want to use phosphate buffer when studying phosphatases (enzymes that
hydrolyze phosphoesters).
When working with solutions containing detergents (as when working with membrane
proteins), buffers should be mixed and their pH should be adjusted BEFORE adding
detergent, as some detergent types interfere with pH measurements.
The following table is taken from Calbiochems booklet Buffers.

39

Acetic acid
Formic acid
CAPS
Phosphoric Acid

pKa = 4.75
pKa = 3.75
pKa = 10.4
pKa1= 2.1

Ammonia

pKa = 9.3

40

Buffers: A Summary
All buffers are weak acids/bases, meaning Ka is finite. For acetic acid in water:
H3C-COOH + H2O

Ka

H3O+

(acid form)

H3C-COO-

pKa = log(1/Ka)

(base form)

All weak acids have weak base conjugate forms and vice versa.
When the pH equals the pKa, the moiety will be 50% in its acid form and 50% in its base form.
When the pH is more than 1 unit lower than the pKa, then >90% will be in the acid form.
When the pH is more than 1 unit higher than the pKa then >90% will be in the base form.
Buffers are effective at controlling pH when:
(1)

the concentration is high enough to soak up

any acid or base produced in the solution

(2)

the target pH is within 0.5-1 units of the buffers pKa.

Chelating Agents
Often, it is desirable to work under conditions in which there are not divalent or trivalent
cations present in solution. This is very easy to do by including a certain amount of a
"chelating agent" in the buffer. Such agents bind to and thereby "tie up" all of the stray
divalent or trivalent metal ions present. These molecules are kind of molecular octopuses
that bind cations because of having several negatively charged carboxyl groups tethered
closely but flexibly together and that "gang up" to bind metal ions:
What should one know about chelating agents?
1. Carboxylates are weak bases (the conjugate bases of carboxylic acids). Thus, when
EDTA or EGTA are dissolved, they can perturb the pH of the solution (depending upon their
original ionic form).
2. Once one or more of the carboxylates becomes protonated, the affinity of the chelator for
metal ion is greatly reduced (because protons are successfully competing with the "ligand
binding sites"). Therefore, be aware that the lower the pH, the harder it will be for a chelator
to do its job.
3. It is important to have more moles of chelator in the solution than moles of
divalent/trivalent metal ions or the chelator will become saturated with free ions left over.
41

4. Chelators have different affinities for different ions. For example, EGTA is has an
especially high affinity for Ca2+, but may be unsuitable for other ions. EDTA is used for most
other divalent and trivalent cations.
5. The following Tables from Sigma/Aldrich product literature summarize the properties of
common chelating agents. The upper table presents metal ion binding affinity in the form of
logKassociation constants. The bottom table gives the pKa.

42

Concentration Units When Working with Membrane Proteins and/or Membrane

Associated Ligands.
When working studying the association of molecules that are both associated with detergent
micelles and/or with lipid vesicles, it is not appropriate to use bulk concentration units
(molarity) to express concentrations and as units for Kd. For membrane associated
molecules what matters is the concentration in the MEMBRANE.
Consider the case where you put 100 molecules of compound A into a single lipid vesicle
that it sitting in a 1 ml solution. In this case the molecules are much more likely to bump into
each other than in the case where there are 100 molecules of compound A distributed into 50
vesicles that are sitting in 1 ml of solution. While in both cases the bulk concentration is the
same (100 molecules/ml), the local concentration in the first case (100 molecules/vesicle) is
much higher than in the second case (2 molecules per vesicle).
What this means is that for meaningful thermodynamic measurements to be made regarding
molecular association in micelles or lipid vesicles, you have to use concentration units that
express the concentration of the molecules of interest within the membrane-mimicking phase.
Usually the units that are used are mole % units. If there are a total of 100 lipids in vesicles
for every molecule of protein X then the concentration of protein X is 1/101 X 100 = 1 mol%.
number of moles of solute A
Mole % of solute A in micelles or vesicles = ------------------------------------------------------------------------total moles of molecules in each micelle or vesicle

X 100

For example, if you have 22 mM DPC micelles that contain 20 mol% POPC (a lipid) and you
solubilize 1 mM of diacylglycerol kinase the mol% of diacylglycerol kinase would be:
molarity of DPC in micelles = 22 mM critical micelle concentration (which is 2 mM for DPC)
= 20 mM
1 mM DAGK
mol% DAGK = ------------------------------------------------------------------------20 mM (micellar DPC) + 5 mM (POPC) + 1 mM (DAGK)

X 100 = 3.8 mol%

For molecular association between two membrane-associated molecules, Kd will therefore be

expressed in mol% units.
Note that when working with micelles or mixed micelles you can assume that the mixing of
component molecules between micelles will be quite rapid on most experimental time scales.
However, for very hydrophobic molecules in lipid vesicles (liposomes) this is often not the
case. If you have two vesicles in solution, the rate at which lipids and integral membrane
proteins hop from one vesicle to the other may be very very slow. This must sometimes be
taken into account in experimental design and data analysis.

43