KINETICS OF

PROTEIN BINDING
KINETICS OF PROTEIN BINDING
 The kinetics of reversible drug-protein binding for a protein
with one simple binding site can be described by

 The law of mass action, an association constant, Ka, can be

expressed as the ratio of the molar concentration of the products
and the molar concentration of the reactants.
This equation assumes only one-binding site per protein
molecule.
The extent of the 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.
Drugs 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 therapeutic
concentration of free drug.
The binding behavior of drugs
Most kinetic studies in vitro use purified albumin as a standard protein source
portion.
The free drug [D] and the protein-bound drug [PD], as well as the total protein
concentration [P] + [PD], may be determined as,
0ne independent binding site for each molecule
of drug.
r= a determinable ratio (binding behavior of drug) is defined,
According to Equation 10.16,

(moles of drug bound)=[PD] = K a[P][D]

by substitution into Equation 10.17, the following expression is obtained:

This equation describes the simplest situation, in which 1 mole of drug binds to 1 mole of
protein in a 1:1 complex.
Identical independent binding sites
per protein molecule
Protein molecules are quite large compared to drug molecules and may
contain more than one type of binding site for the drug
If there are n identical independent binding sites per protein molecule,
then the following is used:
r=
Since Kd = 1/Ka => Ka = 1/Kd

r=

Multiply numerator and demon with Kd

r=

r =
• When n = 1 and the unbound (free) drug concentration is equal to Kd, the protein
binding of the drug is half saturated.
• When [D] is much greater than Kd, the Kd is negligible in Equation 10.20, and r = n
ie, r is independent of concentration or fully saturated).
• When Kd > [D], the [D] is negligible in the denominator of Equation 10.20,
r is dependent on n[D]/Kd.
When Kd > [D]
In this case, the number of sites bound is directly proportional to the
number of binding sites, the association binding constant (also called the
affinity constant), and the free drug concentration.
This relationship also explains why a drug with a higher Ka may not
necessarily have a higher percent of drug bound, because the
number of binding sites, n, may be different from one drug to
another.
If more than one type of binding sites
The numerical subscripts represent different types of binding sites, the K's represent
the binding constants, and the n's represent the number of binding sites per molecule
of albumin.
Assumptions of These equations
Each drug molecule binds to the protein at an independent binding site.
The affinity of a drug for one binding site does not influence binding to
other sites.
Cooperativity
For some drugs, the binding of the first drug molecule at one site on the protein
molecule influences the successive binding of other drug molecules.
The binding of oxygen to hemoglobin is an example of drug cooperativity.
 DETERM OF BINDING CONSTANTS AND BINDING SITES BY GRAPHIC
METHODS
In-Vitro Methods
(Known Protein Concentration)
A plot of the ratio of r (moles of drug bound per mole of protein) versus free drug
concentration [D] . Equation 10.20 shows that

As free drug concentration increases, the 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. .
Determination of Ka and binding
sites(n)
The values for the association constants and the number of binding sites
are obtained by various graphic methods.
1-Double reciprocal plot
2-Scatchard plot
Double reciprocal plot
The reciprocal of Equation 10.19 gives the following equation:
Double reciprocal plot
A graph of 1/r versus 1/[D] is called a double reciprocal plot.
The y intercept is 1/n and the slope is 1/nK a.
From this graph, the number of binding sites may be determined from the 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] does not yield a straight line, then the drug-protein
binding process is probably more complex.
Scatchard plot
• A Scatchard plot is a plot of the ratio of unbound drug versus the bound drug
concentration.
• It is a method for analyzing data for freely reversible drug/protein binding
interactions.
• The Scatchard plot spreads the data to give a better line for the estimation of the
binding constants and binding sites.
•A graph constructed by plotting
r/[D] versus r yields a straight line with
the intercepts and slope.
In-Vivo Methods (Unknown Protein Concentration)
Reciprocal and Scatchard plots cannot be used if the exact nature and amount of
protein in the system is unknown.
The percent of drug bound is often used to describe the extent of drug-protein
binding in the plasma.
The fraction of drug bound is equal to the ratio of the concentration of bound drug,
[Dβ], and the total drug concentration, [DT], in the plasma.
fraction of drug bound = the conc. of bound drug /the total drug conc.
The value of the association constant can be determined, even though the nature of
the plasma proteins binding the drug is unknown.
Conc. of both free and bound drug may be found experimentally
A graph obtained by plotting[Dβ ]/[D] versus [D ] will yield a straight line for which
the slope is the association constant K a.
Above eq. shows that the ratio of bound drug to free drug is influenced by the
1- Affinity constant,
2- The protein concentration, [PT], which may change during disease states,
and
3- 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,
The values for n and Ka give a general estimate of the affinity and binding capacity of
the drug
BECAUSE
Plasma contains a complex mixture of proteins.
The drug-protein binding in plasma may be influenced by competing substances e.g
ions, free fatty acids, drug metabolites, and other drugs.
Measurements of Drug-protein binding should be studied over a wide drug
concentration range;
BECAUSE
 at low drug concentrations a high-affinity, low- capacity binding site might be
missed
at a higher drug concentration, saturation of protein-binding sites might occur.
Relationship between Protein Concentration and Drug Concentration in
Drug-Protein Binding
• The drug concentration, the protein concentration, and the association (affinity)
constant, K a, influence the fraction of drug bound.
• With a constant concentration of protein, only a certain number of binding sites are
available for a drug.
• At low drug concentrations, most of the drug may be bound to the protein, whereas at
high drug concentrations, the protein-binding sites may become saturated, with a
consequent rapid increase in the free drug concentrations
As the protein concentration increases, the percent of drug bound increases to a max.
The shapes of the curves are determined by the association constant of the drug-protein
complex and the drug concentration.
Effect of protein concentration on the percentage of drug bound.
A, B, and C represent hypothetical drugs with respectively
decreasing binding affinity.
Protein Binding and Drug Exposure
 Free plasma drug concentration is more relevant than total plasma
drug concentration.
 When considering drug safety, how high and how long the free
plasma drug level will be sustained .
 This is often measured by the AUC for the free drug concentration.
The effect of changes in f u
Within therapeutic drug concentrations, the effect of changes in f u is apparently not
sufficient to change the efficacy of most drugs and is not of clinical concern.
More potent biological drugs with short elimination half-lives are used, plasma drug
concentrations may potentially fall several-fold and fu may change significantly at
various plasma conc.
Protein binding determination
1- Gel filtration.
2- Equilibrium dialysis.
3-Ultrafiltration.
4- Spectral changes.
Gel filtration
1-use of porous gel
2-seperates components on the basis of size.
3-Low molecular drugs are held on gel.
4-Bound drugs and protein washed away
Equilibrium dialysis
 A specific application of dialysis that is important for the study of the binding of
drugs by proteins.
It is physical and simple.
Protein solution containing drug and a buffer solution are place on opposite sides.
After sufficient time(12-24 hrs) free drug conc on both side will be same.
 membrane

Protein Buffer

Drug
Free drug

buffer Free drug

Ultrafiltration
Quicker method
 forces like pressure or conc. gradients lead to a separation through a semi
permeable membrane.
The equilibrated protein sample is applied to a filtration device and pressure
(centrifugal force) is applied to force the solution through a semi permeable (MW
cutoff) membrane.
Drug and protein solution

Membrane

Free drug filtrate

Spectral changes
Most drugs have distinct UV spectra.
When combines with protein spectrum may be changed due to alteration in
electronic configuration.
These alteration can be measured and extent of drug protein binding may be
determined.