Complexation & Protein Binding

Complexation
• A complex is a species formed by the reversible or irreversible association
of two or more interacting molecules or ions.
• In the context of this course, it will be used to characterize the covalent or
noncovalent interactions between two or more compounds that are
capable of independent existence.
• Complexes have been usually referred to as coordination compounds.

(inorganic-inorganic) (organic-inorganic)
Co+3 + 6NH3=Co(NH3)6+3 Ca-tetracycline

(organic-organic)

Inclusion Type (organic-organic) Inclusion Type (organic-inorganic)

 Complexation
• Complexes, according to the classic definition, result from a donor-
acceptor mechanism or Lewis acid-base reaction between two or more
different chemical constituents.
• A Lewis acid is a molecule or ion that accepts an electron pair to form a
covalent bond. The acceptor, or constituent that accepts a share in the pair
of electrons, is frequently a metallic ion, although it can be a neutral
species.
• A Lewis base is a molecule or ion that donate a pair of unshared electrons
by which the base coordinates with the acid. Any nonmetallic atom or ion,
whether free or contained in a neutral molecule or in an ionic compound,
that can donate an electron pair may serve as the donor.
• Metal ions are electron acceptors so that are capable of binding donating
ligands. Examples include Co3+ , Ni2+, Ag+, Fe2+ and Cr3+.
• Examples of electron donors include :NH3, H2O:, CN:- and Cl:- can act as
ligands.
Coordination covalent bond
 Complex:
Different than staring materials!!
• Once complexation occurs, the physical and chemical properties of the complexing
species are altered (solubility, stability, partitioning, optical absorption/emission and
electrical properties)
• Complex formation usually alters the physical and chemical properties of the drug. For
examples:
(1) chelates of tetracycline with calcium are less water soluble and are poorly absorbed

Milk; antacid, iron-supplement..etc

 Complex:
Different than staring materials!!

• Tetracycline antibiotics are capable of acting as chelating agents and

binding a variety of polyvalent metal ions (Fe2+, Mg2+, Al3+, Bi3+ ).

• The complexation results in changes in both the drugs’ and the

metal ions’ physical and chemical properties.

• The complexation between tetracycline antibiotics and metal ions

either in food (cabbage) or in pharmaceutical preparations (iron
containing supplements) has been found to reduce both the solubility
and bioavailability of the antibiotics.
 Complex:
Different than staring materials!!
• Tetracyclines are contraindicated in pediatric patients since
they are prone to tetracycline complexation of calcium in
teeth and bones resulting in teeth discoloration and bone
growth problems.
 Complex:
Different than staring materials!!
(2) theophylline complexes with ethylenediamine
to form aminophylline , which is more water
soluble (for parenteral and rectal administration)
O O

N N
N H2C NH2 N H2C NH2
N +H C NH2
N
2 H2C NH2
O N O N

2 2
Theophylline Aminophylline
(Water-soluble)
 Complex:
Different than staring materials!!
(2) theophylline complexes with ethylenediamine
to form aminophylline , which is more water
soluble (for parenteral and rectal administration)
 Complex:
Different than staring materials!!
(3) cyclodextrins are used to form complexes with
many drugs to increase their water solubility.

Hydrophobic
drug

Hydrophilic
Hydrophobic
exterior interior
 Complex:
Different than staring materials!!
(4) Quantification of drugs using complexation
The chelating properties of procainamide (Sodium channel
blocker, Class IA antiarrhythmic) has been used as an assay for
its content in pharmaceutical preparations. Complex formation
with Cu2+ results in a colored compound that can be measured
by visible spectrophotometry. Thus calorimetric methods to
assay procainamide in injectable solutions is based on the
formation of a 1:1 complex of procainamide with cupric ion at
pH 4 to 4.5.

+ Cu(II) = Complex
 Classification of Complexes
Complexes may be divided broadly into two classes depending on whether the
acceptor component is a metallic ion or an organic molecule; these are
classified according to one possible arrangement in the following table. A third
class, the inclusion / occlusion compounds, involving the entrapment of one
compound in the molecular framework of another is also included in the table.

1. Metal Ion Complexes:

a. Inorganic Type
b. Chelates
2. Organic molecular Complexes
3. Inclusion/occlusion Complexes

a. channel lattice type

b. layer type
c. clathrates
Metal Ion Complexes
 Classification of Complexes
Intermolecular forces involved in the formation
of complexes are the
• Coordinate covalence is important in metal
complexes
• van der Waals forces of dispersion, charge
transfer and hydrophobic interaction may also
contribute, dipolar and induced dipolar types
and hydrogen bonding provide a significant
force in some molecular complexes
 Metal Complexes: Inorganic Complexes
• Example:
[Co(NH3)6]3+ .3Cl-
– The ammonia molecules in the hexamineocobalt III chloride is
called the ligand.
– The ligands are coordinated to the cobalt ion.
– The coordination number of the cobalt ion is the number of
ammonia molecules coordinated to the ion (6).
– Each ligand donates a pair of electrons to form a coordinate
covalent link between itself and the central ion.
Co3+ + 6(:NH3) = [Co(NH3)6]3+
Metal Complexes: Chelates
– Chelates are complexes that typically
involve a ring-like structure formed by the
interaction between a partial ring and a
metal.
– In chelates, ligands are usually organic
molecules, known as chelating agents,
chelators, chelants or sequestering agents.
– When a ligand provides one group for
attachment to the central ion, then its called
monodentate. Oxaliplatin, Bi-dentate
– Molecules with two or three groups are
called bidentate and tridentate respectively
(multidentate or polydentate).
– If a metal ion binds to two or more sites
on a multidentate ligand, a cyclic
complex is formed; this cyclic complex is
known as a chelate.
 EDTA( hexadentate)

+M

Gadolinium chelate
octadentate
 Metal Complexes: Chelates
• Some of the bonds in a chelate may be ionic or of the
primary covalent type, while others are coordinate
covalent links.

• The formation of chelate complexes is controlled by

stringent steric requirements on both the metal ion and
the ligand. Only cis-coordinated ligands – ligands adjacent
on a molecule – will be readily replaced by reaction with a
chelating agent.
 Metal Complexes: Natural Chelates!!
• Many biologically important molecules (e.g. hemoglobin,
chlorophyll, insulin, cyanocobalamine), are natural chelates. Porphyrin
ring

square
planar

• Other biological chelates include albumin, the most common plasma

protein which acts as a carrier of various metal ions (Cu2+ and Ni2+)
and small molecules in the blood.
Metal Complexes: Vitamin B12, Cyanocobalamin
Metal Complexes: EDTA
• EDTA is a synthetic chelating agent used to sequester ions (iron and
copper) that catalyzes oxidative degradation reactions in drug
preparation.
• EDTA is also widely used to sequester and remove calcium ions from
hard water.
• treating mercury and lead poisoning; remove excess iron from the body.

EDTA( hexadentate)

+M

EDTA: Ethylenediaminetetraacetic acid

 2.4. Organic Molecular Complexes:

• An organic molecular complex is made of constituents held together

by weak forces of:
1) Hydrogen bonds

energy of attraction is less

than 5kcal/mol. the distance
between these components
is usually greater than 5 A.
Weaker than coordination or
covalent or chelates..
 2.4. Organic Molecular Complexes:

• An organic molecular complex is made of constituents held together

by weak forces of:
2) dipole-dipole

Induced Dipole Dipole

For example, the polar nitro

groups of trinitrobenzene
induce a dipole in the readily
polarizable benzene molecule
and the electrostatic
interaction that results leads
to complex formation.
 2.4. Organic Molecular Complexes:

• An organic molecular complex is made of constituents held together

by weak forces of:
2) dipole-dipole

Dipole Dipole

O O

O O
N N N N
O O

O N O N

N N

N N
H H H H

Benzocaine Caffeine
 2.4. Organic Molecular Complexes:
• The incompatibility of certain polymers used in suspensions,
emulsions, ointments and suppositories and certain drugs
may be due to the formation of organic molecular complexes.
The incompatibility may result in precipitate, flocculate,
delayed biological absorption, loss of preservative action, or
other undesirable physical, chemical, and pharmacologic
effect.
 2.5. Inclusion Compounds:
• This class of complexes differ from the previously discussed classes in that
they are mainly the result of the architecture of the molecules rather
than their chemical affinity.
• In this class of complexes, one of the constituents of the complex is
trapped in the open lattice or cage like structure of the other to yield a
stable arrangement.
• Some times they are referred to as occlusion compounds.

A) Channel Lattice Type B) Layer Type

starch and iodine
Bentonite
Inorganic silicates(Al-slicate)
 2.5. Inclusion Compounds:
• This class of complexes differ from the previously discussed classes in that
they are mainly the result of the architecture of the molecules rather
than their chemical affinity.
• In this class of complexes, one of the constituents of the complex is
trapped in the open lattice or cage like structure of the other to yield a
stable arrangement.
• Some times they are referred to as occlusion compounds.

C) Clathrates D) Monomolecular inclusion Compounds

Co-Crystallization
Hydrophobic+
+drug+

Hydrophilic++ Hydrophobic+
exterior+ +interior+
 2.5.4. Monomolecular inclusion Compounds:
• In this class of inclusion compounds, a single guest molecule is entrapped
in the cavity of one host molecule.
• A representative example of such compounds is cyclodextrins.
• Cyclodextrins are cyclic oligosaccharides containing a minimum of six D (+)
glucopyranose units attached by an a-1,4 linkage.
• Cyclodextrins are produced from starch by the action of bacterial amylase.
• The naturally occurring a-CD, b-CD and g-CD contain 6, 7 and 8 units of
glucose respectively.
 2.5.4. Monomolecular inclusion Compounds:
• The interior of the CD cavity is usually hydrophobic because of the
CH2 groups, while the exterior of the cavity is hydrophilic because of
the presence of the hydroxyl groups.

• Complexation with CD does not ordinarily involve the formation of

covalent bonds. Molecules of appropriate size and stereochemistry
can be included in the cyclodextrin cavity by hydrophobic
interaction.

• The aqueous solubility of many lipophilic drugs is improved by

complexation with CD and CD derivatives.
 2.5.4. Monomolecular inclusion Compounds:
• The bioavailability of many of these drugs has been improved as
well.

• CD has been used to improve the organoleptic characteristics (bitter

taste) of oral liquid formulations.

• Hydrophobic CD derivatives has been used as sustained release

drug carriers. Ethylated b-CD has been used to reduce the release
rate of the water soluble drug diltiazem.
 2.6. Methods of Analysis:

• Different applications of complexes in pharmaceutical

sciences require a quantitative knowledge of the
complexation process and product.

– The Stoichiometric Ratio of ligand to metal or donor to

acceptor.
– The Stability Constant of the formed complex.
 2.6.1. Distribution Method:

• Distribution of solute between two

phases can be used to calculate the
stability constant of complexes.
• This depends on the fact that the
distribution coefficient applies only to
the species common to both phases.
Water
• Example: The complexation of iodine (I2) [I2]w
K
[I-]w + [I-3]w
with potassium iodide (I-)can be
represented by the following Ko/w
equilibrium: [I2]o
I2 + I- = I3- Oil
CS2

[I −3 ]w
K= −
[I ]w *[I 2 ]w
Iodine molecule (insoluble)+iodide ion (soluble) =tri-iodide ion (soluble)
 2.6.1. Distribution Method:

• Determine Ko/w
• Add KI to the aqueous layer with known amount Initial [I-]
• Determine [Free iodine in oil] =[I2]o
• Calculate [Free iodine in water] =[I2]w
• Determine [TOTAL iodine in water(free and complexed)] ] =[I2]w + [I-3]w
• Calculate [complexed iodine in water] = [I-3]w= {[I2]w + [I-3]w}-[I2]w
• Calculate [Free iodide in water] = [I-]w=Initial [I-]-consumed [I-]=Initial [I-]-[I-3]w
• Calculate stability constant

Water
K
[I-]w + [I2]w [I-3]w

• Ko/w Ko/w
• Total [I-]
• [I2]o
[I2]o
• [I2]w + [I-3]w Oil
CCl4
 2.6.1. Distribution Method:

- Ko/w 1) From Ko/w and [I2]o, calculate [I2]w

- Initial [I-] 2) From ([I2]w + [I-3]w) and [I2]w, calculate [I-3]w
- [I2]o 3) complexed [I-3]w = complexed [I-]w (I2 in excess)
- Total I2 in water [I2]w+[I-3]w 4) From Initial [I-] and complexed [I-]w,
calculate Free [I-]w =Initial[I-] - complexed [I-]w
Analysis only see
iodine (free and
Water complexed!!)
[I 2 ]o K
K o/w = [I-]w + [I2]w [I-3]w
[I 2 ]w
[I −3 ]w Ko/w
K= −
[I ]w *[I 2 ]w [I2]o
Oil
CCl4
 Example 10-2: Martin’s 6th ed.
Iodine is distributed between
water and chloroform
(Ko/w=625). When iodine is Total I2=
distributed between 0.125 M Water 0.02832M
solution of potassium iodide Initially =
and chloroform, the 0.125 M
K
concentration of iodine in [I-]w + [I2]w [I-3]w
organic phase was found to be Ko/w=625
0.1896 M. When the aqueous [I2]o=0.1896M
phase was analyzed, the Oil
concentration of iodine was CCl4
found to be 0.02832M.
Calculate the stability constant
of the complexation reaction
between iodide and iodine.
 Example 10-2: Martin’s 6th ed.
- Ko/w=652
- Initial [I-]=0.125
- [I2]o=0.1896
- Total I2 in water [I2]w+[I-3]w=0.02832
1) From Ko/w and [I2]o, calculate [I2]w
[I 2 ]o
K o/w = 3) complexed [I-3]w = complexed [I-]w (I2 in excess)
[I 2 ]w
4) From Initial [I-] and complexed [I-]w,
0.1896 calculate Free [I-]w =Initial [I-] - complexed [I-]w
625 =
[I 2 ]w
Complexed[I − ]w = [I −3 ]w = 0.02802
[I 2 ]w, free = 0.000303M
[I − ]w = initial[I − ]w − Complexed[I − ]w
2) From ([I2]w + [I-3]w) and [I2]w,
[I − ]w = 0.125− 0.02802 = 0.09698M
calculate [I-3]w
Finally: Plug into the equation!!
[I 2 ]w,complexed = [I 2 ]w,total − [I 2 ]w, free [I −3 ]w
K= −
[I ]w *[I 2 ]w
[I −3 ]w = 0.02832 − 0.000303
0.02802
−
[I ] = 0.02802 K=
3 w 0.09698* 0.000303
K = 954
 2.6.2. Solubility Method:

Solutions of the Excess solid

complexing agent (Drug) In
in various stoppered
concentrations containers

Bottles are agitated in a

constant temperature bath
until equilibrium is attained

Aliquot portions of the

supernatant liquid are
removed and analyzed
 Point B 2.6.2. Solubility Method:
Saturation of the Drug-Ligand
(solubility limit)

Point C
Molar Concentration of the Drug

All excess solid

Drug converted to
(free of in a complex)

Drug-Ligand
complex

Point A
Solubility of
the Drug

Molar Concentration of the Ligand

Solubility profile of a drug in the presence of a complexing agent

 2.6.2. Solubility Method:
• Point A in the previous graph represents the intrinsic solubility of the drug in
water.
• As we add the ligand, the drug complexes with it and more solid drug is
withdrawn into solution to maintain the free drug concentration constant,
resulting in increased total drug concentration.
• Consequently the concentration increases to reach point B.
• At point B the system is saturated with respect to both the drug and the
complex. Point B
Saturation of the Drug-Ligand
(solubility limit)

Point C
Molar Concentration of the Drug

All excess solid

Drug converted to
Drug-Ligand
complex

Point A
Solubility of
the Drug

Molar Concentration of the Ligand

 2.6.3. Solubility Method:
• In the plateau BC, the complex continues to form and precipitate, however,
the total concentration does not change because of the presence of excess
solid.

• At point C, all the excess solid has been exhausted and turned into the
complex.

• The decline in the total concentration is due to the formation of complexes

that has lower solubility.
Point B
Saturation of the Drug-Ligand
(solubility limit)

Point C
Molar Concentration of the Drug

All excess solid

Drug converted to
Drug-Ligand
complex

Point A
Solubility of
the Drug

Molar Concentration of the Ligand

 Calculation of the ratio of the constituents (B-C plateau ) :

1. The concentration of the drug entering the complex during this plateau is the
solid drug that dissolves during this period (precipitated excess drug)
= total amount of solid added initially __ the amount dissolved at point B
2. calculate the concentration of the ligand entering the complex
throughout the plateau (B-C).

m[ Drug ] + n[ Ligand ] - - - - > [ Drug m - Ligand n ]

y2
B C
m
Ration =
n
m = Drug0 - y2 y1
A

Drug
n = x2 - x1
Drug0 - y2
Ration =
x2 - x1

X1 X2
Ligand
 Calculation of the stability constant (A-B segment) :

– The concentration of the complex = Total Conc of Drug at B-Conc of Drug at A

– The concentration of the free drug is constant through AB= Conc at A.
– The concentration of the free ligand= originally added ligand concentration
to the system- the concentration of the complex.

m[ Drug ] + n[ Ligand ] - - - - > [ Drug m - Ligand n ] B C

[ Drug m - Ligand n ] yL2
K= ( assume : n = m = 1)
[ Drug ] * [ Ligand ]
y1
[ Drug - Ligand ] A
K=
[ Drug ] * [ Ligand ]
[ Drug - Ligand ] = y L - y1 Drug
[ Drug ] = Cons tan t = y1
[ Ligand ] = xL - [ Drug - Ligand ]

xLL#
Ligend
 0 1 2 3 4 5 6
Molar Concentration of the Drug

5 Solubility of drug =3
4 Solubility of complex =2
Free or in a complex

6
3

0 1 2 3 4 5 6 Molar Concentration of added Ligand

 4) What is the concentration of Drug-complex at point A?
5) What is the concentration of Drug-complex at point C at equilibrium?
6) What is the concentration of free drug at point C at equilibrium?
7) What is the concentration of free ligand at point C at equilibrium?
8) Calculate the complexation constant
9) Calculate the stichometry of the complexation
Point A Point B
Molar Concentration of the Drug

9
7
Free or in a complex

5
Point C

0 2 4 6 8 10 12 Molar Concentration of added Ligand

 Example 10-3 (Martin’s 6th ed.): PABA (drug) and caffeine (ligand)

Given
Initial PABA concentration added to the system in excess
[ PABA]0 = 0.073M
y1 = 0.0458M Solubility of PABA in water
y2 = 0.055M Concentration of PABA (free+complexed) in water
x1 = 0.017 M
x2 = 0.035M
y2
B C
Q1: Ratio?
y1
m[ Drug ] + n[ Ligand ] - - - - > [ Drug m - Ligand n ] A

Drug
m
Ration =
n PABA
m = Drug0 - y2 = 0.073 - 0.055 = 0.18M
n = x2 - x1 = 0.035 - 0.017 = 0.18M
Drug0 - y2 0.18 X1 X2
Caffeine
Ration = = =1 Ligand

x2 - x1 0.18
 Example 10-3 (Martin’s 6th ed.): PABA (drug) and caffeine (ligand)

Given

y1 = 0.0458M Solubility of PABA in water

xL = 0.01M Total Caffeine concentration added to the system up to (xL, yL)

y L = 0.0531M Concentration of PABA (free+complexed) in water

Q2: K? B C
[PABA]+[Caff ]− − − − > [PABA − Caff ] yyL2
[PABA − Caff ] y1
K= A
[PABA]*[Caff ]

PABA
[PABA − Caff ] = yL − y1 = 0.0531− 0.0458 = 0.0073M
[PABA] = Cons tan t = y1 = 0.0458M
[Caff ] = xL −[PABA − Caff ] = 0.01− 0.0073 = 0.0027M
0.0073
K= = 59
0.0458* 0.0027 xLL#
 Home work

• The problems to be solved in relation to the solubility

method from the physical pharmacy book – 4th edition
are: 11-4, 11-5, 11-6 – all from page 279. If you have the
fifth edition, solve the following problems in relation to
solubility: 11-4, 11-5, 11-6 – all from page 715.

• Example 11-3 page 265 in the fourth edition from the

physical pharmacy book. If you have the fifth edition, see
example 11-3 page 283.

• If you have the sixth edition of the physical pharmacy

book by Martin, see Example 10-3 page 212 and solve
problem 10-3 in relation to the solubility method.
 5.5.1. Methods of Continuous Variation:
Max/min response occurs when A and B are mixed in the perfect ratio they form in real complex

Complex
(Dielectric Constant)
Additive Property

Curve for no
Complex

100% Composition 100%

A B
Mole fraction

3/1/20 50
 Example
• Using the method of continuous variation, it
was found that the maximum absorbance
(response) was obtained when 5.0mL of A
(0.05 M) was mixed with 5.0 mL of B (0.1 M).
Find the stoichiometric ratio of complexation
 Absorbance (a.u)

Volume of A (0.05 M) in mL 0 1 2 3 4 5 6 7 8 9 10
Volume of B (0.1 M) in mL 10 9 8 7 6 5 4 3 2 1 0
Use the information in the figure below to calculate the concentration of solution A (value for x in

The complexation reaction follows this stichometry: 3A+B A3B

Absorbance (a.u)

Volume of A (x M) in mL 0 1 2 3 4 5 6 7 8 9 10
Volume of B (0.1 M) in mL 10 9 8 7 6 5 4 3 2 1 0
0

0
1

3
3

2
1
0
5.5.1. Methods of Continuous Variation:

• If complexation occurs then the value of the additive property

will decrease or increase depending on the nature of the
complex formed and will pass through either a minima or
maxima.

• The point of inflection represents the ratio of the two

components in the complex.

• For a constant total concentration of both species A and B, the

complex is at its greatest concentration at a point where the
species A and B are mixed at the same ratio in which they
occur in the complex.

3/1/20 55
 5.5.1. Methods of Continuous Variation:

• This method has been applied for the determination of the

stoichiometric ratio of complexation between Cu+2 (blue
solution) with thiobarbiturate (colorless solution) to produce a
green complex.

• In such cases the equation for complexation can be written as

follows:
M + nA Û MAn

• The stability constant is: K=

[MAn ]
[ M ][ A]n

3/1/20 56
2.5.1. Methods of Continuous Variation:

• By taking the log of the previous relation we get:

log [MAn] = log K + log [M] + nlog [A]
• Where
• [MAn] is the concentration of the complex
• [M] is the concentration of the uncomplexed metal ion
• [A] is the concentration of the uncomplexed ligand
• K is the stability constant
• n is the stoichiometric ratio of the metal – ligand in the complex
• We use the above equation to determine the stability
constant for complex formation K.

3/1/20 57
2.5.1. Methods of Continuous Variation:

• The concentration of the metal ion is held constant and the

concentration of the ligand is varied, the corresponding
concentration of the complex [MAn] is obtained by
spectrophotometry.
• Assume n is predetermined and thus known

• If log [MAn] is plotted against log [A] a straight line is obtained

with a slope of “n” or the coordination number and and
intercept of ([M] + log k).

• [M] is constant and so log k can be calculated.

3/1/20 58
2.5.1. Methods of Continuous Variation:

log [MAn]

Slope = n

log [M] + log k

log [A]

3/1/20 59
 Protein Binding:

interaction of drugs
with plasma
proteins (albumin)

Antibody-antigen
recognition
(immunity)
enzyme-substrate
interaction
drug binding to receptor
 Protein Binding:
The binding of drugs to proteins in the body can affect
their actions by:
– Affecting the drug distribution throughout the
body.
– Affecting the activity of the drug by reducing
amount of free drug available to bind the
receptor site.
– Retard the excretion of the drug and increase its
half life.
 Protein Binding:

• Since proteins are molecules

composed of different types of
amino acids, the interactions
between proteins and small
molecules can occur through one
or more than one of the
followings:
– Hydrogen bonding
– Electrostatic interactions
– van der waals interaction
– Hydrophobic interactions
Schematic representation of hydrophobic interaction
 Protein Binding
!Pf!+!Df!!!!!!!!!!!!!!!!!PD!
• The two most important parameters of protein binding are:
– Affinity of binding , expressed using the association constant and is a
measure of the strength of interaction between the protein and drug
molecule.
[PD]
K=
[P] f [D] f
• K: Association constant
• [PD]: The concentration of formed protein-drug
complex = concentration of bound drug
• [P] f: The concentration of free protein(unbound)
• [D]f: The concentration of free drug (unbound)

[ D ]bound [ PD ]
– Ratio of bound drug to total proteins = r= =
[ P ]total [ P ]total
 Protein Binding: Equilibrium Dialysis
Dialysis tubing or sac:
selective diffusion of small molecules through semipermeable membranes

equilibrium dialysis
 Protein Binding: Equilibrium Dialysis
• The protein (e.g. serum albumin or other protein under investigation) at a specific
concentration and drug in various concentrations are placed in a tied cellulose
semipermeable tubing (dialysis bag or sac). The sac is placed in a beaker with proper media
to simulate the physiological one. Drug only start to diffuse and ultimately it will reach
equilibrium (concentration of free drug in sac=concentration of free drug in dialysate=[D]f ).
• If binding occurs, the drug concentration in the sac [D]total containing the protein is greater at
equilibrium than the concentration of the drug in the vessel outside the sac [D]f .
• Samples are collected and analyzed to obtain the concentration of free and bound drug.

[PD]
K=
[P] f [D] f

Obtain [D]f in the beaker

[D]f (in eq. with sac!!)
Analyze [DP]= [D]b=[D]t-[D]f
[P]t (stay constant)
[P]f= [P]total -[DP]

T0 [P]t Teq
Experimental Setup for equilibrium dialysis for analysis of protein-ligand interaction
Protein Binding: Equilibrium Dialysis
[ PD ]
K= ........consider : [ P ] f = [ P ]t - [ PD ]
[ P] f [ D] f
[ PD ]
K=
([ P ]t - [ PD ])[ D ] f
[ PD ] = K [ D ] f ([ P ]t - [ PD ])
[ PD ] + K [ D ] f [ PD ] = K [ D ] f [ P ]t
[ PD ] K [ D] f
r= =
[ P ]t 1 + K [ D ] f
[ D ]bound K [ D ] free
r= =
[ P ]total 1 + K [ D ] free

This equation assumes the

presence of one binding site, K[D f ]
r=v
in case of the presence of v 1+ K[D f ]
independent binding sites,
V: # of drugs bind to a single protein
the equation becomes
Maximum binding capacity
Langmuir isotherm and the double reciprocal plot:

K[D] f
Langmuir isotherm r=v
1+ K[D] f
v
1 1+ K[D] f
=
r

r vK[D] f
1 1 1
= + Double reciprocal plot
[D]f (mole/L)
r vK[D] f v
y = bx + a

slope = 1/vK
1/r

V: # of drugs bind to a single protein

Maximum binding capacity y-intercept = 1/v

1/[D]f (L/mole)
 Example
The number of binding sites and the association constant for the binding of
sulfamethoxypyridazine to albumin at pH 8 can be obtained from the following data:

!
[D] bound ! ! ! !
r! = 0.23! 0.46! 0.66! 0.78!
! [P]total
!
!!!!!!!!!!!!!x10
[D] f %4! 0.10! 0.29! 0.56! 1.00!
!
where [Db] is the concentration of drug bound, also referred to as [PD], and
[Pt] is the total protein concentration. What values are obtained for the
number of binding sites, v, and for the association constant, K?
 Example
D r 1/D 1/r
0.00001 0.23 100000 4.34782609 Slope 3.415e-005 ± 6.598e-007
0.000029 0.46 34482.7586 2.17391304 Y-intercept when X=0.0 0.9437 ± 0.03554
0.000056 0.66 17857.1429 1.51515152
0.0001 0.78 10000 1.28205128 X-intercept when Y=0.0 -27635

R square 0.9993
5

3
1/r
y-Intercept=1/v à v=1.06 (~ 1) 2

Slope=1/vk 0
0 50000 100000 150000
àK=2.93X104 L/mole
1/[D]f (L/mole)
 Home work
The following data were obtained in the in vitro binding study of naproxen, with
human serum albumin at 37 C.

a) Plot the data according to the double-reciprocal model of the protein-

drug binding
b) After linear egression analysis (linear fitting using excel or any other
software), calculate the binding constant K and the number of binding
sites (v) of naproxen to albumin
Answers: v=2.0; K=8.33X108 L/mole