Protein Binding

By: Pharma Tips | Views: 7168 | Date: 15-Jun-2010

Drug protein binding is the reversible interaction of drugs with proteins in plasma.Protein binding values (% fraction bound) are normally given as the percentage of the total plasma concentration of a drug that is bound to all plasma proteins.

Protein Binding

Protein Binding
Drug protein binding is the reversible interaction of drugs with proteins in plasma.
Protein binding values (% fraction bound) are normally given as the percentage of the total plasma concentration of a drug that is bound to all plasma proteins.
In most cases, binding to plasma proteins is reversible, and the concentration of the free and bound species of the drug at equlibrium may be expressed as:
The total plasma concentration of the drug is expressed as the sum of the percent of free drug and the percent bound.

For A drug moiety there are several tissue components comprising of blood and extravascular tissues available in the body for biological interactions to take place. The molecules indulged in such biological or physiological interactions are said to be macromolecules as they are large in structures and quite complex depending upon the type involved. These macromolecules are proteins, DNA, adipose tissues etc. thus the protein binding process is defined as a phenomenon of complex formation following the interaction of drug moiety and the protein molecule. 

The significance of protein binding of drugs is that - As a protein bound drug is neither metabolized nor excreted hence it is pharmacologically inactive due to its pharmacokinetic and Pharmacodynamic inertness. A bound drug always remains confined to a specific tissue or to a particular site with which it possess a greater affinity. The major benefit is the prolonged duration of action of drug as the protein bound-enormous size of drug complex cannot undergo membrane transport. 

Usually weak chemical bonds such as ionic bonds, hydrophobic bonds, hydrogen bonds or Vander wall’s forces are involved in protein binding of drugs and thus are defined as a reversible process. Covalent bond formation though being very rare may result into permanent or so called irreversible binding with a great potential of portending adverse effects like carcinogenicity, terratogenecity, tissue or organ toxicity etc. this has been widely observed in the therapeutic combination of chloroform and paracetamol whose metabolites gradually lead to hepatotoxicity.

Protein binding mechanism and DNA recognition
Transient interactions between both proteins and nucleic acids are ubiquitous and fundamental tomany subcellular processes. In recent studies we have shown that protein binding are robust and owing tothe minimal frustration principle, just as for protein folding, are governed primarily by the protein’snative topology (1-3). The native topology based landscape model, which corresponds to a perfectlyfunneled energy landscape, reproduces many of the grosser and finer structural and kinetic aspects ofvarious binding mechanisms found in the laboratory (3). Not only are our computational resultsconsistent with experiments, they also demonstrate that protein plasticity, as envisioned by the fly-castingmechanism, is more fundamental in protein recognition than traditionally imagined. An asymmetricbinding mechanism is often observed for the formation of the symmetric homodimers where one monomer is more structured at the binding transition state; a fact not directly accessible to inference fromkinetic studies at the ensemble level. In addition, models of this class have suggested a new bindingmechanism of the HIV-1 PR and thus a new way of its inhibition (4). The protein topology wassuccessfully implemented to predictsymmetric complexes that are formed via domain-swapping (5).

The topological-basedmodel has been recently used toresolve t h e longstanding
experimental puzzle on the folding of Rop dimer (6). The folding of Rop (repressor of
primer) dimer is exceptional because some of its mutants with a redesigned hydrophobic core both fold and unfold much faster than the wild-type (wt) protein.This appears to conflict with a simple funneled energy landscape for which topology mainly determines the kinetics. We propose that the mystery of Rop folding can be unraveled by assuming a double funneled energy landscape (Fig. 1A) where there are two basins that correspond to distinct but related topological structures. Owing to the near symmetry of the molecule, mutations can cause a conformational switch to a nearly degenerate yet distinct topology or lead to a mixture of both topologies. The topology predicted to have the lower free energy barrier height for folding was further found by all-atom modeling to give a better structural fit for those mutants with the extreme folding and unfolding rates. Thus, the non-Hammond effects can be understood within energy landscape theory if there are in fact two different but nearly degenerate structures for Rop

In a recent study, native topology-based model supplemented by electrostatics was used to study protein-DNA binding. We have found that the heat capacity for folding is shifted to lower temperatures with decreasing the dielectric constant (Fig. 2). This is accompanied with broadening of the heat capacity plots. Accordingly, strong electrostatics destabilizes the protein as the attractive and repulsive forces pull the protein residues. This can result in changing the folding mechanism by stabilizing intermediates. Moreover, similar to protein-protein binding, flexibility was found to be fundamental in protein-DNA binding where protein flexibility acts in concert with electrostatic long-range forces to maximize efficiency of protein-DNA binding (8).

Figure 2. (A) The complex between Sap-1 protein and DNA (positively and negatively charged residues or phosphate atoms are shown by red and yellow beads, respectively). (B) The specific heat profiles for folding for different values of dielectric constants. (C). The effect of protein flexibility on the binding efficiency to DNA (Q equals 1 means a rigid protein).



(a) Physiochemical characteristics of the drug.
Lipophilicity is the most desirable physiochemical parameter that is perquisite for protein binding to occur. Also an increase in the lipid content of drug moiety eventually enhances the rate as well as extends of protein binding process. As observed in case of
intramuscular. Injection of cloxacillin as attributed to greater lipophilicity displays 95% protein binding.

(b) Concentration of drug in the body.
Alteration in the concentration of drug substance as well as the protein molecules or surfaces subsequently brings alteration in the protein binding process. 

(c) Drug’s affinity towards protein/tissue.
This factor entirely depends upon the degree of attraction or affinity the protein molecule or tissues have towards drug moieties. For Digoxin has more affinity for cardiac muscles proteins as compared to that of proteins of skeletal muscles or those in the plasma like HSA.


(a) Concentration of protein/binding component.
This is the most important tissue related parameter to be given priority. As the human serum plasma proteins constitute the major portion of  the plasma  proteins, a large number of drugs undergo an extensive binding with them as compared to the concentration of other protein molecule

(b) Number of binding sites on the protein
In association to the concentration of proteins molecules available the number of binding sites available in the protein molecules is also significant. Albumin not only possesses large number of binding sites but also has greater potential of carrying out binding process. Numerous drug exhibit multiple site binding with albumin molecules in plasma like fluocloxacillin, ketoprofen, indomethacin etc .

Types of Interactions

Protein-Protein Docking Interactions

protein docking with another protein
Protein-protein interactions occur between two proteins that are similar in size. The interface between the two molecules tend to be flatter and smoother than those in protein-ligand interactions. Protein-protein interactions are usually more rigid; the interfaces of these interactions do not have the ability to alter their conformation in order to improve binding and ease movement. Conformational changes are limited by steric constraints and thus are said to be rigid.

Protein Receptor-Ligand Docking
Also known as the molecular docking technique, protein receptor -ligand docking is used to check the structure, position and orientation of a protein when it interacts with small molecules like ligands. Protein receptor-ligand motifs fit together tightly, and are often referred to as a lock and key mechanism. There is both high specificity and induced fit within these interfaces with specificity increasing with rigidity. Protein receptor-ligand can either have a rigid ligand and a flexible receptor, or a flexible ligand with a rigid receptor.

Rigid Ligand with a Flexible Receptor
The native structure of the rigid ligand flexible receptor often maximizes the interface area between the molecules. They move within respect to one another in a perpendicular direction in respect to the interface. This allows for binding of a receptor with a larger than usual ligand. Normally when there is ligand overlap in the docking interface, energy penalties incur. If the van der Waals forces can be decreased, energy loss in the system will be minimized. This can be accomplished by allowing flexibility in the receptor. Flexible receptors allow for docking of a larger ligand than would be allowed for with a rigid receptor.

Flexible Ligand with a Rigid Receptor
When the fit between the ligand and receptor does not need to be induced, the receptor can retain its rigidity while maintaining the free energy of the system. For successful docking, the parameters of the ligand need to be constant and the ligand must be slightly smaller in size than that of the receptor interface. No docking is completely rigid though; there is intrinsic movement which allows for small conformational adaptation for ligand binding. When the six degrees of freedom for protein movement are taken into consideration (three rotational, three translational), the amount of inherent flexibility allowed by the receptor is even greater. This further offsets any energy penalty between the receptor and ligand, allowing for easier, more energetically favorable binding between the two.
Docking Problems
The search space consists of all possible conformations and configurations. With present computing resources, it would be impossible to exhaustively explore the search space for all possible poses (a pose is the name given to the configuration of the conformation of a molecule in a coordinate system). Needless to say, every docking simulation is a trade-off between accuracy and speed and a good docking tool is expected to maintain a reasonably good balance between the two. See also Protein-Protein Interaction Network Visualization and Prediction Methods for Interactions and Docking.

(a) Competitive binding of drugs.
Displacement interactions are predominant ones among these reactions. In case where two or more drugs have same or identical affinity for a same site then they struggle with one another to bind at the same site.  Consider a drug I is bound to a specific site on the molecule and if a second drug called as Drug II is administered now, then the drug meaty having greater affinity towards the bound site would effectively displace th e former drug. This phenomenon is said to be Displacement reaction. The drug which is been removed from its binding site is said to be displaced drug while the one that does the displacement is called as displacer. 
The best example for such interactions is the competitive protein binding that occurs between Warfarin and phenylbutazone for HSA, as both are potent binders of HAS, where phenylbutazone is displacer while warfarin is displaced. Clinically such reaction acquire importance when  the displaced drug (any) is more than 95% bound to plasma proteins, or occupies small volume of distribution even less than that of 0.15 L/Kg. also when the active drug or the administered pharmacological agent possess narrow therapeutic index. Such situation may also develop in case the displacer drug has greater affinity or at the same time the drug/protein concentration ratio is very high and exhibits a very rapid and significant increase in the plasma concentration of drug.
Using 2 drugs at the same time may affect each other's fraction unbound. For example, assume that Drug A and Drug B are both protein-bound drugs. If Drug A is given, it will bind to the plasma proteins in the blood. If Drug B is also given, it can displace Drug A from the protein, thereby increasing Drug A's fraction unbound. This may increase the effects of Drug A, since only the unbound fraction may exhibit activity. See the example below:
Before Displacement After Displacement  % increase in unbound fraction
Drug A   
% bound 95 90 
% unbound 5 10 +100
Drug B   
% bound 50 45 
% unbound 50 55 +10
Note that for Drug A, the % increase in unbound fraction is 100%-- hence, Drug A's pharmacologic effect has doubled. This change in pharmacologic effect could have adverse consequences.
This effect of protein binding is most significant with drugs that are highly protein-bound (>95%) and have a low therapeutic index, such as warfarin. A low therapeutic index indicates that there is a high risk of toxicity when using the drug. Since warfarin is an anticoagulant with a low therapeutic index, warfarin may cause bleeding if the correct degree of pharmacologic effect is not maintained. If a patient on warfarin takes another drug that displaces warfarin from plasma protein, it could result in an increased risk of bleeding


Patients related factors have their own importance after all the drug has to generate its response on to the administered patient. In this numerous parameters are taken into account like Age, diseased state, pharmacokinetic and Pharmacodynamic characteristics. Protein content and its specific type greatly varies with the age factor. As observed the neonates or newly born babies have very low levels of albumins in the plasma thereby resulting in rebound concentration of drug that is primarily bind to albumin is a major shortcoming. As far as elderly patients are concerned the albumin levels goes down while the concentration of AAG is high enough. 

Disease states: the alterations in protein content and thereby the rate and extend of protein binding is greatly influenced by the albumin which is the major drug binding protein. This may ultimately lead to hypoalbuminemia which eventually with the pace of time completely impairs the entire  protein drug binding process. For such situations the basic pathological conditions of diseases like trauma, burns, renal, cardiac or hepatic failure etc are largely responsible. Pharmacokinetics as well as Pharmacodynamic of drugs greatly influences the distribution, clearance and thus the biotransformation of drugs to a greater extend.   an increased potential of toxicity is observed due to increased concentration of free or the unbound drug.


(A) Binding of drugs to blood components which includes
a. plasma proteins
b. blood cells

(B) Binding to extravascular tissues like proteins, fats, bones etc.


There are several blood components for a drug to be exposed to and undergo protein binding process.

I. Plasma- protein drug binding
Blood components mainly plasma proteins and RBC’s are the major portion of the bulk that actually interacts with the drug moieties as soon as they enter the blood systemic circulation. The plasma proteins being in surplus amounts in the blood undergoes major complexation with drug also this reaction or process is usually reversible due to large variety available in types of proteins. The sequence of protein-drug binding is: Albumin> α1-Acid Glycoprotein > Lipoproteins>Globulins

A drug's efficiency may be affected by the degree to which it binds to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Common blood proteins that drugs bind to are human serum albumin, lipoprotein, glycoprotein, α, β‚ and γ globulins.
A drug in blood exists in two forms: bound and unbound. Depending on a specific drug's affinity for plasma protein, a proportion of the drug may become bound to plasma proteins, with the remainder being unbound. If the protein binding is reversible, then a chemical equilibrium will exist between the bound and unbound states, such that:
Protein + drug ⇌ Protein-drug complex 
Notably, it is the unbound fraction which exhibits pharmacologic effects. It is also the fraction that may be metabolized and/or excreted. For example, the "fraction bound" of the anticoagulant warfarin is 97%. This means that of the amount of warfarin in the blood, 97% is bound to plasma proteins. The remaining 3% (the fraction unbound) is the fraction that is actually active and may be excreted.
Protein binding can influence the drug's biological half-life in the body. The bound portion may act as a reservoir or depot from which the drug is slowly released as the unbound form. Since the unbound form is being metabolized and/or excreted from the body, the bound fraction will be released in order to maintain equilibrium.
Since albumin is basic, acidic and neutral drugs will primarily bind to albumin. If albumin becomes saturated, then these drugs will bind to lipoprotein. Basic drugs will bind to the acidic alpha-1 acid glycoprotein levels of albumin, alpha-1 acid glycoprotein, and lipoprotein.

(a) Binding of drugs to human serum albumins.

The human serum albumin also abbreviated as HSA is a plasma protein with a molecular weight of 650000 and comprises of 59% of total plasma protein content present in the blood. It is most abundantly present in plasma with a very high potential of binding drugs. It has been widely observed that there is no equilibrium  between the concentration of drug and that of HSA, as the administered dose is usually smaller as compared to that of plasma proteins present. Almost all types of drugs whether acidic, basic or neutral drugs undergo significant HSA binding. 

Experimental studies have postulated that any drug can bind to numerous protein binding site .In such circumstances the primary site is the major binding site and the other as the secondary site; for example, for dicoumarol site I is the primary site while site II is secondary. Those drugs having affinity for the same binding sites compete with one another. But the non-competitively binding drugs either promote or inhibit binding of a drug to another site. This is usually accomplished by mechanism of coupling. There are four different sites for the drugs to bind. 
Many substances (generally endogenous compounds) such as tryptophan, saturated fatty acids, unsaturated fatty acids, bile salts or bilirubin etc all exhibit effective albumin binding . This is due to diversity in the structures of proteins,  
the structures of free drug moiety and their affinity towards the protein molecule.

The different sites are:

Site I:To this specific site a large population of drugs bind like Non-Steroidal Anti-Inflammatory Drugs mainly phenylbutazone, indomethacin, many sulfonamides e.g.; sulfamethoxine, sulfamethizole, and even many anti-epileptic drugs like phenytoin etc. this site is also called as Warfarin binding site or as Azapropazone binding site. 
Site II:This is actually said to be Diazepam binding site. benzodiazepines, medium chain fatty acids, ibuprofen, ketoprofen, etc. bind extensively at the very site. This is 
so because due to structural changes the following drugs have high and specific affinity for the site. At both the sites I&II many drugs are known to bind.
Site III:This very protein site is called as Digitoxin binding site
Site IV:This is referred as Tamoxifen binding site.
At the sites III & IV very few drugs are known to bind. 
Characterization of Binding Sites
Protein stability and flexibility are dependent on electrostatic interactions such as hydrogen bonding and van der Waals forces. The energy of the interaction is not evenly distributed between the two proteins at the interface. Rigidity in the interface ensures that the entropy loss is offset and the binding free energy is contributed to in a favorable way by the amino acid residues contained in the interface. Contributions of hot spots to the stability of the protein-protein complex within a hot region is cooperative, however the contributions of independent hot regions are additive.
• Hydrogen Bonding/Hydrophobic Packing 
• Dehydron Bonds 
• Salt Bridges 
• van der Waals 
• Cation-π Interactions 
• Pockets 
• Hot Spots 
• Protein Chips 
Hydrogen Bonding and Hydrophobic Packing
Hydration is very important for the three dimensional structure and activity of proteins. Surface water molecules are held most strongly by the positively charged basic amino acids. Hydrogen bonding adds conformational flexibility, while the level of hydration determines the degree of flexibility in the internal molecular motions. Water molecules in close proximity to peptide hydrogen bonds cause separation in the bonds and a release of tension in the structure. The presence of water molecules also prevents swift conformational fluctuations from occurring. Water interacts with protein surfaces; the breaking of hydrogen bonds between the water molecules and the amino acids has an unfavorable enthalpic result on the system, effecting the overall energy of the protein. Water molecules act as an adhesive, sealing holes in complementary surfaces that would normally lack shape. Hydrogen bonding drastically reinforces other bonding present in the interface between proteins. This type of bonding occurs when an hydrogen atom proximal to a docking facade interacts across the interface with atoms of another molecule. Hydrogen bonding can occur between side chains, backbone groups, and between the two.[1]
Hydrophobic surfaces located at ligand receptor interfaces have greater ambiguity with bonding. Hydrogen bonding between the peptide atoms decreases the hydrophobicity of the backbone. Coincidentally, hydrophobic surfaces can be sealed with an ordered shell of water molecules, creating an entropically favorable state when water molecules are ejected. Water removal increases the thermodynamic benefit in hydrophobic patches. These patches are uncommon though; the patches found in proteins are more often hydrophilic. The secondary structure of the protein is a result of hydrogen bonding since the residues arrange themselves in such a way so that hydrophobicity of the backbone is reduced. α-helices and β-sheet conformations are the way in which is accomplished.[2]
Amino acids in order of hydrophobicity
Least hydrophobic to most hydrophobic:
• Lysine 
• Glutamic Acid, Aspartic Acid 
• Arginine, Glutamine, Serine, Aspargine 
• Proline, Glycine, Threonine 
• Histidine, Alanine 
• Tyrosine 
• Cystine, Valine, Tryptophan, Methionine 
• Leucine, Isoleucine, Phenylalanine 
Dehydron Bonding
The hydrogen bonds in the backbone of a protein usually exist in the form of dehydron bonds, which form between two hydrogen atoms bonded that differ in polarity bonds. The proton acceptor in this case is a metal hydride with the donor being OH or NH. Dehydrons attract hydrophobes, their bonding increases the electrostaticity of hydrogen bonds by increases the surrounding dielectric coeffiecient. The dehydron bonds thus create a hydrophobic shell around the backbone increasing the electorstatic interaction; this desolvated environment is condusive for salt bridge formation increasing structural stability.[3]

Salt Bridges
Salt bridges, also referred to as ionic bonds, are a form of chemical bonding that occur between amino acids of opposite charge. They contribute greatly to the stability of the protein and are usually found in the interior of the protein structure. This stability is dependant on the location of the side chains, and the electrostatic contribution. Hydrophobic environment is conducive to salt bridges. Charged residues within four angstroms to each other have the ability to form salt bridges.[4]
Van der Waals Interactions
Van der Waal forces occur between dipoles. Covalently bonded atoms differ in electronegativity that causes a minute dipole. Since the distribution of electrons is asymmetrical, a dipole effect is created by the van der Waals attraction. These dipoles oscillate, creating a dipole field. This field allows two atoms with identical oscillation frequencies to synchronize, one negative while the other positive. Thus the charge between the two cancel each other out and the net charge is zero. van der Waals bonds are one of the weakest intermolecule forces studied. But van der Waal bonds are important, since it is with these forces that noble gases can achieve bond saturation with the dominant form of interaction between electrically neutral species. Induced dipole interactions cause van der Waal bonding to occur, and the large close surface contact allows van der Waal binding to contribute significantly.[5] The following formula is used to determine van der Waal bonding:
A/(r6) - B/(r12) where r is the distance between A and B.

A protein pocket is a concave surface region containing the active sites of the protein. These areas are solvent accessible, and are characterized as either unfilled or complemented, the partner conformation not being taken under consideration. Compositional difference between complemented pockets and other surface pockets may be useful for predicting complemented pockets from the unbound states. The floor of complemented pockets are often filled with residues that are ionizable and/or polar. In a hydrophobic environment, these residues provide molecules for salt bridge formation and hydrogen bonding which are essential for binding stability. Pockets are crucial for binding site flexibility for large interfaces reduce binding stability, and allow for ligand movement.
Cation-π Interactions
Cation-π pairs are frequently present in the interface of proteins, probably due to their low energy conformation. They play an important role in specificity, causing a need for conformational changes to provide the correct orientation for binding. Cation-π interactions are found most frequently in homodimers. The abundance of this form of bonding is tied to the occurrence of specific amino acids. In homodimers, arginine bound to phenylalanine forms the most frequent cation-π bonds, while in the other protein complexes arginine-tyrosine and arginine-tryptophan cation-π bonding is a more likely occurrence.
Hot Spots
A hot spot is an area of high energy and binding around an amino acid residue. Surrounded by hydrophobic pockets, hot spots are found in clusters, rather than scattered throughout the interface. This clustering assists with the elimination of water molecules, and increases the bond strength and available bond energy through increases charge-charge relations. Hot spots usually do not include hydrogen bonding, electrostatic interactions, although there is a slight preference of non-polar residues. Packing around hot spots is significantly tighter that the other residues found in the interface. Since hot spots contribute significantly to binding, the association of neighboring residue size, charge, and interaction must be taken under consideration when considering binding affinity and docking. It has been established that a linear relationship between the number of hot spots and the interface size exists; the larger the interface the higher the number of hot spots present.
protein Chips
Much like DNA microarrays, and gene chips, protein chips have revolutionized the method and speed with which scientists can discover differences in gene expression levels, protein chips are a relatively new addition to proteomics that brings high-throughput efficiency to discovering protein-protein interactions. Earlier protein chips emulated existing proteomic techniques by requiring labeling of proteins, a requirement that resulted in only revealing certain proteins or classes of proteins. Recent developments have put forth label-free techniques that couple the original protein chip idea with MALDI-TOF mass spectrometry, and other forms of mass spectrometry, to produce a protein chip technology that does not require proteins to be labeled, consequently widening the range of protein-protein interactions detected with each chip.[6][7] While protein chip technology does not directly contribute to binding site characterization, it is a useful method for gaining insight into how proteins and classes of proteins function through their interactions with many other proteins. See chapter on protein chips more more information.

b) Drugs Binding to α1-Acid Glycoprotein.
The acid glycoprotein exists with a 44,000 molecular weight and comprises of 0.04 to 0.1 g% of the total plasma concentration of proteins. It is actually called as the Oromucoid as it mainly binds to basic drugs like Imipramine, Desipramine, lidocaine, Quinidine, etc.

(c) Binding of drugs to lipoproteins
Lipoproteins are those macromolecules present in plasma which portends a greater capacity of forming hydrophobic bonds. The major reason attributed to this is the larger lipid content present in them. But the plasma concentration of lipoproteins is very limited as compared to that of HSA and AAG.
The lipid core of Lipoproteins has their outer core to be made of apoproteins while the internal core is a potpourri containing triglycerides and esters of cholesterol.
The acidic drugs like diclofenac bind extensively to lipoproteins. Similar response are generated by basic drugs like chlorpromazine and neutral drugs like cyclosporine A.
Lipophilic drugs (basic) exhibit greater affinity for such type of protein binding. Lipoprotein binding is predominant when larger quantity of drugs bind to them and also in circumstances revealing lower plasma levels of HSA and AAG.  The process of binding involves the dissolution of drug molecules into the lipid core of the protein and hence the binding capacity is in direct relation with the lipid content. This binding of drugs to lipoproteins is noncompetitive in nature.  The lipoproteins have molecular weights ranging from 2 to 3 lakhs. This in turn is proportional to the chemical 
composition of the molecules. They are classified on the basis of their density. The 4 classes of lipoproteins are observed depending upon their variations in density:

1. Very Low Density Lipoproteins (VLDL)
2. Low Density Lipoproteins (LDL)
3. High Density Lipoproteins (HDL).
4. Chylomicrons

(c) Binding of drugs to Globulins
Different types of plasma proteins are labeled here below:
(i) α1-globulins : This is called as CBG  elaborated as corticosteroid binding globulin as it largely binds to steroidal drugs such as cortisone, hydrocortisone, prednisone etc. Thyroxin and Cyanocobalamin are also effectively bound to above proteins.
(ii) α2-globulins: It binds vitamins A, D,E and K and cupric ions and hence is    
broadly called as Ceruloplasmin
(iii) β1-globulins: As it mainly binds to ferrous ions or ferric compounds are
known as Transferring binding site.
(iv) β2-globulins: Carotinoids binding is mainly observed.
(v) γ-globulins: Specific antigens binding is observed due to high selectivity and   
specificity of the site.

RBC’s are the major blood cells which rates about 40% of total blood. The red blood corpuscles  constitute 95% of the total blood cells concentration in the body. The diameter or the width of the RBC’s is 500 times higher than that of plasma proteins. The 3 compartments which being the major portion of red blood cells to which drugs can bind are: 

(a) Hemoglobin: this has molecular weight & structural similarity to that of HAS but the concentration is much higher than of albumins in blood. Examples of drugs that bind are phenytoin, pentobarbital etc.

(b) Carbonic Anhydrase: Carbonic anhydrase inhibitors mainly bind to the site like chlorthaizine.

(c) Red Blood cell membrane: basic drugs like imipramine are known to bind to RBC membrane.Both the hydrophilic and lipophilic drugs can enter RBCs but the lipophilic drugs can do to a greater extend.

The tissue binding of drugs are also very significant processes occurring in the body. Unlike HSA, the body tissues constitute100 times that of HAS i.e; about  40% of the total body weight . Multiple tissue drug binding are feasible. Tissue drug binding is very essential and vital process as it assists in enhancing the apparent volume of distribution for drugs as this follows a direct relation with the ratio of concentration of drug in body to-free or unbound drug in plasma.
Also it results in prolonged duration of action due to increase in half life reason being the localization of drug at a specific site in the tissues. Studies also reveal that a very large population of drugs no matter acidic, basic or neutral undergoes reversible binding whereas the plasma protein drug binding exhibits vice-versa. The order of binding to extravascular tissues is given as:
Liver> Kidney> Lung> Muscle.lets have an overview of some tissue drug binding. 

1. Liver: Irreversible binding of drugs like paracetamol and their epoxide-metabolites to liver tissues results in hepatotoxicity.

2. Lungs: like imipramine, chlorpromazine and antihistamines accumulation of drugs like imipramine, desipramine or other drugs in lungs eventually leads to congestion in heart or may even produce severe lungs cancer.
3. Kidneys: the protein called as metallothion is widely present in kidneys which have a tendency to undergo complexation with heavy metals such as lead, mercury and cadmium. This gradually paves path for  the major renal failures or renal toxicity.

4. Skin: Many drugs are known to accumulate in skin with subsequent reaction with melanin which can ultimately result in skin diseases. Drugs such as chloroquine , phenothiazines are usually involved in this.

5. Eyes: the retinal pigments of the eye also contain melanin. Drugs like chlorquine are responsible for retinopathy as these drugs they interact with the melanin present in the retinal pigments.

6. Bones: bones are made up of calcium and most of the antibiotics mainly like tetracycline exhibits extensive binding to bones and teeth. The permanent brown-yellow discoloration of teeth is an adverse effect of administration of such antibiotics to newly born babies or infants. Similarly lead also generates similar responses with teeth and bones.

Structural Biochemistry/Protein function/Ligand
A ligand is a small molecule that is able to bind to proteins by weak interactions such as ionic bonds, hydrogen bonds, Van der Waal, and hydrophobic effects. In some cases, a ligand also serves as a signal triggering molecule.
For example, oxygen is the ligand that binds to both hemoglobin and myoglobin.

Binding site: a region of the protein that is complementary to a specific molecule or ion. This site usually exhibits specificity to ligands. The weak interactions of the primary structure of protein, specifically the side chains to the ligand, usually initiates a response. The concentration at which all binding sites are bound to a ligand is termed the point of saturation.
Induced fit is the concept that an enzyme is a flexible rather than a rigid entity. Interactions between the active site and substrate continually reshape the tertiary structure slightly. Instead of the substrate simply binding to the active site, the enzyme and substrate mold to induce a fit similar to that of a lock and key. This allows the substrate to be in the precise position to enable a catalytic response.
Dissociation constant: Kd is the tendency for a ligand to bind to a binding site. It is measured by the ratio of concentrations of the ligand and enzyme over the concentration of the Enzyme-ligand complex. It is equal to the concentration of the ligand at which the total binding sites are half occupied. Association constant is equal to the reciprocal of the dissociation constant.
Cooperativity: Allosterism: Hill equation:
A molecule, atom, or ion that is charged or neutral and of non-bonding pairs of electrons as electron donors or Lewis bases that form bond to a central metal atom or ion to be as complex ion; it is important for control of chemical reactivity of the complex of ligands and metal; monofunctional ligands are complex ions that have one non-bonding pair of electrons, polyfunctional or known as chelates, two or more. Biological ligands are mostly electron-donating groups; important one of biological system is heme that is of nitrogen donating groups and forms chelate structure.
When a ligand binds to the protein, the chemical conformation of the protein changes. The tertiary structure of the protein is altered. The conformation of the protein determines the function of the protein, as structure often denotes a lot about the function. The tendency in which the ligand binds to the protein is known as the term affinity.
The binding affinity depends on the interaction of the binding site with the ligand. When the interaction of the intermolecular forces between the ligand and binding site are high, the affinity is increased. Similarly, when the intermolecular forces between the ligand and binding site are weak, the affinity is low.
When the affinity is high for the ligand binding, the concentration of the ligand does not need to be high in order for the ligand to bind to its maximum potential. Similarly, when the affinity is low, the concentration of the ligand must be large in order for the ligand to bind properly to the binding site.
Introduction about ligand
A ligand is a substance that has the ability to bind to and form complexes with other biomolecules in order to perform biological processes. Essentially, it is a molecule that triggers signals and binds to the active site of a protein through intermolecular forces (ionic bonds, hydrogen bonds, Van der Waals forces). The docking (association) is usually a reversible reaction (dissociation). Within biological systems, it is rare to find irreversible covalent bonds between the ligand and its target molecule. The chemical conformation is changed when the ligand bonds to its receptors. For example, the three dimensional shape of the receptor protein is change upon the binding of the ligand. Also, the conformational state of a receptor protein will cause variations in the functional state of a receptor. The strength/tendency of the ligand binding is known as affinity. Different types of ligands include substrates, inhibitors, activators, and neurotransmitters.
Receptor/Ligand Binding Affinity
Binding affinity is the interaction of ligands with their binding sites. A higher affinity ligand binding is usually the result of greater intermolecular force between the ligand and its receptor and involves a longer residence time for the ligand at its receptor binding site. A lower affinity ligand binding, though, involves less intermolecular force between the ligand and its receptor and involves a shorter residence time for the ligand at its receptor binding site. High affinity is essential to the physiology of humans when the binding energy can be used to cause a conformational change in the receptor, which results in altered behavior of an associated ion channel or enzyme. An agonist for any receptor is a ligand that can alter the function of the receptor and cause a physiological response. Agonist binding to a receptor is characterized by how much physiological response can be caused as well as its concentration required to produce the physiological response. High affinity ligand binding means that a relatively low concentration of a ligand will be needed to maximally occupy a ligand binding site and cause a physiological response. Low affinity ligand binding means that a relatively high concentration of a ligand is needed prior to maximizing the occupancy of the binding site and the maximum physiological response to the ligand is achieved. An agonist that is able to partially activate the physiological response is called a "partial agonist." Ligands that bind to a receptor, but do not activate the physiological response, are receptor "antagonists." Ligand binding is usually characterized by the concentration of ligand at which half of the receptor binding sites are occupied, known as the dissociation constant (kd). Binding affinity is usually determined through the use of a radiolabeled ligand called a "hot ligand." Homologous competitive binding experiments use the binding-site competition between a hot and cold ligand.


Drug Targeting:-
Targeted drug delivery is the major research area in this era of medical and life sciences. The binding of drugs to lipid containing proteins called as lipoproteins is effectively utilized for controlled and site-specific drug delivery. For this the hydrophilic drug moieties are of great priority. The major application of delivering the drug at a predetermined rate is highly beneficial in treatment and appreciable management of cancer therapies. 
The best requirement of site-specific drug delivery is revealed in cancer treatment with that of estramustine. Thus binding of suitable antineoplastic agents like vincristine or vinblastine with the LDL is desirable to prevent the normal cells from damage caused by the administered drug. 
The protein drug binding also portends an advantage of efficient drug distribution, absorption and finally prolonged duration of action for longer treatment of chronic diseased conditions.

The absorption equllibrium is attained by transfer of free drug from the site of administration e systemic circulation and when the concentration in these  two compartment becomes equal.following equilibrium the process may stop.however binding of the absorbed drug to plasma protein decreases free drug concentration and disturbs such an equilibrium.thus sink condition and the concentration gradiant are reestablished which now act as the driving force for further absorption.thise is particularly useful in case of ionized drugs which are transported with difficulty.

Systemic solubility of drugs:-
Water insoluble drugs,neutral endogeneous macromolecules such as heparin and several steroids and oil soluble vitamins are circulated and distributed to tissue by binding especially to lipoprotens which act as a vehical for such hydrophobic compound.

Plasma protein binding restricts the entry of drugs that have specific affinity for certain tissues.Thise prevents accumulation of a large fraction of drug in such tissues and thus,subsequent toxic reactions.Plasma protein drug binding thus favors uniform distribution of drug throughout the body by its buffer function.maintains equilibrium between the free and the bound drug.a protein bound drug in particular does not cross the BBB the placental barrier and the glomerulus.


If P represant and D the drug,then applying law of mass action to reversible protein-drug binding,we can write:

P+D  PD ………………………………………………….(1)

At equilibrium,

Ka = [PD]/[P] [D] ……………………………………………….(2)

[PD] = Ka [P] [D] …………………………………………...…..(3)


[P]=concentration of free protein
[D]= concentration of free drug
[PD]= concentration of preotein-drug complex
Ka= concentration rate constant
Kd= concentration rate constant

Ka>Kb 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]…….…………...………………………………..(4)

If r is the number of moles of drug bound to total moles of protein, 

r  = [PD] / [PT]

= [PD] / [PD] + [P]………………………………..…………(5)

Substituting the value of [PD] from equation (3) in (5),We get

r = Ka [P] [D] / Ka [P] [D] + [P]

= Ka [D] / Ka [D] + 1…………………...……………………..(6)

Equition (6)_ holds when there is only one binding site on the protein and the protein-drug complex is 1:1 complx.If more than one or N number of binding sites are available per mole of the protein then:

r = N Ka [D] / Ka [D] + 1 …………………………...………….(7)

The value of association constant Ka and the number of binding sites N can be obtained by plotting equation (7) in three different ways as shown below.

Is made by plotting r versus [D] as shown below

Is made by transforming equ. (7) in to a liniar form,thus

r = N Ka [D] / Ka [D] + 1

r + r Ka [D] = N Ka [D]

r = N Ka [D] - r Ka [D]


r / [D] = N Ka - r Ka

A plot of r/[D] versus r yield a straight line.slop of the line= -Ka,y-intercept =NKa 
x-intercept = N

The reciprocal of equation (7) yield:

1/r = 1/ N Ka [D] + 1/N ………………………………………(8)

A plot of 1/r versus 1/[D] yield a straight line with slop 1/NKa and y-intercept 1/N

Plasma Protein Binding Assays(9,15,24,28)
The pharmacokinetic and pharmacodynamic properties of drugs are largely a function of the reversible binding of drugs to plasma or serum proteins. Such proteins include albumin, α1-acid glycoprotein, lipoproteins and α, ß‚ and γ globulins. Generally, only the unbound drug is available for diffusion or transport across cell membranes, and for interaction with a pharmacological target (e.g. receptor, ion channel, transporter, enzyme). As a result, the extent of plasma protein binding of a drug influences the drug’s action as well as its distribution and elimination. Highly plasma protein bound drugs are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs and tissues, resulting in large volumes of distribution. The binding of drugs to proteins both in the vascular space and/or the extravascular space results in a decrease in drug clearance and a prolonged drug half-life. Only the unbound drug is available for glomerular filtration and, in some cases, hepatic clearance. However, for high extraction ratio drugs, clearance is relatively independent of protein binding.
NoAb BioDiscoveries offers plasma or serum protein binding assays using the equilibrium dialysis method. Equilibrium dialysis studies are offered using a 96-well Teflon dialysis unit. Combined with LC-MS/MS analysis for test compound quantification, the 96-well format is a rapid throughput method that is excellent for screening compounds to identify those with high, medium and low binding fractions. The 
system can be used to determine the fraction of a compound bound and unbound to plasma proteins and the effect of concentration on the extent of binding. 

Key Features of the Assay
• can use whole plasma or an individual plasma protein
• plasma from difference species are available, including human, dog, rabbit, rat, mouse        
• using the 96-well format, the assay achieves a higher throughput and can be used to screen small libraries of compounds

Assay Applications
• early prioritization of compounds or compound series
• assessment of plasma proteins which contribute to the binding of the compound
• early determination of species or sex differences
• estimation of dose level for in vivo studies.

Assay Principle
The assay is performed in a 96-well Teflon dialysis unit. Each well consists of 2 chambers separated by a vertically aligned dialysis membrane of predetermined pore size. Plasma spiked with the compound of interest is added to 1 chamber and buffer to the other chamber. Over time, free compound diffuses from the plasma chamber to the buffer chamber until an equilibrium is reached. The unbound fraction is calculated as the concentration in the buffer side divided by the total concentration in the plasma side.

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11) Gibson, C.G.  And Skett, P.: Introduction to Drug Metabolism, London, Chapman and Hall, 1986.
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