The document discusses the mechanisms and significance of protein-drug binding, highlighting the types of binding (reversible and irreversible) and the various proteins involved, such as plasma proteins and blood cells. It details factors affecting drug binding, including drug characteristics, patient-related factors, and the impact of diseases. Additionally, the document covers the clinical implications of protein binding on drug metabolism, distribution, and action, emphasizing the importance of understanding these interactions in pharmacotherapy.

1. Protein Binding
Dr. Rameshwar Dass
Guru Gobind Singh College of Pharmacy,
Yamunanagar, Haryana

2. Contents
• Introduction
• Mechanisms of protein drug binding
• Classes of protein drug binding.
• Binding of drug to blood components.
• Plasma proteins
• Blood cells
• Binding of drug to extravascular tissue protein
• Factors affecting protein drug binding
• Significance of protein/tissue binding of drug

3. Introduction
The interacting molecules are generally the macromolecules such as protein,
DNA or adipose. The protein are particularly responsible for such an
interaction.
P + D ⇌ P-D complex
• The phenomenon of complex formation of drug with protein is called as protein binding of drug
• The protein bound drug is neither metabolized nor excreted hence it is pharmacologically
inactive.
• P-binding divided into: Intracellular and Extracellular

4. Mechanisms of P-D Binding
Binding of drugs to proteins is generally of reversible & irreversible.
Reversible generally involves weak chemical bond such as:
• Hydrogen bonds
• Hydrophobic bonds
• Ionic bonds
• Van der waal’s forces
Irreversible drug binding
• It is rare
• Covalent binding
• It is responsible for the carcinogenicity or tissue toxicity

5. Reversibl
e P-D
Binding

6. P-D Binding
To Blood components
Plasma Proteins
Blood cells
To Extra vascular Tissues
Proteins
Fats
Bones

7. Plasma P-D
Binding
Reversible
Order : Albumin > α1 AG > Lipoproteins >
Globulins.
• Human serum
• Albumin.
• α1 Acid Glycoprotein,
• Lipoproteins,
• Globulins
Involves binding to :

8. HSA P-D
Binding
•59%
•M.W. of 65,000 with large
drug binding capacity

9. α1-Acid glycoprotein P-D Binding
• M.W. 44,000
• Plasma conc. 0.04 to 0.1 g%.
• It binds to no. of basic drugs like imipramine, lidocaine,
propranolol, quinidine

10. Lipoproteins P-D Binding
:
Binding by: Hydrophobic Bonds, Non-
competative. M W. 2-34 Lacks Dalton.
Lipid core composed of:
Inside: triglyceride & cholesteryl esters.
Outside: Apoprotein.
Example:
Acidic: Diclofenac.
Neutral: Cyclosporin A.
Basic: Chlorpromazine.
LDL HDL
VLDL
Chylomicrons
Types

11. Globulin P-D Binding
Globulin Synonym Binds to
1. α1 Globulin
Transcortine/
Corticosteroid
globulin Binding
Steroidal drugs, Thyroxin &
Cyanocobalamine.
2. α2 Globulin Ceruloplasmine Vitamin A,D,E,K.
3. β1Globulin Transferin Ferrous ions
4. β2Globulin --- Carotinoids
5. γ Globulin --- Antigens

12. Blood Cells -D Binding
• In blood 40% of blood cells
• Major component is RBC (95%).
• The RBC - 500 times in diameter as the albumin.
• RBC binding more for lipophilic drugs.
• The RBC comprises of 3 components.
• Hemoglobin: M.W. 64,500 Dal.
• Drugs binds phenytoin, pentobarbital.
• Carbonic anhydrase: Carbonic anhydrase inhibitors drugs are bind
to it e.g. acetazolamide & chlorthalidone.
• Cell membrane:
• Imipramine & chlorpromazine bind with the RBC membrane.

13. Extravascular Tissue P-D Binding
Binding order: Liver › Kidney › Lung › Muscles
Tissue Drug examples
Liver Irreversible binding of Epoxides of Halogenated Hydrocarbon & Paracetamol
Lungs Basic drugs: Imipramine, Chlorpromazine, & Anti-histaminics
Kidney
Metallothionine protein binds to Heavy metals & results in Renal accumulation
and toxicity.
Skin Chloroquine & Phenothiazine binds to Melanin
Eye
Chloroquine & Phenothiazine also binds to Eye Melanin & results in
Retinopathy.
Hairs Arsenicals, Chloroquine, & Phenothiazine.
Bones
Tetracycline(yellow discoloration of teeth), Lead(replaces Ca & cause
brittleness)
Fats Lipophilic drugs (thiopental), Pesticides (DDT)
Nucleic Acid Chloroquine & Quinacrine.

14. Factors Affecting P-D Binding
Drug related Factors and Effect
Physicochemical
characteristics
Directly related to the lipophilicity of drug and increase in lipophilicity
increases the extent of binding
Concentration
The conc. of drug substance as well as the protein molecules or surfaces
subsequently brings alteration in the P-binding process.
Drug Affinity to
protein
The degree of attraction or affinity of the protein molecule or tissues have
towards drug moieties. Digoxin has more affinity for cardiac muscles
proteins than other protein.
Protein/ tissue related factors and Effect
Physicochemical
characteristics
Lipoproteins & adipose tissue tend to bind lipophilic drug by
dissolving them in their lipid core.
pH determines the presence of active anionic & cationic groups on the
albumin to bind a variety of drug.
Concentration
Binding predominantly depends on conc. Like albumin.
Proteins and tissue components conc. May be changes during disease state

15. Factors Affecting P-D Binding
Drug interactions and Effect
Allosteric changes in P
Alteration of the protein structure by the drug or it’s metabolite changes its binding capacity
Aspirin acetylates lysine fraction of albumin thereby modifying its capacity to bind
NSAIDs like phenylbutazone
Drug-drug competition for
binding sites
Administration of phenylbutazone to a patient on Warfarin therapy results in Haemorrhagic
reaction
Competition b/w D&F The free fatty acids are known to interact with a no. of drugs that binds primarily to HSA
Patient-related factors
Age Neonates: Low albumin content as compare to adults: More free drug
Inter-subjects variability Due to genetics & environmental factors
Disease states
Renal and hepatic failure Decrease albumin conc. And less binding of acidic drugs; neutral and basic drugs are un
affected
Inflammatory states i.e,
truama
Increase AAG levels and inc. binding of basic drugs; neutral and acidic drugs are unaffected

16. Apparent volume of Distribution
• Different tissue have diff. amount of drug and Vd cannot have a true physiologic
meaning. But it depends on physiologic mechanism (primary parameter)
• The relationship between tissue-drug binding and volume of distribution
(Amount of drug in body) α ( Conc. of drug in plasma)
X α C
X = Vd.C Vd =X/C
Definition : Hypothetical Vol. of body fluid into which drug is dissolved or
distributed.
• It is Apparent Vd: Because all parts of body equilibrated with drug do not
have equal amount.
• Extensively blood component bind drugs have low vol. of distribution
• Extra-vascular binding of drug show large vol. of distribution.
• Tissue binding of drug acts as depot/storage site.

17. Apparent volume of Distribution
• It depends on physiologic mechanism (primary parameter) Vd =X/C
• Example: Vd 2 L/kg for lumefantrine
Body fluid
Volume
(lit.)
% of
Body wt.
% of TWB
1.Vascular fluid 6 9 15
(plasma) (3) (4.5) (7.5)
2.Extracellular fluid (excluding plasma) 12 14 28
3.Intracellular fluid (excluding blood
cells)
24 34 57
Total Body 42 60 100

18. Kinetics of P-D Binding
• The bound drug is neither metabolized nor excreted due to its pharmacokinetic
and pharmacodynamic inertness.
Protein + Drug = Protein-Drug complex
Note: applying law of mass action.
P+D PD
At equilibrium,
Where, [P]=concentration of free protein
[D]=concentration of free drug
[PD]=concentration of protein-drug complex
Ka = association rate constant and Kd =dissociation constant
𝐾 𝑑
𝐾 𝑎

19. Kinetics of P-D Binding
Ka > Kd indicates forward reaction i.e. protein-drug binding is favored. If Pt
is the total concentration of protein present , bound and unbound, then :
Pt =[PD]+[P]………….(2)
If r is the number of moles of drug bound to total moles of protein, then,
……….(3)
Substituting the value of [PD] from equation (1) in equation (3)
r =
=
………(4)
Equation (4) holds when there is only one binding site on the protein drug
complex is 1:1 complex

20. Direct Plot Kinetics
It is made by plotting r vs [D]. Note
that all binding sites are occupied by
the drug.
The protein is saturated and plateau is
reached
r = N where r = N/2 , [D]=1/ka

21. Scat-chard Plot
It is made by transforming equation (5)
into linear form
r=
r+r ka [D]
r=N ka [D]-r ka [D]
therefore,
=N ka -r ka
A plot of r/[D] vs r yields a straight line
slope line=- ka
y-intercept=N ka and x- intercept=N

22. Klotz Plot/Lineweaver- Burke Plot
The reciprocal of equation
=N
A plot of 1/r vs 1/[D] yields a straight line
Slope = -1/N ka and
y-intercept 1/N

23. Hitchcock Plot
It is made by re-writing equation (5) as
=1+ ka [D]
Dividing both sides with N ka gives
=

24. Clinical Significance P-D Binding
Absorption
The conventional dosage form follow first order kinetics. So when there is more protein
binding then it disturbs the absorption equilibrium and 9ncrease the absorption.
Distribution
A protein bound drug in particular does not cross the BBB, the placental barrier, the
glomerulus. It decreases the distribution of drugs
Metabolism
Protein binding decreases the metabolism of drugs & enhances the biological half life.
Example: Phenylbutazone & Sulfonamide
Elimination
Only the unbound drug is capable of being eliminated.
Prevent the drug to metabolize (liver ) & glomerulus filtration (Tetracycline)
The patients with acute hepatitis, protein binding of the glucocorticoids will be reduced
and peak concentrations of administered glucocorticoids increased

25. Clinical Significance P-D Binding
Drug action
Protein binding inactivates the drugs because P-D not bind to the receptor site for
action (Naphthoquinone)
Aspirin reduces ketoprofen protein binding and increases its clearance
Sustain release
The complex of drug protein in the blood act as a reservoir & continuously supply
the free drug.
Diagnosis
The Cl atom of chloroquine replaced with radiolabelled I131
can be used to visualize-
melanomas of eye & disorders of thyroid gland
Systemic solubility of drug
Lipoprotein act as vehicle for hydrophobic drugs like steroids, heparin, oil soluble
vitamins