IMA Unit-5 Affinity Chromatography.pptx

Affinity Chromatography

1. Subject Name: Instrumental Methods of Analysis Unit Name: Chromatographic Techniques

2. History of Affinity Chromatography 1930s, first developed by A. Wilhelm Tiselius-a Swedish biochemist, won the Nobel Prize in 1948 Used to study enzymes and other proteins Relies on the affinity of various biochemical compounds with specific properties

3. Introduction to Affinity Chromatography Affinity chromatography is one of the most diverse and powerful chromatographic methods for purification of a specific molecule or a group of molecules from complex mixtures. It is a method of separating a mixture of proteins or nucleic acids by specific interactions of those molecules with a component known as a ligand, which is immobilized on a support. Mixture of proteins is passed through the column, one of the proteins binds to the ligand on the basis of specificity and high affinity (they fit together like a lock and key).

4. The other proteins in the solution wash through the column because they were not able to bind to the ligand. Introduction to Affinity Chromatography

5. It is based on highly specific biological interactions between two molecules such as interactions between enzyme and substrate, receptor and ligand, or antibody and antigen. These interactions which are typically reversible are used for purification by placing one of the interacting molecules referred to as affinity ligand onto a solid matrix to create a stationary phase while a target molecule is in the mobile phase. Many of the commonly used ligands coupled to affinity matrices are now commercially available and are ready to use. Introduction to Affinity Chromatography

6. The Sample is injected into the equilibrated affinity chromatography column Only the substance with affinity for the ligand are retained on the column The substance with no affinity to the ligand will elute off The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent Introduction to Affinity Chromatography

7. Specificity of Affinity Chromatography Specificity is based on three aspects of affinity: Matrix: for ligand attachment. Matrix should be chemically and physically inert. Spacer Arm: used to improve binding between ligand and target molecule by overcoming any effects of steric hindrance. Ligand: molecule that binds reversibly to a specific target molecule(site of interaction).

8. 1. Matrix The matrix simply provides a structure to increase the surface area to which the molecule can bind. Amino, hydroxyl and carbonyl groups located with the matrix serve as ligand binding sites. Matrix are made up of agarose and other polysaccharides. The matrix must be activated for the ligand to bind to it but still able to retain it’s own activation towards the target molecule. The matrix also must be able to withstand the decontamination process of rinsing with sodium hydroxide or urea.

9. 1. Matrix For having an effective matrix, it must have certain characters: It must be insoluble in solvents and buffers employed in the process. It must be chemically and mechanically stable. It must be easily coupled to a ligand or spacer arm onto which the ligand can be attached. It must exhibit good flow properties and have a relatively large surface area for attachment.

10. Types of Matrix Used Solid Support: 1. Carbohydrate P olymers : ex, agarose, cellulose, dextran . Commercially available agarose in 1-12% is usually adequate. cross linking with divinylsulfone (DVS) or 1,4-butanediol diglycidyl ether increases both the chemical and thermal stability of the gel but may diminish the binding capacity. DVS increases the gel’s rigidity. Cross linked agarose tolerates any solvents except strong acids and oxidizers but may be damaged by some rare enzymes and must not be frozen or dried. Agarose gel activates complement system in human blood and must never be used in vivo ( e.g., for affinity removing of toxins from blood).

11. Dextran: It consists of two types of gels. Sephadex S ephacryl Sephacryl are used in the chromatography of biomolecules. Sephacryl S-1000 is especially suitable for affinity chromatography of very large molecules. At low pH Sephacryl S-200 adsorbs proteins. Sephadex is used mainly as a glucose polymer (example for the purification of lectins ) it is mechanically to weak or has insufficient porosity. It can be activated in aqueous media with non-cross-linking reagents. Types of Matrix Used

12. B. Synthetic Polymers: P olyacrylamide(PAA) is synthesized by copolymerization of acrylamide and a cross linking agent. Primary disadvantage is that the gels are either soft or have small pores. PAA gel is resistant against enzymatic attacks and does not adsorb biomolecules. Hydroxy Alkyl Methacrylate G els: The gel is chemically and mechanically more stable than PAA gel but is more hydrophobic and thus it is inappropriate for many applications. Trisacryl : It is hydrophilic, biologically inert, rigid, and macroporous . Despite its low working volume( it contains more than 30% of solids) it is used for large scale preparations. Types of Matrix Used

13. C. Mixed G els: Polyacrylamide-agarose gels ( ultrogel AcA ) were developed for gel filtration and tested for affinity chromatography. D. Inorganic M aterials: When extreme rigidity of the support is needed , as for HPLC and large-scale applications, inorganic materials are used ( irregular porous glass or silica particles). They are soluble pH >8, although a coating of zirconium will enhance their stability. otherwise, glass or silica may be coated with an inert polymer. E. Magnetic C arriers : Magnetic sorbent particles ( magnogel AcA 44 and Actmagnogel AcA 44) are useful for batch extraction from large volumes of diluted and/or turbid solutions. Types of Matrix Used

14. 2. Spacer Arm T he binding sites of the target molecule are sometimes deeply located and difficult to access due to steric hindrance, a spacer arm is often incorporated between the matrix ( agarose bead as solid support) and ligand to facilitate efficient binding and create a more effective and better binding environment.

15. 2. Spacer Arm This inhibitor with a hydrocarbon chain is commonly known as the spacer between the agarose bead and the target molecule. The length of these spacer arms is critical. Too short or too long arms may lead to failure of binding or even non-specific binding. In general, the spacer arms are used when coupling molecules less than 1000 Da.

16. 2. Spacer Arm

17. 3. Ligand The Ligand binds only to the desired molecule within the solution. It attaches to the matrix which is made up of an inert substance. It should only interact with the desired molecule and form a temporary bond. The ligand/molecule complex will remain in the column, eluting everything else off. The ligand/molecule complex dissociates by changing the pH. The ligand can be selected only after the nature of the macromolecule to be isolated is known. The chosen ligand must bind strongly to the molecule of interest.

18. If the ligand can bind to more than one molecule in the sample a technique, negative affinity is performed. this is the removal of all ligands, leaving the molecule of interest in the column. done by adding different ligands to bind to the ligands within the column. Examples: For antibody isolation, an antigen may be used as ligand. If an enzyme is to be purified, a substrate analog, inhibitor, cofactor, or effector may be used as a the immobilized ligand. 3. Ligand

19. The selection of the ligand for affinity chromatography is influenced by two factors: the ligand must exhibit specific and reversible binding affinity for the target substance(s) and it must have chemically modifiable groups that allow it to be attached to the matrix without destroying binding activity. 3. Ligand

20. Immobilization of ligand Immobilization of the affinity ligand is also very important when designing an affinity chromatography method for biomolecule purification. Activity of the affinity ligand can be affected by multi-site attachment, Multi-site attachment occurs when an affinity ligand is attached through more than one functional group on a single ligand molecule. O rientation of the affinity ligand, and If these multiple attachment sites cause the affinity ligand to become denatured or distorted, multisite attachment can lead to reduced binding affinity. However, in some instances, the additional attachment sites can result in more stable ligand attachment steric hindrance. 3. Ligand

21. Covalent Immobilization Covalent immobilization is one of the most common ways of attaching an affinity ligand to a solid support material. There is a wide range of coupling chemistries available when considering covalent immobilization methods. Amine, sulfhydryl, hydroxyl, aldehyde, and carboxyl groups have been used to link affinity ligands. C ovalent attachment methods are more selective than other immobilization methods, they generally require more steps and chemical reagents onto support materials. 3. Ligand

22. Adsorption of Affinity L igands : A dsorption can be either nonspecific or specific. Nonspecific Adsorption: In nonspecific adsorption the affinity ligand simply adsorbs to the surface of the support material and is a result of Coulombic interactions, hydrogen bonding and/or hydrophobic interactions. Bio Specific A dsorption: it is commonly performed by using avidin or streptavidin for the adsorption of biotin containing affinity ligands or protein A or protein G for the adsorption of antibodies in, and/or hydrophobic interactions. 3. Ligand

23. The principle of Affinity Chromatography Sample is injected into the equilibrated affinity chromatography column. Only the substance with affinity for the ligand are retained on the column. The substance with no affinity to the ligand will elute off. The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent

24. The principle of Affinity Chromatography

25. The principle of Affinity Chromatography Affinity Chromatography have 3 main part:

26. Procedure Step-1 Attach ligand to column matrix Binding of the selected ligand to the matrix requires that a covalent bond be formed between them. Most ligands are attached first to spacer arms which are then bonded to the matrix. The ligand-matrix gel is then loaded into an elution column.

27. Step-2 Load protein mixture onto column Once the column has been prepared, the mixture containing isolate is poured into the elution column. Gravity pulls the solution through the gel, because most of the proteins do not bind to the ligand-matrix complex. When ligand is recognized substrate passes through the gel, it binds to the ligand-matrix complex, halting its passage through the gel. Some of the impurities flow through the gel due to gravity, but most remain, unbound, in the gel column Procedure

28. Procedure Step-3 Proteins bind to ligand In order to remove these unbound impurities, a wash of extreme pH, salt concentration, or temperature is run through the gel. It is important to use a strong wash so that all the impurities are removed. Once the impurities are washed-out, the only remaining part of the protein mixture should be the desired isolates.

29. Procedure Step-4 Wash column to remove unwanted material In ally to collect isolate, which is still bound to the ligand-matrix in the gel, a stronger second wash is run through the column.

30. Procedure Step-5 Wash off proteins that bind loosely This second wash relies on the reversible binding properties of the ligand, which allows the bound protein to dissociate from its ligand in the presence of this stronger wash.

31. Procedure Step-6 Elute Proteins that bind tightly to ligand and collect purified protein of interest The protein is then free to run through the gel and be collected.

32. Adsorbed compounds can be washed off the column in two ways: by specific (concurrent) or by non specific elution. Specific elution is based on direct interruption by analogs of either ligand or adsorbate in to the complex formed on the affinity resin. N on specific elution is by changing the media atmosphere (e.g. changing the ionic strength, pH or polarity) If the affinity interaction is mainly hydrophobic, elution with detergent or mixed solvents( up to 50% ethylene glycol or isopropanol) is promising but if the interaction is ionic, high salt concentration or pH alteration are used. Elution from antigen-antibody complexes can be modelled on polystyrene microplates : immune complexes are dissociated without eluting the adsorbed antigen, while all parameters are controlled by ELISA technique. Elution Process

33. pH elution: A change in pH alters the degree of ionization of charged groups on the ligand and/or the bound protein. A step decrease in pH is the most common way to elute bound substances. The chemical stability of the matrix, ligand and target protein determines the limit of pH that may be used. Ionic strength elution: The exact mechanism for elution by changes in ionic strength will depend upon the specific interaction between the ligand and target protein. This is a mild elution using a buffer with increased ionic strength (usually NaCl), applied as a line. e.g., Enzymes usually elute at a concentration of 1 M NaCl or lesser gradient or in steps. Elution Process

34. Competitive Elution: Selective eluents are often used to separate substances on a group specific medium or when the binding affinity of the ligand/target protein interaction is relatively high. The eluting agent competes either for binding to the target protein or for binding to the ligand. Substances may be eluted either by a concentration gradient of a single eluent. Elution Process

35. Different Types of Affinity Chromatography

36. Advantages Extremely high specificity High degrees of purity can be obtained The process is very reproducible The binding sites of biological molecules can be simply investigated Disadvantages: Expensive ligands Leakage of ligand Degradation of the solid support Limited lifetime Non-specific adsorption Affinity Chromatography

37. Applications of Affinity Chromatography Immunoglobulin Purification ( A ntibody Immobilization): Used to purify antibody against a specific antigen Ex: Immunoglobulins Antibodies can be immobilized by both covalent and adsorption methods. Random covalent immobilization methods generally link antibodies to the solid support via their free amine groups using cyanogen bromide, N- hydroxysuccinimide , N,N’- carbonyldiimidazole , tresyl chloride, or tosyl chloride. As these are random immobilization methods, the antibody binding sites may be blocked due to improper orientation, multi-site attachment or steric hindrance. They can also be immobilized by adsorbing them onto secondary ligands.

38. Recombinant T agged P roteins: Purification of proteins can be easier and simpler if the protein of interest is tagged with a known sequence commonly referred to as a tag. This tag can range from a short sequence of amino acids to entire domains or even whole proteins. Tags can act both as a marker for protein expression and to help facilitate protein purification. M ost commonly used tags are glutathione-S-transferase (GST), histidine fusion (His or poly His tag) and protein A fusion tags. Other types of fusion tags are also available including maltose-binding protein, thioredoxin, NusA ,GB1 domain for protein G Applications of Affinity Chromatography

39. GST T agged P urification : The purification method is based on the high affinity of GST for glutathione. When applied to the affinity resin, GST-tagged proteins bind to the glutathione ligand, and impurities are removed by washing with binding buffer. Tagged proteins are then eluted from the chromatography resin under mild, non-denaturing conditions that preserve both protein structure and function. GST Buffer Kit contains prepared buffer concentrates for binding, washing, and elution of GST tagged protein detection Applications of Affinity Chromatography

40. His-tagged protein purification H istidine-tagged recombinant protein purification using immobilized (IMAC). Ni 2+ Sepharose resins are pre-charged with nickel ions (Ni 2+ ) metal ion affinity chromatography. Ni 2+ Sepharose excel is especially suitable for purification of histidine tagged proteins secreted into eukaryotic cell culture supernatants. Applications of Affinity Chromatography

41. His-tagged protein purification IMAC resins charged with Ni2+ and Co2+ are the most commonly used methods for the purification of histidine tagged proteins. However, in some cases, other metal ions may be more suitable, for example copper (Cu2+) or zinc (Zn2+). In these cases, uncharged IMAC resins can be conveniently charged with the metal ion of your choice. Applications of Affinity Chromatography

42. Protein A, G, and L Purification: Proteins A, G, and L are native or recombinant proteins of microbial origin which bind specifically to immunoglobulins including immunoglobulin G (IgG). The most popular matrixes or supports for affinity applications which utilize protein A, G, or L is beaded agarose (e.g. Sepharose CL-4B; agarose cross-linked with 2,3 dibromopropanol and desulphated by alkaline hydrolysis under reductive conditions), polyacrylamide, and magnetic beads. Applications of Affinity Chromatography

43. Biotin and biotinylated molecules purification: Biotin: (vitamin H or B7) cofactor in the metabolism of fatty acids and leucine, and in gluconeogenesis. In affinity chromatography it is often used an affinity tag due to its very strong interactions with avidin and streptavidin. One advantage of using biotin as an affinity tag is that it has a minimal effect on the activity of a large biomolecule due to its small size (244 Da). Applications of Affinity Chromatography

44. Streptavidin is a large protein (60 kDa ) that can be obtained from Streptomyces avidinii and bind biotin. Avidin is a slightly larger glycoprotein (66 kDa ) with slightly stronger binding to biotin . Both avidin and streptavidin have four subunits that can each bind one biotin molecule. Due to the strong interaction between biotin and ( strept )avidin, harsh elution conditions are required to disrupt the binding. Applications of Affinity Chromatography

45. Lectin Affinity C hromatography: Lectins are carbohydrate binding proteins that contain two or more carbohydrate binding sites and can be classified into five groups according to their specificity to the monosaccharide. They exhibit the highest affinity for: mannose, galactose/ Nacetylgalactosamine , Nacetylglucosamine , fructose, and N-acetylneuraminic acid. In this affinity technique, protein is bound to an immobilized lectin through its sugar moieties . Once the glycosylated protein is bound to the affinity support, the unbound contaminants are washed away, and the purified protein is eluted. Applications of Affinity Chromatography

46. Nucleic acid separation using immobilized metal affinity chromatography (IMAC) The method can be used to purify compounds containing purine or pyrimidine moieties where the purine and pyrimidine moieties are shielded from interaction with the column matrix from compounds containing a non-shielded purine or pyrimidine moiety or group. Thus, double-stranded plasmid and genomic DNA, which has no low binding affinity can be easily separated from RNA or oligonucleotides which bind strongly to metalcharged chelating matrices IMAC columns clarify plasmid DNA from bacterial alkaline lysates, purify a ribozyme, and remove primers and other contaminants from PCR reactions Applications of Affinity Chromatography

47. Reversed Phase C hromatography Reversed phase chromatography is a kind of affinity interaction between a biomolecule dissolved in a solvent (mobile phase) that has some hydrophobicity (e.g. proteins, peptides, and nucleic acids) and an immobilized hydrophobic ligand (stationary phase). When using reversed phase chromatography, the most polar macromolecules are eluted first and the most nonpolar macromolecules are eluted last: the more polar (hydrophilic) a solute is, the faster the elution and vice versa. I nitial step of reversed phase separation involves equilibration of the column under suitable conditions (pH, ionic strength and polarity.) Next, sample is applied and bound to the immobilized matrix. Applications of Affinity Chromatography

48. Following this step, desorption and elution of the biomolecules is achieved by decreasing the polarity of the mobile phase (by increasing the percentage of organic modifier in the mobile phase). At the end of the separation, the mobile phase should be nearly 100% organic to ensure complete removal of all bound substances. Applications of Affinity Chromatography

49. Industrial Application Affinity chromatography is widely used in the pharmaceutical industry to purify and extract molecules of interest from complex mixtures. These molecules tend to be enzymes, proteins or amino acids, but other biological species can be selectively retained. Applications

50. Applications Other Pregnancy test Allergy test Immune assay Kinetic studies Qualitative measurement of substrate.

51. Vogel’s, Text Book of Quantitative Chemical Analysis R. Chatwal, Instrumental Methods of Chemical Analysis A.H. Beckett & J.B. Stenlake , Practical Pharmaceutical Chemistry Dr. G. Devala Rao, Pharmaceutical Analysis Dr. S. Ravi Sankar, Pharmaceutical Analysis Dr. A.V. Kasturi, Pharmaceutical Analysis Egon Stahl, Thin Layer Chromatography B.K. Sharma, Instrumental Methods of Chemical Analysis Skoog, Instrumental Analysis Ashutosh Kar, Pharmaceutical Drug Analysis References:

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