1. PROTEIN BINDING
presented by :
Nimra Zulfiqar
Faiza Aslam
Zeenat Fatima
Fakhra Bukhari
Zubaira Afzal
Biopharmaceutics
The University of Faisalabad

2. "Many drugs interact with plasma or tissue
proteins or with other macromolecules, such
as melanin and DNA ,to form a drug-
macromolecule complex. The formation of a
drug protein complex is named as drug
protein binding."

3. Binding of drug to proteins may:
Facilitate the distribution of drugs
Inactivate the drug by not enabling a
sufficient concentration of free drug to
develop at a receptor site
be a reversible or an irreversible process.

4. Binding of drug:

5. Two Important Plasma Proteins
ALBUMINALBUMIN
Is the most important protein that binds to drug
molecule due to its high concentration compared
with other proteins
It binds both acidic and basic
Constitute 5% of the total plasma

6. Two Important Plasma Protein
∂∂1-ACIDGLYCOPROTEIN1-ACIDGLYCOPROTEIN
Also known as orosomucoid (∂1-globulin)
Binds to numerous drugs
Have greater affinity for basic than acidic
drugs molecules
Binds only basic and highly lipophilic
drugs

7. Drugs may bind to protein through:
Hydrophobic Interaction
Proposed by Kauzmann
tendency to develop of hydrophobic molecules or
parts of molecules to avoid water because they are
not readily accommodated in the H-bond structure of
water

8. Drugs may bind to protein through:
Self-Association
Some drug may self dissociate to form dimers,
trimers or aggregates of larger size
Dimers or trimers - is a reaction product of two or
three identical molecules
May affect solubility, diffusion, transport, therapeutic
action of drugs

9. Amino Acids
A. Basic Group
Arginine
Histidine
Lysine
bind
Acidic Drugs
Amino Acids
B. Acidic Group
Aspartic Acid
Glutamic
Acid
bin
d Basic Drugs

10. Protein binding is determined by:
Dialysis
Ultracentrifugation
Ultrafiltration
Sephadex-gel filtration
Molecular filtration
Electrophoresis
Agar plate test

11. Factors affecting protein-Binding
1. Factors relating to the drug
2. The Protein
3. Factors relating to the protein and other binding
component
4. Drug interactions
5. Patient related factors

12. 1. DRUG RELATED FACTORS
Physicochemical Characteristics of the DrugPhysicochemical Characteristics of the Drug
 E.g. Cloxacillin 95% bound, Ampicillin 20% bound; hence
Cloxacillan is released slowly after i.m. injection.
 Increase in lipophilicity increases the extend of binding.
 Acidic/anionic drugs bind to HSA; basic/cationic drugs to
AAG; neutral/unionized drugs to lipoproteins.

13. Concentration of Drug in the Body
At low concentrations, most drugs may be bound to
proteins
At high concentrations, more free drugs may be present
owing to saturation of binding sites on protein

14. Drug-Protein/Tissue Affinity
Lidocaine has greater affinity for AAG than for HSA.
Digoxin has more affinity for proteins of cardiac muscles
than those of skeletal muscles or plasma.
Iophenoxic acid has half life of 2 ½ yrs due to its high
affinity to plasma proteins.

15. 2. PROTEIN RELATED FACTORS
Physicochemical Properties of Protein/Binding
Component
o Lipophilicitylipoproteins bind with lipophilic drugs.
o Albumindepend on physiological pH.
Concentration of Protein/Binding Components
o Disease states affect the concentration of proteins in
blood.

16. Number of Binding Sites on the Protein
o Albumin has a large number of binding sites as
compared to other proteins.
o Indomethacin binds to 3 sites on albumin.
o AAG is a protein with limited binding capacity due to low
concentration & molecular size.
o Lidocaine binds to 2 sites on AAG in presence of HSA.

17. 3. DRUG INTERACTIONS
 Displacement Interactions
Competition between
Drugs for the Binding
Sites.
e.g. warfarin and
phenyl butazone

18. Interactions will result when:
The displaced drug (E.g. warfarin)—
a) is more than 95% bound
b) has a small volume of distribution
c) shows a rapid onset of therapeutic or adverse effects
d) has a narrow therapeutic index

19. The displacer drug (E.g. phenyl butazone)—
a) competes for the same binding sites
b) the drug/protein concentration ratio is high
c) shows a rapid and large increase in plasma drug
concentration
d) has a high degree of affinity as the drug to be displaced

20. Competition between Drugs and
Normal Body Constituents:
a) Interaction with free fatty acids.
b) Free fatty acid levels are increased during fasting,
diabetes, MI, etc.
c) Eg.: Interaction of sod. salicylate with bilirubin in
neonates.

21. Allosteric Changes in Protein
Molecule:
involves alteration of the protein structure by the
drug/metabolite modify binding capacity.
Eg.: Aspirin acetylation of albumin; modify the binding
capacity of NSAIDs (increased affinity).

22. 4. PATIENT RELATED FACTORS
Age:
Neonates
Change in albumin content affects the drugs
binding to it.
Infants
Elderly
Intersubject Variations:
Disease States:
Hypoalbuminemia severely impair protein-drug
binding.

24. Influence of Disease States on
Protein-Drug Binding

25. Clinical Example
Macfie et al (1992) studied the disposition of intravenous
dosing of alfentanil in six patients who suffered 10% to 30%
surface area burns compared with a control group of six
patients matched for age, sex, and wieght.
Alfentanil binding to plasma proteins was meisured by
equilibrium dialysis. The burn patients had significantly
greater concentration of AAG and smaller concentration of
albumin.

26. The mean alfentanil binding was 94.2%± 0.05 (SEM) in
the burn group and 90.7% ± 0.4 in the control group
(p=0.004).
A good correlation was found between AAG
concentration and protein binding.
The greater AAG concentration in the burn group
corresponded with significantly reduced volume of
distribution and total clearance of alfentanil.
The clearance of unbound drug and half life of alfentanil
were not decreased.

27. The Pharmacokinetic Importance
of Protein Binding
Drug-protein binding influences the
distribution equilibrium of the drug
Plasma proteins exert a buffer and
transport function in the distribution
process
Only free and unbound drug acts can
leave the circulatory system and diffuse
into the tissue

28. Disease and Protein Binding
The reduced albumin concentration and binding
capacity is due to:
Change in albumin molecule
 presence of endogenous binding
inhibitors such as free fatty acids, and
metabolic acidosis.
Hypoalbuminemia may result in patients with
cancer, burms, cardiac failure, cystic fibrosis,
enteropathy, inflammations, liver impairment,
malabsorption, nephrotic syndrome, renal
failure, sepsis and trauma.

29. Binding of Drugs to RBC
Lipophilic molecules dissolved in the lipid
material of the RBC membrane
Anions can be attracted to and enter the
positively charged pores of RBC.
 Drugs absorbed in the RBC membrane
inhibits the deformity of RBC thus
becoming lodged in the capillaries

30. Kinetics of protein binding:-
The kinetics of reversible drug-protein binding for a
protein with one simple binding site can be described by
law of mass action, as follows :-
Protein + drug drug protein-complex
or,
[P] + [D] [PD ] ……………. 1
From eq. 1 and law of mass action , an association
constant, Ka, can be expressed as the ratio of molar
concentration of product and the molar concentration of
the reactants. The assumes only one binding site per
protein molecule.
Ka =
[PD]
[P] [D]
………………..2

31.  The extent of drug-protein complex formed is
dependent on the association binding constant
Ka.
 The magnitude of Ka yields information on
the degree of drug protein binding. Drug
strongly bound to protein have a very large Ka
and exist mostly as the drug-protein complex.
With such drugs , a large dose may be needed
to obtain a reasonable concentration of free
drug.

32. 
Most kinetic studies in vitro use purified
albumin as a standard protein source, b’coz
these protein is responsible for the major
portion of plasma drug-protein binding.
 Experimentally, both the free drug [D] and the
protein bound drug [PD], as well as the total
protein concentration
[P] + [PD], may be determined. To study the
binding behavior of drugs, a determinable ratio
( r ) is defined, as follows;
r = Moles of drug bound
Total moles of protein

33. B’coz moles of drug bound is PD and the total moles of protein is
P + PD, this eq. becomes
According to eq.2 , [PD] = Ka [P][D]; by substitution into eq.3, the
following eq. is obtain,

34. This equation describes the simplest situation, in which 1
mole of drug binds to 1 mole of protein in a 1:1 complex.
This case assumes only one independent binding site for
each molecule of drug. If there are n identical
independent binding sites per protein molecule, then the
following is used:
In terms of Kd, which is 1/ Ka, eq. 5 reduces to:-

35. 
protein molecule are quit large than drug
molecules and contain more than one type of
binding site for the drug. If there is more than
one type of the binding site with its own
association constants, eq. 6 expands
where the subscripts represents different types
of binding sites, the K’s represents binding
constants, and the n’s represents the number of
binding sites per molecule of albumin.

36. These eq. assumes that each drug molecule binds to
the protein at an independent binding site, and the
affinity of a drug at one binding site does not
influence binding to other sites. In reality Drug-
protein binding sometimes exhibits a phenomenon of
co-operativiy. For these drugs the binding of the first
drug molecule at one site on protein molecule
influences the successive binding of other drug
molecule. E.g. binding of oxygen to hemoglobin. Each
method for the investigation of drug-protein binding
in vitro has advantages and disadvantages like; cost,
ease of measurement, time, instrumentation and other
considerations. Drug-protein binding kinetics yields
valuable information concerning proper therapeutic
use of the drug and prediction of possible drug
interactions.

37. Determination of Binding Constants and
Binding Sites by Graphic Methods
1. in vitro methods ( known protein
concentration)
2.in vivo methods ( unknown protein
concentration)

38. In vitro methods
1.Direct plot :
A plot of the ratio of r ( moles of drug bound per mole
of protein) versus free drug concentration increases, number
of moles of drug bound per mole of protein becomes
saturated and plateaus. Thus drug protein binding resembles
a Langmuir adsorption isotherm, which is also similar to
adsorption of a drug to an adsorbent becoming saturated as
the drug concentration increases. B’coz of nonlinearity in
drug-protein binding, eq. 6 is rearrange for the estimation of
n and Ka, using various graphic methods.

40. Scatchard plot :
It is a rearrangement of eq. 6. The scatcherd
plot spreads the data give a better line for the
estimation of the binding constants and binding
sites. From eq. 6 , we obtain,
The graph constructed by plotting r / [D] verses r
yields a straight line with the intercept and

41. References:-
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