Affinities chromatography

Chromatography may be considered to have two extremes. At one extreme, long, efficient columns of low-to-moderate selectivity are used for the separation of 

As an example of the steps involved in affinity chromatography, we will consider the purification of an enzyme present in a mixture of proteins, using an immobilized competitive inhibitor as an affinity ligand. The first step after column preparation is the application of the sample during this step, the enzyme that binds to the inhibitor is retained, or adsorbed on the column. This is followed by a rinse step, whereby all nonbinding species are removed. The third step involves elution, and this step 

TABLE 14.3. General Affinity Ligands and Their Binding Partners  

Cibachron blue dye, or derivatives of AMP, the NADH, or NADPH Lectins concanavalin A, lentil lectin, wheat germ lectin configurations Trypsin inhibitor, methyl esters of amino acids, or D-amino acids Phenylboronic acid 

Dehydrogenase enzymes, through binding at NAD(P)H binding site 

The photoaffinity label O -(ethyl-2-diazomalonyl)adenosine-3, 5 -cyclic phosphate (17) has been used to label rabbit muscle phosphofructokinase. If 

Affinity chromatography involves precisely the same kind of electrostatic, hydrophobic, dipolar, and hydrogen-bonding interactions described above, but the specificity of binding is extraordinarily high. Demands on the homogeneity of the stationary phase and on the rigidity of the support are often 

Affinity chromatography has become one of the standard techniques for the separation and purification of enzymes. It is based on the idea of utilizing the specific interaction between an enzyme and an immobilized substrate or inhibitor. The substrate is chemically bound to an inert support material, and when a sample is added to the column the enzyme binds to it to an extent depending on the strength of the interaction between the two. Other enzymes and proteins which do not interact with the substrate pass through the system with little or no retention. Removal of the enzyme from the stationary support is accomplished by changing the eluent so as to alter the enzyme-substrate interaction. The 

Affinity chromatography is the most specific chromatographic method. The interaction is biochemical in nature, e.g.  

The sample is specifically retained by the stationary phase, whereas the 

Practical High-Performance Liquid Chromatography, Fifth edition Veronika R. Meyer 

With stronger ot/inity due to - ond + charges stead of only 8 - and 8 + 

With pH chonge so that S loses its S - chorges (the some thing happens when L loses its 8-charg s) 

Affinity chromatography has the highest specificity and selectivity of all chromatographic methods and is a powerful method for the purification and isolation of biomolecules even at low concentrations. The target molecule can be picked selectively from complex mixtures such as blood or serum. 

The process can be divided into the following steps (1) sample introduction, 

The highly specific nature of these interactions is due to the fact that the two participating compounds are ideally suited to each other both spatially and electrostatically. One component (ligand) is bonded to a support (in a similar way to a phase chemically bonded to silica) and the other (sample) is adsorbed from solution, the process being reversible (Fig. 16.1). 

Practical High-Performance Liquid Chromatography, Fourth edition Veronika R. Meyer 2004 John Wiley Sons, Ltd ISBN 0-470-09377-3 (Hardback) 0-470-09378-1 (Paperback) 

With stronger affinity due to - and + charges instead of only S and 8 + 

Affinity chromatography is a special type of adsorption chromatography for the isolation and purification of biologically active macromolecules. It was used successfully for the first time in 1968 for the purification of enzymes [168]. Since then, innumerable proteins (e.g., enzymes. 

The high selectivity of affinity chromatography is based on biospecific interactions, such as those occurring between two molecules in natural biological processes. One interaction partner (ligand) is covalently attached (immobilization) to an insoluble carrier, while the corresponding partner (frequently a protein) is reversibly adsorbed by the ligand because of its complementary biospecific properties. 

In laboratory practice, an affinity matrix (stationary phase) is tailor-made for the protein to be purified and filled into a chromatography column. For process applications many different affinity gels are commercially available. The raw extract is then passed through the column by using a physiological buffer as the mobile phase. In this process, the desired product is adsorbed selectively by the ligand and all unwanted components are washed away. 

The chemical composition of the mobile phase is then changed to permit desorption and isolation (elution) of the protein (Fig. 51). 

1) A hydrophilic neutral surface—avoidance of nonspecific adsorption 

Affinity chromatography is a type of liquid chromatography in which a biologically related agent is used in a column as a stationary phase to purify or analyze the components of a sample [1—4]. The ability of this method to selectively bind and purify its target compounds is based on 

FIGURE 1.1 Typical scheme for the application of a sample to an affinity column, elution of the retained targets, and regeneration of the affinity column [1]. 

Antibody affinity chromatography is employed to isolate antigen-specific antibodies. The most common affinity matrix for coupling of molecules is cyanogen bromide-activated 

Sepharose. The following procedure can be used to purify antibodies raised against a particular protein (Javois, 1999). 

Sprinkle 1 g of cyanogen bromide-activated Sepharose 4B (Pharmacia-LKB, Piscataway, NJ) over 20 ml of 10 mM HC1. The gel swells immediately. One gram of dry gel yields 3.5 ml of hydrated matrix. 

Wash the gel on a 50-ml coarse, sintered glass funnel four times with 50 ml of 10 mM HC1 by repeatedly suspending the matrix in the HC1 solution, and then drain using vacuum suction. These washing steps remove the additives present in the dry matrix. 

Add this supension to the gel and mix by inversion overnight at 4°C in a capped 15-ml polycarbonate tube. Avoid mechanically stirring the gel to avoid damaging the gel matrix. 

The power of affinity chromatography lies in its selectivity. In the clinical laboratory, affinity chromatography has 

until recently, affinity chromatography has been limited to low pressure operations. However, as described above for size 

This mode of separation, as the name suggests, uses stationary phases with a special affinity for a specific analyte. The affinity ligand immobilized on the stationary phase varies dramatically from peptide, to protein, to oligonucleotide, to monoclonal antibody. In some cases the target molecule is labelled with an affinity tag to simplify the separation. This approach is common in the synthesis of recombinant proteins where the system can be engineered so that the target biomolecule expresses a tag such as polyhistidine. A stationary phase functionalized with aminodiacetic acid and nickel chelate is then used to fish out the required molecule by chelating with the polyhistidine tag. 

Existing stationary phases used in this area are usually soft gels that often suffer from low loading capacity brought about by the inability of biological macromolecules to penetrate the matrix. The most likely progress in this arena over the forthcoming 

The technique of affinity chromatography was described in some detail by Lowe and Dean in a text published in 1974[316]. 

Huot et al. [38] used affinity chromatography to identify and partially purify an amiloride-binding protein with characteristics of the renal brush border Na /H exchanger. The high-affinity amiloride analog A35 (5-A-(3-aminophenyl)amiloride) was coupled to Sepharose CL-4B through a triglycine spacer. Rabbit renal brush border membranes were solubilized with 0.6% Triton X-100, incubated with the 

A35 affinity matrix, and eluted with various media. A 25-kDa protein bound to the affinity matrix and was completely eluted with 5 mM free amiloride. The abundance of the 25-kDa protein in brush border and basolateral membranes correlated closely with Na /H exchange activity. Importantly, binding of the 25-kDa protein to the affinity matrix was blocked by MIA amiloride benzamil, a rank order identical to that for inhibition of Na /H exchange activity, which suggested strongly that the 25-kDa protein was a structural component of the transporter. 

Subsequently, proteolytic fragments of the rabbit renal 25-kDa amiloride-binding protein were micro-sequenced and found to have high sequence homology with rat and human NAD(P)H quinone oxidoreductase. Indeed, enzymatic assays revealed that renal brush border membrane vesicles contain significant NADPH quinone oxidoreductase activity. Presumably NAD(P)H quinone oxidoreductase coincidentally binds amiloride analogs with the same rank order as the Na /H exchanger [39]. 

In a preliminary report, Ross et al. [40] used affinity chromatography to identify a putative bovine renal brush border Na /H exchanger. Brush border membranes were solubilized with Triton X-100 and chromatographed sequentially over lentil lectin Sepharose 4B and 5-(A-benzyl-iV-ethyl)amiloride coupled to epoxy-activated Sepharose 6B. The eluant contained 178- and 146-kDa proteins that were susceptible to Endo-F. Moreover, the eluants reacted on dot blot immunoassays with antisera to a 20-amino acid peptide of a human Na /H exchanger vide infra). The relationship between these proteins and the 66-kDa protein previously identified by the same investigators using amiloride photolabeling is presently unclear. 

Whereas the above studies have attempted to identify the Na /H exchanger in renal brush border membranes (a resistant-type), at least one study has reported possible identification of a sensitive-type transport protein [49]. The Na /H exchanger in lymphocytes (a sensitive-type) can be activated by either 12-0-tetradeca-noylphorbol 13-acetate (TPA) or osmotic shrinkage. TPA or osmotic shrinkage 

High performance liquid affinity chromatography (HPLAC) is a recently introduced form of HPLC and few commercial stationary phases are currently available the Beckman Fast Affinity stationary phase, stationary phases from LKB and Biorad and the Pierce High Performance Affinity stationary phase. Only a few reports are available of their use and in the past most affinity stationary phases have been prepared by proprietary methods in individual laboratories. An 

A very interesting application of affinity chromatography to the purification of halophilic enzymes was reported by Sundquist and Fahey (1988). These authors have purified the enzymes bis-y-glu-tamylcysteine reductase and dihydrolipoamide dehydrogenase from H. halohium using immobilized metal ion affinity chromatography in high-salt buffers. 

As the name suggests, the principle is the use of a moiety or molecule which has high affinity for the protein of interest. 

These molecules could either be co-factors, modified substrates, inhibitors or carbohydrates. This strategy of purification is used mostly in the later stages where the protein is relatively pure, and more specific approaches are required for addifional purification. The affinity moiety or molecule is coupled to the matrix and used as a bait to fish the protein of interesf (Fig. A.4). The protein could either be eluted with high sait in some cases or with increased amount of fhe affinify molecule ifself. 

It is not normally prudent to employ biospecific affinity chromatography as an initial purification step, as various enzymatic activities present in the crude fractions may modify or degrade the expensive affinity gels. However, it should be utilized as early as possible in the purification procedure in order to accrue the full benefit afforded by its high specificity. 

A molecularly imprinted polymer is one that is polymerized in the presence of a template molecule to which components of the polymer have some affinity. When the template is removed, the 

Separation of theophylline and caffeine by column containing polymer imprinted with theophylline. Caffeine washed right through with CHCI3 solvent. Addition of 20 id. of CH3OH to the solvent breaks hydrogen bonds between theophylline and the polymer and elutes theophylline in a volume of t mL [From W. M. Mulleti and P C. Lai. Anal. Chem. 1998, 70.3636.] 

Complementary structures of biological materials, especially those of proteins, often result in specific recognitions and various types of biological affinity. These include many pairs of substances, such as enzyme-inhibitor, enzyme-substrate (analog), enzyme-coenzyme, hormone-receptor, and antigen-antibody, as summarized in Table 11.2. Thus, bioaffinity represents a useful approach to separating specific biological materials. 

Histidine-containing protein Chelated heavy-metal 

Estimate the equilibrium concentration of the solution. An adsorption isotherm of the Freundlich-type is given as 

2 By single- or three-stage adsorption with an adsorbent, 95% of an adsorbate A in an aqueous solution of CA0 needs to be recovered. When the linear adsorption equilibrium is given as 

3 Derive the following adsorption isotherm for an A-B gas mixture of two components, each of which follows the Langmuir-type isotherm  

The first reaction in which substrate (S) binds to the enzyme (E) to yield an enzyme-substrate complex (ES) represents the recognition needed for efficient biological function and is the step of interest here P is the normal product of the reaction. The basis of affinity chromatographic techniques 

Careful planning must be done before specific laboratory procedures are performed. Major considerations include (1) the type of matrix used, 

Ordinary chromatographic techniques depend for their effectiveness on differences (often small) in adsorption, partition, ionic charge, or size, between solute molecules of similar chemical character. A modified gel technique in which use is made of the high specificity of biochemical reactions is affinity chromatography [54,55]. 

Studies on the dephosphorylation of nucleoside 5 -triphosphates catalysed by Mn2+, Ni2+, and Zn2+ have been performed.89 The ions Mn2+ and Ni2+ co-ordinate to 

From this, we can see we have three types of peaks 1) the exclusion peak, containing all molecules of a certain size or larger 2) resolved peaks of intermediate diameter and 3) the inclusion peak containing all compounds of a given diameter and smaller. In a crude mixture of compounds, we are forced to suspect that both the exclusion and inclusion peaks contain multiple components. 

Size separation columns are available with silica, zirconium, and heavily cross-linked organic polymer backbones. The polymer columns show the same pressure and solvent fragility described for ion exchange columns. Silica size columns must be protected from pH changes like partition columns, which must be used with a pH between 2.5 and 7.5. Zirconium columns are not pH or temperature sensitive, but possess chelation properties that must be chemically masked to prevent interference with the size separation. 

Much less commonly used than partition, ion exchange, and size columns, affinity columns are of growing interest in the HPLC purification of proteins 

In practice, affinity column recognition specificity is never as complete as described in theory. Usually a range or class of similar compounds can be attracted and retained. The recognizer must be bound to the column for each target compound and after that point the column must be dedicated for that separation. Usually there is no possibility of removing the recognizer and reusing the column for a different separation. 

Polymer types Typical solvent system Typical column packings6 Supplier 

Proteins, polypeptides Aqueous buffers Zorbax Bio Series GF Du Pont 

Biopolymers, viruses, DNA, RNA Aqueous buffers TSK-G-PW Superose Toya Soda Pharmacia 

Cellulose derivatives, polyvinyl alcohol, polysaccharides Aqueous buffer, salts TSK-G-SW Toya Soda 

Many polar noncrystalline synthetic polymers, some crystalline polymers, small molecules Tetrahydrofuran Ultra-Styragel Waters 

That some biological molecules had affinities for other biological molecules was first observed by Starkenstein in 1919, who noticed that amalyase binds tightly to insoluble starch. In 1953, Lerman used an azo dye immobilized on cellulose to separate mushroom tyrosinase from other proteins. 

000 publications and several books referring to affinity chromatography. 

Several commercial beads are available. Of more importance is the fact that by using a few well- established reactions, any one of hundreds of ligands can be added to the spacer arm. A few of these reactions will be discussed later. 

The matrix should (1) be stable to the eluant, (2) be mechanical and chemically stable, (3) have a large surface area, (4) be easily derivatized, and (5) have good flow characteristics. Currently used matrix materials are agarose, 

Agarose is obtained from sea kelp and is a linear polysaccharide consisting of alternating residues of a-1,3 and P-1,4 linkages plus a few carboxylate and sulfate ionic residues. The ionic residues must be removed, because they interfere with most separations. This can be done by a NaBH4 reduction. The unpurified material is commonly called agar. 

Sarnesto, A., et al. (1990). Purification of H Gene Encoded 3-galactoside al -2-fucosyltransferase from Human Serum, Rio/. Chem. 265 15067-15075. 

and Karger, B. (1996). Hydrophobic Interaction Chromatography of Proteins, Methods Enzymol. 270 27-47. 

my love of the blue gel is based more on the nice color than on success with it. I feel that the blue looks pretty among all the white in the column forest. In the United States, at a VA medical center, I once saw a Bavarian colleague cry in front of his white-blue column grove. I do not know whether it was because of homesickness (white and blue are the state colors of Bavaria) or because he had lost his sample. After all, the purification effect is quite low. If you achieve an emichment of 5 with a blue gel and only lose half your sought-after protein you may pat yourself on the back. Recommendation use small columns (a volume of 1 to 1.5 ml or less is enough). 

The blue gel is recommended if you want to rid media at BSA. Depending on matrix and substitution degree, BSA binds in amounts between 8 and 15 mg/ml in the blue gel. Even if you know or suspect that your sought-after protein has a dinucleotide pocket, you can do chromatography on blue gel. This, in any case, is true when nothing better occurs to you. I would not risk the entire preparation. It is best to test the purification effect first with an aliquot. It happens occasionally that a protein completely disappears in the blue depths. 

Depending on the protein, the blue gel is eluded with high ion strengths (e.g., 1.5 M KCl) or with detergents. You can also try using NADH for eluding proteins with dinucleotide pockets. 

Maitre, L. Ossola, and P. Mandel, Biochim. Biophys. Acta, 1978, 524, 26. V. Berariu, R. deck, and C. Woenckhaus, Justus Liebigs Ann. Chem., 1978(1), 118. 

Hoshino, R. Miyajima, S. Murao, and K. Mitsugi, Agric. Biol. Chem., 1977,41,709. 

Watenabe, N. Kondo, T. Fujii, and T. Noguchi, Plant Cell Physiol., 1977,18, 387. 

Saldeen, and R. Wallin, Thromb. Haemostasis, 1978, 39, 97. 

Hackenthal, R. Hackenthal, and U. Hilgenfeldt, Biochim. Biophys. Acta., 1978, 522, 561. 

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Affinity chromatography is used in the preparation of more highly purified Factor IX concentrates (53—55) as well as in the preparation of products such as antithrombin III [9000-94-6] (56,57). Heparin [9005-49-6], a sulfated polysaccharide (58), is the ligand used most commonly in these appHcations because it possesses specific binding sites for a number of plasma proteins (59,60). 

Another by-product of Factor VIII processing having clinical value is von Wikebrand factor. It has been recovered from side fractions using ion-exchange and affinity chromatography (196). 

Alpha-1-proteinase inhibitor and antithrombin III are used to treat people with hereditary deficiencies of these proteins. Both can be recovered from Cohn Fraction IV (Table 7) using ion-exchange chromatography (52) and affinity chromatography (197), respectively. Some manufacturers recover antithrombin III directiy from the plasma stream by affinity adsorption (56,198,199). 

Fig. 4. Example of antibody purification from animal or culture sources. In some cases, affinity chromatography may be used directly with the source...

Affinity Chromatography. This technique involves the use of a bioselective stationary phase placed in contact with the material to be purified, the ligate. Because of its rather selective interaction, sometimes called a lock-and-key mechanism, this method is more selective than other lc systems based on differential solubiHty. Affinity chromatography is sometimes called bioselective adsorption. 

Ligand exchange Equihbrium Chromatographic separation of glucose-fructose mixtures with Ca-form resins Removal of hea y metals with chelating resins Affinity chromatography... 

Sepharose (e.g. Sepharose CL and Bio-Gel A) is a bead form of agarose gel which is useful for the fractionation of high molecular weight substances, for molecular weight determinations of large molecules (molecular weight > 5000), and for the immobilisation of enzymes, antibodies, hormones and receptors usually for affinity chromatography applications. 

P. Bailon, G.K. Ehrlich, W. Fung and W. Berthold (Eds), Affinity Chromatography Methods and Protocols, Humana Press, Totowa, 2000. ISBN 0896036944. 

I. A. Chaiken, Analytical Affinity Chromatography, CRC Press Inc, Florida, 1987. ISBN 084935658X. 

P.D.G. Dean, W.S. Johnson and F. Middle (Eds), Affinity Chromatography A Practical Approach, IRL Press, Oxford, 1985. ISBN 0896036944. 

Angiotensin (from rat brain) [70937-97-2 M 1524.8. Purified using extraction, affinity chromatography and HPLC [Hermann et al. Anal Biochem 159 295 1986],... 

Avidin (from egg white) [1405-69-2] Mr -70,000. Purified by chromatography of an ammonium acetate soln on CM-cellulose [Green Biochem J 101 774 1966]. Also purified by affinity chromatography on 2-iminobiotin-6-aminohexyl-Sepharose 4B [Orr 7 Bio/C/iew 256 761 1981]. It is a biotin binding protein. 

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