Plasma protein binding

  • Drug protein binding is the reversible interaction of drugs with proteins in plasma.
  • Free drug + free protein à drug-protein complex
  • Drug protein binding may be:
    • Reversible: Hydrogen bonds or vander Walls forces
    • Irreversible: covalent bond
    • E.g.- Alkylating agents rarely                                                                               
      • Hepatotoxicity of acetaminophen (high doses)

Binding of drug

  • 1) Blood components:  a) plasma proteins b) blood cells
  • 2) Extra vascular tissues:  a) proteins b) bones c) fats


  • Each albumin molecule has at least 6 distinct binding sites for drugs and endogenous compounds.
  • Two of these very tightly and specifically bind long chain fatty acids.
  • There is another site which selectively binds bilirubin.
  • There are two major drug binding sites called site I and site II which mainly bind acidic drugs.
  •  Site I binds drugs such as warfarin and phenylbutazone, whereas site II binds drugs such as diazepam and ibuprofen.
  • Drugs may be categorized into two groups with respect to albumin binding:
  • Class I: Drugs that have a low dose/albumin binding ratio.  Albumin binding sites exceed the availability of the drug. The bound fraction consists of a significant proportion of the total drug.  Many clinically useful drugs are Class I types.
  • Class II:  Drugs that have a high dose/albumin capacity ratio.  The majority of the drug exists in the free state, bound drug is a small proportion of the total drug.  Class II drugs can displace Class I drugs from albumin dramatically increasing the amount of free (active) drug.


  • Reversible
  • Irreversible  eg.Chemical carcinogenesis                                          Acetaminophen hepatotoxicity

Drug Displacement

  • Competitive –drug bind to same site
  • Non-competitive-inhibitory drug causing conformational change in protein molecule that inhibit binding of first drug
  • Drug acting as a displacing agent has to be present in1)high conc. 2)high affinity to protein
  • Acidic drugs do not displace basic drugs & vice versa.

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
  • Lipophilic drugs may be absorbed to RBC membrane due to change of:
    • Change of shape of membrane and membrane proteins
    • Membrane extension which may lead to change of RBC shape

The RBC binding sites are:

  • Intracellular proteins
    • Hemoglobin
    • Carbonic anhydrase
    • Cell membrane
    • ATPase

Binding of drug to blood cells

  • hemoglobin – bind to phenytoin, pentobarbital , phenothiazine
  • carbonic anhydrase- drug bind  like acetazolamide , chlorthalidone
  • cell membrane – imipramine , chlorpramazine bind to RBCs cell membrane

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 drug can leave the circulatory system and diffuse into the tissue.
  • When free drug is eliminated by the body, some bound drug is released from protein binding. 
  • Some drugs persist in the body for three days by this mechanism.
  • 2 drugs given concurrently & highly bound to the same site on a plasma protein will compete for the binding site resulting in a greater proportion of free drug.
  • This effect may increase the freedrug totoxic levels.

Binding & interaction of drugs to protein

   Binding of drugs may :

  • Facilitate the distribution of drugs
  • Inactivate the drug by not enabling a sufficient concentration of free drug to develop at a receptor site
  • Retard the excretion of a drug

    Interaction of drugs may cause:

  • Displacement of body hormones or co-administered agent
  • Change the configuration of protein to another structure capable of binding a co-administered agent
  • Inactivates the drug biologically by forming a drug-protein complex
  • Many drugs bind to the same receptor site but drugs with higher affinity will replace those drugs with lower affinity by competition
  • Only free and unbound drugs exert therapeutic effect by interacting with receptors

Factor affecting drug protein binding

1. Factors relating to the drug

  1. Physicochemical characteristic of drug
  2. Concentration of drug in the body
  3. Affinity of drug for a particular protein.

2. Factor relating to the protein

  1. Physicochemical characteristic of the protein
  2. Concentration of protein
  3. No. of binding site on protein

3. Drug interaction

4. Patient related factor

Displacement of drugs from Protein Binding is due to:

  • Total amount of protein-bound drug in that body
  • Extent of tissue binding structure
  •  Apparent volume of distribution

Methods for studying Drug-Protein binding

  • Equilibrium dialysis
  • Dynamic dialysis
  • Diafiltration
  • Ultrafiltration
  • Ultracentrifugation
  • Gel chromatography
  • Spectrophotometry
  • Electrophoresis
  • Optical rotatory dispersion and circulatory dichroism

the most suitable in-vitro experimental methods are equilibrium dialysis, ultracentrifugation, and ultrafiltration.                                                           The concentration of drug in saliva,tears or the erythrocyte/plasma drug concentration ratio may give a useful measure of in-vivo binding of selected drugs-for example, phenytoin. Unfortunately, none of these methods is suitable for large-scale routine use at present.

Regulatory Guidelines

  • Before initiating human trials: “In vitro” PPB data for animals and humans should be evaluated
  • If extensively protein bound, the risk of displacement of other drugs should be investigated

Examples related to protein binding of drugs

  • Aspirin acetylates the lysine residue of albumin, which changes the binding capacity of this protein for anti-inflammatory drugs.
  • In infants, displacement of bilirubin by sulfonamides cause kernictirus
  • Digoxin binds to cardiac tissues of heart. Quinine causes rise in steady state plasma level of digoxin due to displacement of tissue bound digoxin
  • Warfarin, when co-administered with phenylbutazone, causes bleeding due to displacement reaction.
  • Diazepam is considered restrictively eliminated while propranolol non-restrictively eliminated. 
  • Plasma protein binding acts as a carrier mechanism to hasten drug elimination e.g. excretion of penicillin, metabolism of lignocaine.

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