Complexation and protein binding

What is complexation Classification of complexes Metal Ion complexes Organic molecular complexes No-bond complexes or inclusion compound Concept on Protein binding Advantage of Protein binding Mechanism of protein binding

Complexation can defined as an association of two or more species capable of independent existence . Complexes generally result from a donor acceptor mechanism The donor compound is a non metallic atom or ion, which can donate an electron pair. The acceptor is usually a metallic ion or a neutral atom which is capable of accepting a pair of electrons Intermolecular forces involved in the formation of Complexation are Van der Waals forces, dipolar and induced dipolar types.

Classification of Complexes: Complexes are classified according to the type complex which is formed: Metal Ion Complexes: A. Inorganic type B. Chelates C. Olefin type D. Aromatic type i. Pi bond complexes ii. Sigma bond complexes Sandwich compounds

2. Organic Molecular Complexes A. Quinhydrone type B. Picric acid type C. Caffeine and other drug complexes (hydrogen bonded complexes) D. Polymer type

3. No-Bond Complexes or Inclusion Compounds A. Clathrate B. Channel lattice type C. Layer type D. Monomolecular type E. Macromolecular type

Metal- Ion Complexes Inorganic Complexes In this type of complex, the central atom or acceptor in the complex is a metal or a metal ion which accepts electrons from the donor. The donor compound is also known as the ligand and is said to be coordinated with the acceptor molecule. The type of bonding between the metal and the ligand may be electrostatic or covalent. Example:
3 Hexamine cobalt III chloride Co NH 6 Cl formed by reaction between ammonia and cobalt chloride.

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Electron spin resonance spectroscopy and magnetic susceptibility techniques are used to find out the number of unpaired electrons in a compound

Chelates (Greek: kelos meaning claw). A substance containing two or more donor groups may combine with a metal ion to form a complex known as chelate The bonds in the chelate may be ionic or primary covalent type or coordinate covalent type. Ligands may have more than one group capable of bonding with the metal ion. When the ligand provides two centres for attachment to the central metal ion, the chelate is known as bidentate. A molecule with three donor groups is called tridentate and so on.

EDTA

Ethylenediamine tetraacetic acid (EDTA) is a popular chelating agent. It has six points for attachment for the metal ion (two nitrogen and four oxygen donor groups) and is therefore called hexadentate. If the ligand forms a stable, water soluble metal chelate, it is called a sequestering agent. Sequestration is used in analysis and in the removal of unwanted ions in solution. For example, EDTA is extensively employed as a sequestering agent to remove calcium ions from hard water.

In the pharmaceutical field, EDTA has been used to prevent discolouration (due to traces of metal ions) in antibiotics and in anti histaminic and anaesthetic preparations. It is useful for preventing oxidation (by chelating the metal ion which may induce oxidation) in cosmetic creams or lotions.

Olefin Complexes Aqueous solutions of certain metal ions such as platinum, iron, palladium, mercury and silver can absorb olefin such as ethylene to yield water soluble complexes. these have been employed as catalysts in the polymerization of unsaturated hydrocarbons such as ethylene and propylene to form polyethylene and polypropylene respectively.

Protein Binding

Plasma proteins such as albumin, globulin and acid glycoprotein or lipoproteins present in the body have been known to bind with a large number of drug molecules. This protein binding alters the biological properties of the drug molecule as free drug concentration is reduced. Effect of protein binding: (i) Facilitate the distribution of drugs throughout the body.

(ii) Inactivate a drug by binding so firmly that sufficient concentration is not available at the receptor site. (iii) Retard the excretion of a drug which may accumulate in the body. (iv) Alter the duration of action of a drug. (v) Displace body hormones (vi) Bring about a configurational change in the protein which may become capable of binding other agents. vii) Alter the therapeutic effect by forming a drugprotein complex which is itself biologically active.

Mechanism of protein binding: A protein molecule is a macromolecule composed of many hundreds of aminoacids linked together. The functional groups present in the side chains of the aminoacids provides sites for binding of small drug molecules. Protein binding may be considered to be an adsorption process obeying the law of mass action.

The interaction between a protein (P) and a drug molecule (D) for a simple case of 1: 1 protein drug complex can be represented as: P+D PD

Applying the law of mass action, the expression becomes: K = [PD] / [P] [D] Or
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[PD] = K [P] [D]

Where, K is the association constant [P] is the concentration of unbound protein in terms of free binding sites [D] is the concentration of unbound drug [PD] is the concentration of protein-drug complex

If the protein concentration in the body is designated as [Pt], we can write, [Pt] = [P] + [PD] Since the protein concentration is the sum of the unbound protein and the protein present in the complex. [P] = [Pt] -[PD]

Substituting for [P] in the above equation, we get

[PD] = K[D] ([Pt] [PD] ) Or Or Or Or [PD] = K[D] [Pt] [ K[D] [PD] [PD] + K[D] [PD] = K [D] [Pt] [PD] (1+ K [D] ) = K [D] [Pt] [PD] / [Pt] = K [D] / 1+ K [D]

Where [PD]/ [Pt] represent the average number of drug molecules bound per mole of protein [Pt] . Replacing [PD]/ [Pt] by r, we get, r = K [D] / 1+ K [D] So far, we have assumed that only one binding site exists per molecule of protein . Suppose there are n number of independent binding sites, then: r = n .K [D] / 1+ K [D]---------------I

The equation (1) can be modified to obtained a plot, known as the Scatchard plot r (1+K [D]) = nK[D] or or or r r r + rK[D] = nK[D] = nK[D] - rK[D] = [D] (nK- rK)

or r/ [D] = nK rK .(2)

r= Boud drug/Total protein

In scatchard plot, r/D is plotted against r to give a straight line if only one class of binding site is present.

Scatchard plot for binding of bishydroxycoumarin to human serum albumin

Experimental method for determining Protein Binding

Equilibrium dialysis, ultrafiltration and electrophoresis are used for the determination of protein binding . The other methods include gel filtration, and nuclear magnetic resonance.

Aim of experimental methods: (i) To know the solubility behavior of a drug in the presence and absence of a protein . This may be used to assess the degree of interaction between various species, solvent, drug and protein. (ii) To study the influence of protein on the partitioning behavior of drug molecules between an aqueous and an organic solvent that are immiscible. To study change in spectral characteristics of drug molecules for assessing the degree of binding.

Equilibrium Dialysis Method  In this method, a protein solution is enclosed within a semipermeable membrane such as cellophane which is permeable to small drug molecules and not to macromolecules, the protein molecules. The protein solution within the membrane is then immersed in a drug solution. The solution is agitated slightly until equilibrium has been achieved.
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Sample from both sides of the membrane are withdrawn and analysed.  If the concentration of drug within the membrane and outside the membrane are same, it indicates that no protein binding has occurred. If the concentration within the membrane is more, it indicates that protein binding has occurred because both bound and unbound drug is present within the membrane

Ultrafiltration Ultrafiltration method is similar to equilibrium dialysis method in that the protein and the drug solution are separated by a semi-permeable membrane. However, hydraulic pressure or centrifugation is used in Ultrafiltration to force the solvent and the free drug across the membrane Ultrafiltration method is more convenient than other methods for routine determinations since thus method is less time consuming.

Dynamic dialysis This is a kinetic method for studying the protein binding of drugs. It is relatively rapid, requires very small quantities of protein The method is based on the rate of disappearance of drug from dialysis cell, the rate of disappearance being proportional to the concentration of unbound drug.

The apparatus consists of a jacketed (temperature controlled) beaker into which a buffer solution is placed. A cellophane dialysis bag containing the drug or drugprotein solution is suspended in the buffer solution. Both the solutions are stirred continuously and are periodically removed from outside the dialysis bag and analysed spectrophotometrically. An equal quantity of buffer solution is replaced to the external solution.

The dialysis process follows the rate law: - d [ Dt]/dt Where, [ Dt] is the total drug concentration, [Df] is the concentration of free or unbound drug in the dialysis bag, -d [ Dt]/dt is the rate of loss of drug from the bag and k is the first order rate constant representative of the diffusion process. The concentration of unbound drug in the bag can be calculated using the above equation if k and - d [ Dt]/dt known. = k[Df]