Protein binding

1. PROTEIN BINDING
PROTEIN BINDING
Presented by:-
Presented by:-
Chhavi Nagar
Chhavi Nagar

2. CONTENT
CONTENT
Introduction
Introduction
Mechanism of protein binding
Mechanism of protein binding
Factors affecting protein binding
Factors affecting protein binding
Binding of drug with blood plasma protein
Binding of drug with blood plasma protein
Kinetics of protein binding
Kinetics of protein binding
Classical method to study protein binding..
Classical method to study protein binding..
.......
.......

3. INTRODUCTION
INTRODUCTION
❖
❖ The phenomenon of complex
The phenomenon of complex
formation of drug with protein is
formation of drug with protein is
called as
called as protein binding of drug.
protein binding of drug.
❖
❖ The interacting molecules are
The interacting molecules are
generally the
generally the macromolecules
macromolecules such
such
as
as protein
protein,
, DNA
DNA or
or adipose
adipose.
.
❖
❖ The proteins are particularly
The proteins are particularly
responsible for such an interaction.
responsible for such an interaction.

4. ❖
❖ Protein binding may be divided into -
Protein binding may be divided into -
Intracellular binding
Intracellular binding and
and Extracellular
Extracellular
binding
binding.
.
❖
❖ Protein binding
Protein binding alter the biological
alter the biological
properties
properties of drugs due to reduce in conc.
of drugs due to reduce in conc.
of free drug.
of free drug.

6. # Protein binding may :-
# Protein binding may :-
⚫
⚫ Facilitate distribution of drug throughout
Facilitate distribution of drug throughout
the body
the body
⚫
⚫ Inactivate drug of binding not available at
Inactivate drug of binding not available at
receptor site.
receptor site.
⚫
⚫ Retard excretion of a drug- accumulate in
Retard excretion of a drug- accumulate in
body.
body.
⚫
⚫ Alter duration of action of drug.
Alter duration of action of drug.
⚫
⚫ Displace body hormone.
Displace body hormone.
⚫
⚫ alter therapeutic effect by forming drug
alter therapeutic effect by forming drug
protein complex.
protein complex.

7. MECHANISM OF PROTEIN DRUG BINDING
MECHANISM OF PROTEIN DRUG BINDING
›
› Binding of drugs to proteins is generally of
Binding of drugs to proteins is generally of
reversible and irreversible.
reversible and irreversible.
›
› Reversible
Reversible generally involves weak
generally involves weak
chemical bond such as :
chemical bond such as : Hydrogen bonds
Hydrogen bonds,
,
Hydrophobic bonds
Hydrophobic bonds ,
,Ionic bonds
Ionic bonds,
, Van
Van
der Waal’s forces
der Waal’s forces.
.
›
› Irreversible
Irreversible drug binding, arises as a result
drug binding, arises as a result
of
of covalent binding
covalent binding and is often a reason
and is often a reason
for the carcinogenicity or tissue toxicity of
for the carcinogenicity or tissue toxicity of
the drug.
the drug.

8. #
# Mechanism of protein drug binding
Mechanism of protein drug binding
include
include:-
:-
⚫
⚫ Absorption
Absorption
⚫
⚫ Distribution
Distribution
⚫
⚫ Metabolism
Metabolism
⚫
⚫ Elimination
Elimination
⚫
⚫ Systemic solubility of drug
Systemic solubility of drug
⚫
⚫ Drug action
Drug action
⚫
⚫ Sustain release
Sustain release
⚫
⚫ Diagnosis
Diagnosis

9. Schematic representation of protein
Schematic representation of protein
binding
binding

10. FACTORS AFFECTING DRUG BINDING
FACTORS AFFECTING DRUG BINDING
1) Drug-related factors.
1) Drug-related factors.
a) Physicochemical characteristics of the drug.
a) Physicochemical characteristics of the drug.
b) Concentration of the drug in the body.
b) Concentration of the drug in the body.
c) Affinity of a drug for a particular binding component.
c) Affinity of a drug for a particular binding component.
2) Protein/ tissue related factors:
2) Protein/ tissue related factors:
a) Physicochemical characteristics of protein or binding
a) Physicochemical characteristics of protein or binding
agent.
agent.
b) Concentration of protein or binding component.
b) Concentration of protein or binding component.

11. 3) Drug interactions:
3) Drug interactions:
a) Competition between drugs for the binding sites[
a) Competition between drugs for the binding sites[
Displacement interactions.
Displacement interactions.
b) Competition between drug & normal body
b) Competition between drug & normal body
constituents.
constituents.
c) Allosteric changes in protein molecules.
c) Allosteric changes in protein molecules.
4) Patient-related factors:
4) Patient-related factors:
a) Age, Sex, Body Weight:
a) Age, Sex, Body Weight:
b) Intersubject variability.
b) Intersubject variability.
c) Disease states.
c) Disease states.

12. BINDING OF DRUG WITH BLOOD PLASMA
BINDING OF DRUG WITH BLOOD PLASMA
PROTEINS
PROTEINS
⚫
⚫ The binding of drugs to plasma proteins
The binding of drugs to plasma proteins
is reversible.
is reversible.
⚫
⚫ The extent or order of binding of drug
The extent or order of binding of drug
to plasma proteins is:
to plasma proteins is: Albumin
Albumin ›
› ὰ
ὰ1-
1-
Acid glycoprotein
Acid glycoprotein ›
› Lipoproteins
Lipoproteins ›
›
Globulins
Globulins.
.

13. # Binding of drug to human serum
# Binding of drug to human serum
Albumin
Albumin
⚫
⚫ It is the most abundant plasma protein (59 %).
It is the most abundant plasma protein (59 %).
⚫
⚫ Having M.W. of 65,000 with large drug binding
Having M.W. of 65,000 with large drug binding
capacity.
capacity.
⚫
⚫ Both endogenous compounds such as fatty
Both endogenous compounds such as fatty
acid, bilirubin as well as drug bind to HSA.
acid, bilirubin as well as drug bind to HSA.
⚫
⚫ Four different sites on HSA for drug binding.
Four different sites on HSA for drug binding.
- Site I:
- Site I: warfarin and azapropazone
warfarin and azapropazone
binding site.
binding site.
- Site II:
- Site II: diazepam binding site
diazepam binding site.
.
- Site III:
- Site III: digitoxin binding site
digitoxin binding site.
.
- Site IV:
- Site IV: tamoxifen binding site
tamoxifen binding site.
.

14. # Binding of drug to lipoprotein
# Binding of drug to lipoprotein
⚫
⚫ Binding by: Hydrophobic Bonds,
Binding by: Hydrophobic Bonds,
Non - competative.
Non - competative.
⚫
⚫ Mol wt: 2-34 Lacks dalton.
Mol wt: 2-34 Lacks dalton.
⚫
⚫ Lipid core composed of:
Lipid core composed of:
Inside: triglyceride & cholesterol esters.
Inside: triglyceride & cholesterol esters.
Outside : Apoprotein.
Outside : Apoprotein.
e.g. Acidic:
e.g. Acidic: Diclofenac
Diclofenac.
.
Neutral:
Neutral: CyclosporinA
CyclosporinA.
.
Basic:
Basic: Chlorpromazine
Chlorpromazine.
.

15. # Binding of drug to α1-Acid
# Binding of drug to α1-Acid
glycoprotein
glycoprotein
⚫
⚫ It is called as orosomucoid.
It is called as orosomucoid.
⚫
⚫ It has a M.W. 44,000.
It has a M.W. 44,000.
⚫
⚫ Its plasma concentration range of 0.04 to
Its plasma concentration range of 0.04 to
0.1 g %.
0.1 g %.
⚫
⚫ It binds to no. of basic drugs like
It binds to no. of basic drugs like
imipramine
imipramine,
, lidocaine
lidocaine,
, propranolol
propranolol,
,
and
and quinidine
quinidine.
.

16. # Binding of drug to Globulins
# Binding of drug to Globulins
⚫
⚫ Its
Its molecular weight is about 150,000.
molecular weight is about 150,000.
⚫
⚫ It is of 5 types:-
It is of 5 types:-

17. KINETICS OF PROTEIN BINDING
KINETICS OF PROTEIN BINDING
▪
▪ An equation relating reaction velocity to
An equation relating reaction velocity to
Drug concentration (Mol/L) for a system
Drug concentration (Mol/L) for a system
where a Drug D binds reversibly to an
where a Drug D binds reversibly to an
Protein P of to form an Protein-Drug
Protein P of to form an Protein-Drug
complex .
complex .
P + DF ======= PD
P + DF ======= PD
▪
▪ Applying the law of mass action, the
Applying the law of mass action, the
equilibrium or association constant (K) is;
equilibrium or association constant (K) is;
K = [PD]/ [P] [DF]
K = [PD]/ [P] [DF]
▪
▪ The [PD], [P] and [D] are the
The [PD], [P] and [D] are the
concentration of protein-drug complex,
concentration of protein-drug complex,
protein and drug in Mol/L
protein and drug in Mol/L

18. K[P][ DF] = [PD]
K[P][ DF] = [PD] -----------1
-----------1
Free protein concentration can obtain as;
Free protein concentration can obtain as;
[PT] = [P] + [PD] = [PT] - [PD]
[PT] = [P] + [PD] = [PT] - [PD]
[PT] is the total protein.
[PT] is the total protein.
Substituting the [P] in equation 1
Substituting the [P] in equation 1
K ([PT] – [PD]) [DF] = [PD]
K ([PT] – [PD]) [DF] = [PD]
Where, DF is the free drug.
Where, DF is the free drug.
K [PT] [DF] – K [PD] [DF] = [PD]
K [PT] [DF] – K [PD] [DF] = [PD]
K [PT] [DF] = [PD] + K [PD] [DF]
K [PT] [DF] = [PD] + K [PD] [DF]
K [PT] [DF] = [PD] (1+ K [DF])
K [PT] [DF] = [PD] (1+ K [DF])
[PD] = (K [PT] [DF])/ (1+ K [DF])
[PD] = (K [PT] [DF])/ (1+ K [DF])
[PD]/ [PT] = K [DF]/ 1+ K [DF]
[PD]/ [PT] = K [DF]/ 1+ K [DF]

19. ▪
▪ Let R be expressed as moles of drug bound
Let R be expressed as moles of drug bound
[PD] per mole of total protein[PT]
[PD] per mole of total protein[PT]
R = [PD]/ [PT] = K [DF]/ 1+ K [DF]
R = [PD]/ [PT] = K [DF]/ 1+ K [DF]
▪
▪ If V is the number of independent binding
If V is the number of independent binding
sites available then R,
sites available then R,
R = V (K [DF]/ 1+ K [DF])
R = V (K [DF]/ 1+ K [DF])
1/R = 1/VK[DF] + 1/V
1/R = 1/VK[DF] + 1/V

20. ▪
▪ The graph is plotted between 1/R versus 1/
The graph is plotted between 1/R versus 1/
[DF], called
[DF], called Klotz reciprocal plot
Klotz reciprocal plot, gives a
, gives a
straight line
straight line whose slope is 1/VK and
whose slope is 1/VK and
intercept is V
intercept is V
R + R K [DF] = V K [DF]
R + R K [DF] = V K [DF]
R/[DF] = VK - RK
R/[DF] = VK - RK

21. METHOD TO STUDY PROTEIN
METHOD TO STUDY PROTEIN
BINDING
BINDING
⚫
⚫ Equilibrium dialysis method
Equilibrium dialysis method
⚫
⚫ Dynamic dialysis method
Dynamic dialysis method

22. EQUILIBRIUM
EQUILIBRIUM
DIALYSIS METHOD
DIALYSIS METHOD
In this method the protein under
In this method the protein under
investigation is placed in a cellulose tubing
investigation is placed in a cellulose tubing
or a similar dialyzing memberane .
or a similar dialyzing memberane .
The tubes are tied securely and suspended
The tubes are tied securely and suspended
in vessels containing the drugs in various
in vessels containing the drugs in various
concentration .
concentration .
If binding occurs, the drug concentration in
If binding occurs, the drug concentration in
sac containing the protein is greater at
sac containing the protein is greater at
equilibrium than the concentration of the
equilibrium than the concentration of the
drug in the vessel outside the sac.
drug in the vessel outside the sac.

23. ⚫
⚫ Samples are removed and analysed to
Samples are removed and analysed to
obtain the concentration of free and
obtain the concentration of free and
complexed drug.
complexed drug.

25. DYNAMIC DIALYSIS
DYNAMIC DIALYSIS
METHOD
METHOD
⚫
⚫ This is the kinetic method for Studying
This is the kinetic method for Studying
protein binding of drugs.
protein binding of drugs.
⚫
⚫ This method is advantageous in that it is
This method is advantageous in that it is
relatively rapid, require very small
relatively rapid, require very small
quantities of protein and can be readily
quantities of protein and can be readily
applied for studying the competitive
applied for studying the competitive
inhibition of protein binding.
inhibition of protein binding.
⚫
⚫ It is based on rate of disappearance being
It is based on rate of disappearance being
proportional to the concentration of
proportional to the concentration of
unbound drug.
unbound drug.

26. ⚫
⚫ A cellophane dialysis bag containing the
A cellophane dialysis bag containing the
drug or drug protein solution is suspended
drug or drug protein solution is suspended
in buffer solution.
in buffer solution.
⚫
⚫ Both the solution are stirred continuously
Both the solution are stirred continuously
and are periodically removed from outside
and are periodically removed from outside
the dialysis bag and analysed
the dialysis bag and analysed
spectrophotometrically.
spectrophotometrically.
⚫
⚫ It follows the rate law
It follows the rate law