Protein Binding Of Drugs Biology Essay

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Formation of a protein drug complex is termed as drug-protein binding. Drug protein binding may be a reversible or an irreversible process. Irreversible drug protein binding is usually as a result of chemical activation of drug, when then attaches strongly to the protein or macromolecule by covalent chemical bonding. Reversible drug protein binding implies that the drug binds the protein with weaker chemical bonds such as hydrogen bonds or vanderwaals forces. The amino acids that compose the protein chain have hydroxyl, carboxyl or other sites available for reversible drug interaction.

Drugs may bind to various macromolecular components in the blood including albumin, α1 acid glycoprotein, lipoproteins, immunoglobulins (IgG) and erythrocytes (RBC).Albumin is a protein synthesised by the liver with a molecular weight of 65,000 to 69,000d.Albumin is the major component of plasma proteins responsible for reversible drug binding. In the body, albumin is distributed in the plasma and in the extra cellular fluids of skin, muscle and various other tissues.

Albumin is responsible for maintaining osmotic pressure of the blood and for the transport of endogenous and exogenous substances. Lipoproteins are macromolecular complexes of lipids and proteins. Lipoproteins are responsible for the transport of plasma lipids and may be responsible for binding of the drugs if the albumin sites become saturated.

Reversible drug-protein binding is of major interest in pharmacokinetics. The protein bound drug is a large complex that cannot easily cross cell membranes and therefore has a restricted distribution. The protein bound drug is usually pharmacologically inactive. In contrast, the free or unbound drug crosses cell membranes and is therapeutically active.

Drug protein binding is influenced by a number of factors like:-

The drug


Affinity between drug and protein

Drug interactions

The pathophysiologic condition of patient


The kinetics of reversible drug-protein binding site can be described by law of mass action as follows.

Protein + drug → Drug-Protein complex

[P] + [D] → [PD] --------------1

From this equation and law of mass action, an association constant, Ka can be expressed as the ratio of molar concentration of the products and molar concentrations of the reactants. It assumes only one binding site per protein molecule.

Ka = --------------2

To study the binding behaviour of drugs, a determinable ratio, r is defined as follows.

r =

r = -----------------3

According to eqn 2 [PD] =Ka [P] [D] by substitution into eqn 3.

r =

r = --------------------4

The eqn describes 1mole of drug binds to 1 mole of protein in a 1:1 complex. Therefore this assumes only one independent binding site for each molecule of drug.

If there are "n" independent identical binding sites the equation 4 becomes

r = ---------------------5

In terms of Kd =1/Ka eqn 5 reduces to

r =

Since protein molecules are large in size, more than one type of binding site is present and drugs bind independently on each binding site with its own association constant and hence eqn 6 becomes;-

r =

Where, 1, 2:- different binding sites

K: - binding constants

n :- number of binding sites

As per the equations we assume that each drug binds to the protein at an independent binding site and the affinity of a drug for one binding site does not influence binding to other sites.

In reality a drug protein binding sometimes exhibits a phenomenon called cooperativity. For these drugs the binding of first drug molecule at one site on the protein molecule influences successive binding of other drug molecules.

Ex:-Binding of oxygen to haemoglobin



r = --------------1

As per the equation quoted above, as 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 absorbent becoming saturated as drug concentration increases.

Reciprocal of equation written above gives

1 = ---------------------------2

1 = ---------------------------3

A graph of 1/r versus 1/[D] is called as double reciprocal plot. The y intercept is 1/n and slope is 1/nKa.From this graph, the number of binding sites may be determined from y intercept and the association constant may be determined from the slope if the value for n is known.

If the graph of 1/r versus 1/[D] doesn't yield a straight line then the drug protein binding process is probably more complex.eqn 1 assumes one type of binding site and no interaction among the binding sites.


Reciprocal plots cannot be used if the exact amount and nature of protein in the experimental system is unknown.the percent of drug bound is often to used to describe the extent of drug protein binding in the plasma.the fraction of drug bound,β,can be determined experimentally and is equal to the ratio of the concentration of bound drug,D β ,and the total drug concentration of,Dr,in the plasma as follows.

β =

The value of the association constants can be determined even though the nature of the plasma proteins binding the drug is unknown by rearranging the equation above as

r = =


D β:-bound drug concentration

D : - free drug concentration

PT :- total protein concentration

Rearrangement of the above equation yields the expression

D β = nKaPT-KaD β

Concentrations of both free and bound drug may be found experimentally and a graph obtained by plotting D β/D versus D β will yield a straight line from which the slope is the association constant Ka.

The above equation shows that the ratio of bound Cp to free Cp is influenced by the affinity constant, the protein concentration Pt which may change during disease states and with the drug concentration in the body.

The values for n and Ka give a general estimate of the affinity and binding capacity of the drug as plasma contains a complex mixture of proteins. The drug protein binding in plasma may be influenced by competing substances such as ions, free fatty acids, drug metabolites and other drugs. Measurements of drug protein binding should be obtained over a wide range of concentration range, because at low drug concentrations a high affinity low capacity binding site might be missed or at a higher drug concentration saturation of the protein binding sites may occur.

Clinical significance of drug-protein binding.

Most drugs bind to plasma proteins to some extent. When the clinical significance of the fraction of drug bound is considered it is important to know whether the study was performed using pharmacological or therapeutic plasma drug concentrations. The fraction of drug bound can change with plasma drug concentration and dose of drug administered.

When a highly protein bound drug is displaced from binding by a second drug or agent, a sharp increase in the free drug concentration in the plasma may occur leading to toxicity. Displacement occurs when a second drug is taken that competes for the same binding site in the protein as initial drug. This will lead to increased apparent volme of distribution and an increased half life, but clearance will remain unaffected. If administered by multiple doses, the mean steady state remain unaffected; however the mean steady state free drug level will be increased due to displacement. The therapeutic effect will therefore increase. (Leon shargel, Susanna wu-pong, 2005).


Drug-drug interaction refers to an alteration of the effect of one drug caused by the presence of a second drug. Drug-nutrient interactions similarly refer to the alteration of the effect of a drug or nutrient caused by the presence of a second agent. Drug interactions can be beneficial or detrimental. One example would be administering a drug product like carbidopa/levodopa (Sinemet®). Levodopa is converted to dopamine in the central nervous system (CNS), thereby exerting an effect against symptoms of Parkinson's disease. Carbidopa acts as a chemical decoy, which binds to the enzyme that converts levodopa to dopamine outside the CNS. This increases dopamine levels in the CNS while limiting side effects of increased dopamine in peripheral tissues.

In combination, the paired drugs produce additive effects. Patients with numerous disease states may require treatment with interacting drugs. Where these interactions cannot be avoided, the fact is taken into account when planning therapy. Many times dosing is not altered at all, but usual monitoring is increased.

The risk of having drug interactions will be increased as the number of medications taken by an individual increases. This also implies a greater risk for the elderly and the chronically ill as they will be using more medications than the general population. Risks also increase when a patient's regimen originates from multiple prescribers. Filling all prescriptions in a single pharmacy may decrease the risk of undetected interactions. (Beverly J. McCabe, Eric H. Frankel,2004)

Types of Drug Interactions

Drug interactions are often classified as either pharmacodynamic or pharmacokinetic interactions. Pharmacodynamic interactions include those that result in additive or antagonistic pharmacological effects. Pharmacokinetic interactions involve induction or inhibition of metabolizing enzymes in the liver or elsewhere, displacement of drug from plasma protein binding sites, alterations in gastrointestinal absorption, or competition for active renal secretion. The frequency and prevalence of interactions is dependent upon the number of concomitant medications and the complexity of the regimens. The prevalence is also dependent upon other variables, such as patient adherence, hydration and nutritional status, degree of renal or hepatic impairment, smoking and alcohol use, genetics and drug dosing. Additionally, some patients may exhibit evidence of a particular drug interaction, while others with the same drug combination do not.

Pharmacodynamic interactions

This can be defined depending only on the pharmacology of the given drug.


Interactions Resulting from Alterations in Gastrointestinal Absorption.

The rate and extent of drug absorption after oral administration may be grossly altered by other agents. Absorption of a drug is a function of the drug's ability to diffuse from the lumen of the gastrointestinal tract into the systemic circulation .Changes in intestinal pH may profoundly affect drug diffusion as well as dissolution of the dosage form. For example, the absorption of ketoconazole is reduced by the co-administration of antacids or H2-blockers.

Interactions Resulting from Alterations in Metabolizing Enzymes

The liver is the major, though not exclusive, site for drug metabolism. Other sites include the kidney and the lining of the gastrointestinal tract. The two main types of hepatic drug metabolism are phase I and phase II reactions. Phase I oxidative reactions are the initial step in drug biotransformation, and are mediated by the cytochrome P-450 (CYP) system. This complex super family of enzymes has been subclassified into numerous enzymatic subfamilies. The most common CYP subfamilies include CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. These enzymes may be induced or inhibited by other agents, thereby leading to an increase or decrease in the metabolism of the primary drug. Phase II reactions occur following Phase I reactions. In this process, drug metabolites are converted into more water-soluble compounds that can be more easily eliminated by the kidneys.

Enzyme induction may result in increased CYP enzyme synthesis, faster drug metabolism, subtherapeutic drug concentrations and the risk for ineffective drug therapy. The rapidity of the enzyme induction is dependent upon the half-life of the inducing drug as well as the rate of synthesis of new enzymes. Examples of drugs that cause enzyme induction are the barbiturates, some anticonvulsants.

Enzyme inhibition may result from noncompetitive or competitive inhibition of CYP enzymes by a second drug, an effect that may occur rapidly. Examples of hepatic enzyme inhibitors include cimetidine, fluconazole and erythromycin. The result of noncompetitive enzyme inhibition by addition of a second agent is slower metabolism of the first drug, higher plasma drug concentrations, and a risk for toxicity. In the case of competitive inhibition, the metabolism of both drugs can be reduced, resulting in higher than expected concentrations of each drug.

Interactions Resulting from Alterations in Protein Binding

Drugs may exist in plasma either reversibly bound to plasma proteins or in the free (unbound) state. The primary drug-binding plasma proteins are albumin and α1-acid glycoprotein. It is free drug that exerts the pharmacological effect. Drugs may compete with each other for plasma protein binding sites, and when this occurs, one drug may displace another that was previously bound to the protein. Displacement of a drug from its binding sites will therefore increase that agent's unbound concentrations, perhaps resulting in toxicity.

Interactions Resulting from Changes in Renal Excretion

The majority of renally eliminated drugs are excreted via passive glomerular filtration. Some drugs are eliminated via active tubular secretion, such as penicillins, cephalosporins, and most diuretics. The active secretion may be inhibited by secondary agents, such as cimetidine, nonsteroidal anti-inflammatory agents and probenecid, with resulting elevations in the serum drug concentrations and reduced urinary drug concentrations. In some cases, the interaction is desirable, while others may lead to adverse therapeutic outcomes.( Beverly J. McCabe ,Eric H. Frankel,2004).