Dye-ligand Affinity Systems

.J. Biochem. Biophys. Methods 49 2001 391416www.elsevier.comrlocaterjbbm

Review

Dye-ligand affinity systemsAdil Denizli a, Erhan Piskin b,)

a Biochemistry Diision, Department of Chemistry, Hacettepe Uniersity, 06532 Beytepe, Ankara, Turkeyb Chemical Engineering Department and Bioengineering Diision, Hacettepe Uniersity, 06532 Beytepe,

Ankara, Turkey

Abstract

Dye-ligands have been considered as one of the important alternatives to natural counterpartsfor specific affinity chromatography. Dye-ligands are able to bind most types of proteins, in somecases in a remarkably specific manner. They are commercially available, inexpensive, and caneasily be immobilized, especially on matrices bearing hydroxyl groups. Although dyes are allsynthetic in nature, they are still classified as affinity ligands because they interact with the activesites of many proteins mimicking the structure of the substrates, cofactors, or binding agents forthose proteins. A number of textile dyes, known as reactive dyes, have been used for protein

purification. Most of these reactive dyes consist of a chromophore either azo dyes, anthraquinone,. .or phathalocyanine , linked to a reactive group often a mono- or dichlorotriazine ring . The

interaction between the dye ligand and proteins can be by complex combination of electrostatic,hydrophobic, hydrogen bonding. Selection of the supporting matrix is the first important consider-ation in dye-affinity systems. There are several methods for immobilization of dye molecules ontothe support matrix, in which usually several intermediate steps are followed. Both the adsorptionand elution steps should carefully be optimizedrdesigned for a successful separation. Dye-affinitysystems in the form of spherical sorbents or as affinity membranes have been used in proteinseparation. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Dye-affinity; Dye-protein interactions; Matrix selection and use; Dye immobilization;Adsorptionelution conditions; Selected uses

1. Introduction

Affinity chromatography is already a well-established method for the identification,purification, and separation of macromolecules, and based on highly specific molecularrecognition. As demonstrated in Fig. 1, in this method, a molecule having specific

) Corresponding author. Tel.: q90-312-2977-473; fax: q90-312-2992-124. .E-mail address: [email protected] E. Piskin .

0165-022Xr01r$ - see front matter q2001 Elsevier Science B.V. All rights reserved. .PII: S0165-022X 01 00209-3

( )A. Denizli, E. PiskinrJ. Biochem. Biophys. Methods 49 2001 391416392

Fig. 1. Principle of affinity chromatography.

.recognition capability AligandB or AbinderB is immobilized on a suitable insoluble .support AmatrixB or AcarrierB , which is usually a polymeric material in bead or

.membrane form. The molecule to be isolated AanalyteB or AtargetB is selectively .captured AadsorbedB by the complementary ligand immobilized on the matrix by

simply passing the solution containing the target through the chromatographic column .under favorable conditions. The target molecules are then eluted AdesorbedB by using

proper elutants under conditions favoring desorption, by adjusting the pH, ionic strengthor temperature, using specific solvents or competitive free ligands, so that the interactionbetween the ligand and target is broken and the target molecules are obtained in a

w xpurified form. Since its first introduction by Cuatrecasas et al. 1 in 1968, thousands ofdifferent molecules enzymes, antibodies, hormones, vitamins, receptors, many variety

.of other proteins and glycoproteins, RNA, DNA, etc. , even bacteria, viruses, and cellsw xhave been separatedrpurified by affinity chromatography 25 .

2. Dyes as affinity ligands

A wide variety of functional molecules, including enzymes, coenzymes, cofactors,antibodies, amino acids, oligopeptides, proteins, nucleic acids, and oligonucleotides may

w xbe used as ligands in the design of novel sorbents 610 . These ligands are extremelyspecific in most cases. However, they are expensive, due to high cost of productionandror extensive purification steps. In the process of the preparation of specificsorbents, it is difficult to immobilize certain ligands on the supporting matrix withretention of their original biological activity. Precautions are also required in their use .at sorption and elution steps and storage.

Dye-ligands have been considered as one of the important alternatives to naturalcounterparts for specific affinity chromatography to circumvent many of their draw-backs, mentioned above. Dye-ligands are able to bind most types of proteins, especiallyenzymes, in some cases in a remarkably specific manner. They are commerciallyavailable, inexpensive, and can easily be immobilized, especially on matrices bearinghydroxyl groups. Although dyes are all synthetic in nature, they are still classified asaffinity ligands because they interact with the active sites of many proteins bymimicking the structure of the substrates, cofactors, or binding agents for those proteins.

( )A. Denizli, E. PiskinrJ. Biochem. Biophys. Methods 49 2001 391416 393

2.1. A brief history

Dye-affinity chromatography was initiated with the observation of the unexpectedinteractions between Blue Dextran Cibaron Blue and dextran conjugate, a void marker

. w xused in size-exclusion chromatography and certain kinases 11 . In earlier studies,several proteins e.g., erythrocyte pyruvate kinase, phosphofructokinase, glutathione

.reductase, and several coagulation factors were purified by size-exclusion chromatogra-w xphy with Blue Dextran 1214 . These studies revealed that the reactive dye, Cibacron

w xBlue F3G-A, is responsible for binding of proteins. Roschlau and Hess 15 were first toimmobilize covalently Cibacron Blue on Sephadex G-200 directly and to purify yeast

w xpyruvate kinase with this affinity sorbent 15 . After that, this concept has been appliedto a variety of protein purifications with different matrices carrying the blue ligand, as

w xextensively reviewed elsewhere 1620 .

2.2. Chemical structure of dye-ligands

A number of textile dyes, known as reactive dyes, have been used for proteinpurification in dye-ligand affinity systems, since they bind a variety of proteins in aselective and reversible manner. Most of the reactive dyes used in dye-affinity systems

.consist of a chromophore either azo dyes, anthraquinone, or phathalocyanine , linked to .a reactive group often a mono- or dichlorotriazine ring . They also have sulfonic acid

groups to provide the desired solubility of the molecule in aqueous media. These groupsare negatively charged at all pH values. Some dyes contain carboxyl, amino, chloride, ormetal complexing groups; most contain nitrogen both in or outside on aromatic ring.

Today, triazinyl-based reactive dyes are most widely used in protein purification. .Cyanuric chloride 1,3,5-trichloro-sym-triazine is the basic substance used in the

.synthesis of these dyes Fig. 2a . The presence of electronegative atoms makes the threecarbon atoms highly positive, and therefore very susceptible to nucleophilic attacks.Chromophore molecules are easily attached to this molecule to form the dichlorotri-

.azinyl dyes. The Procion MX series from Imperial Chemical Industries is a typical .example of this type of dyes Fig. 2b . By further reactions of these molecules with .other nucleophilic substituents such as aniline or sulfanilates , monochlorotriazinyl dyes

. .are synthesized. Cibacron from Ciba-Geigy and Procion H from ICI , shown in Fig.2c, are two examples of monochlorotriazinyl dyes. The only difference betwen Cibacronand Procion H series are the position of sulfonate group on the aniline ring, which is inortho-position on Cibacron, but in meta- or para-position in Procion H series.

Two dichlorotriazinyl molecules can be coupled with a bifunctional molecule e.g.,. diaminobenzene to form bifunctional triazinyl dyes. An example is Procion H-E from

.ICI is shown in Fig. 2d. Some other examples of triazinyl dyes are monofluoro-triazinyl . .Cibacron, Ciba-Geigy , trichloropyrimidnyl Drimarene, Sandoz , and difluo-

.rochloropyrimidnyl Lavafix, Bayer and Drimarene, Sandoz , which are shown in Fig.2eg, respectively. Note that, when the chloride atoms on the triazinyl ring are replacedwith other groups, the reactivity of the dye is reduced, substantially. Dye-molecules

.having more chloride or fluoride atoms can easily react with the nucleophilic groups


. . . .Fig. 2. Structure of some of the reactive dye molecules; a cyanuric chloride; b Procion MX series ICI ; c . . . . .Cibacron Ciba-Geigy and Procion H ICI ; d Procion H-E ICI ; e Monofluorotriazinyl, Cibacron,

. .Ciba-Geigy; f Trichloropyrimidnyl, Drimarene, Sandoz; g Difluorochloro-pyrimidnyl, Levafix, Bayer and .Drimarene, Sandoz; h Sulfatoethyl sulfone, Remazol, Hoechst.

on the matrix at the ligand-immobilization step. One interesting group of dyes not basedon triazinyl groups is the Remazol series from Hoechst, which to attach the matrix with

vinyl sulfone active groups, have found use as dye-ligands in protein purification Fig..2h .An important strategy is to tailor-make, or redesign the dye structure to improve the

specificity of textile dyes for target proteins. This new type of ligand is called .Abiomimetic dyeB. It carries all the advantages of the parent unmodified dye includingw xhigh specificity. This concept was first applied by Lowe et al. 21,22 early in the 1980s

and then successfully used by them and also by others for specific enzyme recovery, asw xrecently reviewed by Clonis et al. 23 .

The first biomimetic dye was prepared by linking benzamidine to the reactivechlorotriazine ring via a diaminomethylbenzene group. It was used for the specific


w xseparation of trypsin from chymotrypsin 24 . Dye-ligands having two recognitionmoieties on the triazine ring were designed to isolate kallikrein from a crude pancreatic

w x extract 25 . By using biomimetic Cibacron Blue dye phosphonated via a p-aminoben-.zyl ring , it was possible to purify alkaline phosphates from calf intestinal extract

280330-fold in one chromatographic step after specific elution with inorganic phos-w xphate 26 . A similar biomimetic dye, prepared by using a diaminohexane spacer, was

w xused to purify the same enzyme from the same source 120- to 140-fold 27 . A similarsuccess was reported for the biomimetic-dye-affinity separation of alcohol dehydroge-nase from horse liver by using Cibacron Blue 3GA bearing sulfonate, carboxylate,phosphonate, alcoholic, amido and trimethylammonium groups as terminal-ring substi-

w xtutes 28 .Developments in computational technology, especially in contemporary molecular

modeling and bioinformatics, greatly improved the design of new series of biomimeticdye ligands. It was earlier recognized that anthraquinone-moiety-containing aromaticsulfonated dyes, such as Cibacron Blue 3GA, Procion Blue H-B and MX-R andVilmafix Blue A-R tend to bind preferentially to the nucleotide-binding site of several

proteins and mimic the binding of naturally occurring anionic coenzymes e.g., NADH,. .FAD . Anthraquinone dichlorotriazine dyes such as VBAR also act as affinity labels ofw x w xMDH 29 and LDH 30 . A three-dimensional structural model of LDH as a guide,

appropriate structure changes of the dye molecules have allowed a biomimetic design ofw xthe ligand to improve the purification of L-lactate dehydrogenase 31 . The terminal

biomimetic moiety bears a carboxyl group or a ketoacid structure linked to the triazinering, thus mimicking natural ligands of L-malate dehydrogenase and these dyes have

w xshown high specificity in the affinity purification of this enzyme 32,33 . Ketoacid-grouprecognizing enzymes i.e., formate dehydogenase, oxaloacetate decarboxylase and ox-

. alate oxidase were purified by using biomimetic ligands mercaptopyruvic-, m-amino-. w xbenzoic-, and amino-ethyloxamic-biomimetic dyes 3436 . Molecular modeling has

w xrecently been applied for the design of triazine non-dye ligands for Protein A 37 ,w x w xhuman IgG 38 , and insulin precursor 39 .

2.3. Interactions between dye-ligands and proteins

The binding site of a protein is a unique stereochemical arrangement of ionic, polar,and hydrophobic groups in its three-dimensional structure, and where the polypeptidechains probably exhibit greatest flexibility. The dye-ligand molecules participate innon-covalent interaction with the protein to achieve tight and specific binding.

It has been shown in many kinetic studies that triazinyl dyes interact with an enzymein a way involving the binding site the substrate or coenzyme binding site, or the

. q qAactive siteB for a natural biological ligand NADH, NADPH, NAD , NADP , GTP,.IMP, ATP, HMG-CoA, folate, etc. of that enzyme so that this natural ligand cannot

w xbind 17,4044 . Many form of inhibition, including competitive, non-competitive, andmixed inhibition have been observed in these interactions.

Triazine dyes, polysulphonated aromatic chromophores, mimic the naturally occuringheterocycles such as nucleotide mono-, di-, and triphosphates, NAD, NADH, flavins,


acethyl-CoA and folic acid and inactivate typical nucleotide-dependent enzymes withw xdifferent efficacy 45 . Thus, they can be used as affinity ligands for glycosyltrans-

ferases.Several spectrophotometric techniques including UV visible, FTIR, NMR, ESR, and

circular dichroism, have been utilized to explain dye protein interactions, the existence . of competitive ligands e.g., substrates and coenzymes and perturbing solutes e.g., salts

. w xand organic solvents 4649 . These studies have revealed that confirmation of both thedye and enzyme is important, and the interactions might be a mixture of electrostatic andhydrophobic forces, and also at discrete sites rather than in an indiscriminate fashion.

.Interactions of the parent dyes especially Cibacron Blue F3G-A and their analogswith several oxidoreductases, phosphokinases, and ATPases have been investigatedw x50,51 . These studies have shown that both the anthraquinone and the adjacent benzenesulfonate rings on these dyes are important in binding to the enzymes. They do bind tothe enzyme molecules at a similar position and in a way similar to the AMP moiety ofthe coenzyme. Molecular models have shown a rough resemblance between CibacronBlue F3G-A and NADq, but the most important similarities are with the planar ringstructure and the negative charge groups. It has been shown by X-ray crystallographythat this blue dye binds to liver alcohol dehydrogenase at an NADq site, withcorrespondences of the adenine and ribose rings but not the nicotinamide. Thus, it wasproposed that the dye is an analog of ADP-ribose, and it interacts with the AnucleotidefoldB found in AMP, IMP, ATP, NADq, NADPq, and CTP binding sites of thecorresponding enzymes. Cibacron Blue F3G-A have been an ideal dye-ligand forespecially nucleotide-binding proteins.

.The monochlorotriazinyl dyes e.g., Cibacron Blue F3G-A, Procion Blue H-B areusually not sufficiently reactive to inactivate irreversibly. But there are some exceptionsw x 2q 2q 2q.52 . It was also observed that divalent metal ions e.g., Zn , Mg , Ca mayincrease considerably the inhibition of enzymes with these dyes by binding onto both the

w x substrate and coenzyme binding sites 53 . Dichlorotriazinyl dyes e.g., Procion Blue.MX-R have a greater reactivity, and exhibit irreversible inactivation of the enzymes

.e.g., alcohol dehydrogenase at the coenzyme-binding site.The interaction between the dye ligand and proteins can be concluded as follows:

Dye molecules mimic natural ligands, and bind some protein molecules very specificallyat their active points. However, under same conditions all proteins can be adsorbed ontodye-ligand affinity sorbents, which means that these ligands provide numerous opportu-nities for other interactions with other parts of the proteins. Most proteins are boundnonspecifically by complex combination of electrostatic, hydrophobic, hydrogen bond-ing, and charge-transfer interactions, all of which are possible considering the structuralnature of the dyes.

2.4. The matrix

Selection of the supporting matrix is the first important consideration in affinitysystems. The matrix must show extremely low nonspecific adsorption, which may bedue to charged or hydrophobic groups on its surface, which compromise the specificityof the affinity sorbent. This is essential because the power of affinity sorption relies on


specific interaction between the immobilized ligand and the target molecules within theadsorption medium.

.The matrix must have functional surface groups hydroxyl, carboxyl, amide, etc. forfurther derivatization and immobilization of ligands.

The matrix should be highly porous to allow high amount of ligand immobilization,and therefore, high enough adsorption capacity for the target, which is defined as theamount of molecules specifically bound per unit weight or volume of the sorbent.However, it should be carefully noted that a high level of matrix substitution is notalways indicative higher adsorbent capacity. The pores should be large, because in mostof the cases, the ligand andror target are large size proteins. This loose structure allowsthe target molecules easily diffuse in and out during the separation steps, which meansfast sorptionelution.

Most of the applications of affinity chromatography are performed under conditionsof low pressure, using spherical and rigid sorbent beads of a size range of 50400 mm.The bead form provides excellent flow through properties with minimal channeling inthe column applications.

Expanded bed procedures are becoming increasingly popular in bioseparation as away of avoiding the need for clarification techniques such as centrifugation and filtrationw x54,55 . Expanded bed chromatography is a technique that not only isolates and purifiestarget proteins on a preparative scale directly from crude broths containing suspended

w xmaterials but also reduces the processing time significantly 56,57 . The optimal liquidphase flow rates in the expanded-bed affinity columns are in the range of 100300

.cmrh. The size and density are two important parameters to be controlled or selectedin order to have correct fluidization in the columns at these flow rates. Dense particlescan be smaller, which means higher outer surface area that is of course desirableproperty of the affinity sorbent.

The matrix should also be physically and chemically stable under a wide range ofconditions such as high and low pHs, high and low temperatures, in situations which

require organic solvents, detergents and disruptive eluents e.g., guanidine hydrochlo-.ride especially for difficult elution or regeneration steps.

The matrix should preferentially be hydrophilic, which not only reduces the undesir-able nonspecific adsorption, but also allows the matrix swells in the aqueous mediumand reaches a loose internal bulk structure having openings larger than the preexistingpores in its dry state.

A large number of polymeric bead support materials for affinity chromatographyseparation are commercially available, as exemplified in Table 1. The basic properties ofthese matrices are briefly presented below; full details of the products should beobtained from each company and their related literature.

By far, the most popular support used is spherical cross-linked agarose beads withmolecular exclusion sizes about 107 Daltons. It may be due to its introduction at anearly stage in the development of affinity chromatography and also a large supportingliterature about its applications. This natural polymer is usually purified from marinealgae. Agarose has hydroxyl groups available for ligand immobilization. In order to

increase its physical and chemical strength and also to control the swellability therefore,.the size of the internal opennings in the matrix , agarose beads are cross-linked by


Table 1Some commercially available affinity support materialsSupport material Supplier Trade name

Conentional affinity chromatographyAgarose Pharmacia LKB, Sweden Sepharose

Bio-Rad, USA Bio-gelBio-Rad Affi-gel blue

Cellulose Amicon, USA Matrex CellufineDex tran Pharmacia LKB SephadexAgaroserPolyacrylamide IBF, France UltrogelPolyacrylamiderdextran Pharmacia LKB SephacrylPolyacrylamide Rohm Pharma, Germany Eupergit C

IBF TrisacrylBio-Rad Affi-gel

PHEMA Tessek, Czechoslovakia Separon H 1000Methacrylate Merck, Germany TSK-Gel Toyopearl

Separon Alltech, USAControlled pore glass Pierce, USA CPG

High performance liquid affinity chromatographyPolymer-clad silica J.T. Baker, USA PrepscaleSilica Dupont, USA Zorbax

Shandon, UK Hypersil WP300Merck LichrospherBeckman, USA UltrasphereWaters, USA Spheron

Methacrylate Alltech EupergitSynthetic polymer Dyno Particles, Norway DynospheresVinyl polymer Merck ToyopearlPolystyrene PerSeptive Biosystems, USA Poros-50

Membrane affinity chromatographySilica-PVA FMC Acti-DiskGlass Schott Glass Bioran-M

.covalent linkages with various cross-linkers e.g., epichlorohydrin . Sepharose andSuperose series from Pharmacia and the Bio-Gel A series from Bio-Rad are the mostpopular commercial agarose products.

Cellulose, which is a linear natural polymer consists of b-1,4 linked D-glucose unitsand contains hydroxyl groups for coupling of ligand molecules. Beaded cellulose fromMerck with high porosity, good mechanical stability and a pronounced hydrophiliccharacter has been considered as a useful support matrix.

The commercial product, Sephadex, is a cross-linked dextran in the bead formprepared also using epichlorohydrin as cross-linker. The low degrees of porosity andmechanical stability are its main disadvantages. They also show more pronounced ligandleakage and poorer flow properties.

Polyacrylamide gels are composed of a skeleton which carriers carboxyamide groups.Bio-Gel P series marketed by Bio-Rad Laboratories are one of the main productsprepared by copolymerization of acrylamide and N, N X-methylenebisacrylamide. They


are available with various pore sizes. A commercial variation on pre-activated polyacryl-amide is Enzacryl series from Koch-Light, which are produced especially for enzymeimmobilization.

Trisacryl synthetic carrier produced by LKB are derived from polymerization ofN-acryloyl-2-amino-2-hydroxymethyl 1,3-propane diol. This hydrophilic carrier hasbeen found suitable for the separation of biological macromolecules such as proteins andalso for cells.

With their high biocompatibility, hydroxyalkylmethacrylates have been considered asw xone of the most suitable biomaterials in medical applications 5860 . Spheron by

Waters Chemical with excellent chemical and physical stability have been found to beamong the most promising bioaffinity carrier matrix.

Eupergit C manufactured by Rohm Pharma consists of oxirane acrylic beads havebeen obtained by copolymerization of methacrylamide, methylenebisacrylamide, gly-cidylmethacrylate andror allyglycidylether. It is hydrophilic and exhibits high chemicalstability over a pH range of 1.012.0. These beads have high binding capacity due to ahigh epoxide content and large pore structure accessible for immobilization of largeligand molecules.

Controlled pore glass by Pierce Chemical and by Electro-Nucleonics, with differentsurface properties are the most commonly employed inorganic matrices for the immobi-lization of biological molecules. They have excellent physical properties for columnapplications. However, their use in affinity chromatography is relatively limited.

2.5. Ligand immobilization

There are many methods for immobilization of ligand molecules onto the supportw xmatrix, in which usually several intermediate steps are followed 24 . The main points

for a successful ligand immobilization are given below. Note that the correct choice ofcoupling method and conditions depend on both the matrix and the ligand.

First of all immobilization should be attempted through the least critical region not.from the active site of the ligand molecule, to ensure minimal interference on the

specific interaction between the immobilized ligand and the target molecules. Note thatchemicals and experimental conditions applied may cause deleteriotion of the ligand

.molecules means lost of their activity or functionality during activation or couplingsteps, therefore should carefully be selected.

The active sites of biological molecules are often located deep within the three-di-mensional structure of the molecule, which may cause an important steric hindrancebetween complementary ligand and target molecules. In these circumstances spacerarms, usually short alkyl chains, are frequently imposed between the matrix and theligand to ensure their accessibility to the target. Two alternative procedures may befollowed, schematically shown in Fig. 3. The matrix is first activated with an activationagent, then the spacer arm is attached covalently to the matrix through the active points.The ligand is then reacted with the other end of the spacer molecules. Alternatively, theligand-spacer arm conjugate is first synthesized and then attached to the carrier in onesingle step.


. .Fig. 3. Strategies for coupling of ligands to the support matrix; A coupling through spacer arm; B couplingthrough spacer arm-ligand conjugates.

The linkage between the matrix and ligand should be stable during the sorption andelution steps for expected repeated use of the affinity sorbents.

Many of the reactive dyes are immobilized onto matrix by direct reactions between .the reactive groups mainly hydroxyl groups on the matrix and the dye molecules

.through chloride or fluoride atoms on triazinyl groups. Nonreactive dyes can becoupled to the matrix by the usual activation procedures, and the subject has been

w xextensively reviewed 61 .Direct coupling of reactive triazinyl dyes to the matrices bearing hydroxyl groups is a

w xsimple, inexpensive and safe method 6266 . Coupling is achieved at alkaline condi-tions by nucleophilic substitution of hydroxyl groups with the reactive chlorine on the

.dye molecules Fig. 4 . Nucleophiles are generated by the high pH, which promotes the .ionisation of the matrix hydroxyl groups. Note that high pH usually above 12 may

cause hydrolysis of the chlorotriazines in the aqueous media, therefore very high pHvalues should be avoided. To couple the reactive dyes to the matrix hydroxyl groups, thematrix is incubated within the aqueous medium containing about 0.2% dye at pH 1011 . .adjusted with 0.1 M NaHCO , 1% Na CO or 0.1 M NaOH . A salt NaCl, 2% is also3 2 3included in the incubation medium, which salts-out and adsorbed of the dye moleculeson the matrix surface, and therefore allows favourable hydrolysis and immobilization.

.Coupling can be achieved at room temperature 2030 8C at pH: 1012 in about 23 .days with monochlorotriazinyl dyes e.g., Cibacron and Procion H series . However,


Fig. 4. Coupling of triazinyl dyes to the matrix bearing hydroxyl groups.

because of their higher reactivity, 12 h may be sufficient for dichlorotriazinyl dyes .e.g., Procion MX-series at the same conditions. It has been found also that a similar

substitution can be achieved with monochlorotriazinyl dyes at higher temperatures e.g.,.8090 8C .

After immobilization or use of these sorbents, in order to remove any uncovalently .interacting dye after dye immobilization and all strongly bound protein molecules

.after interaction with protein molecules , sorbents are treated with first water and thenone of the followings: 12 M salt, 6 M urea in 0.5 M NaOH, 8 M urea, dimethylsulph-oxide, 110 mgrcm3 BSA, ethylene glycol, 20% ethanol in water. The dye-immobi-lized adsorbents should be stored in a dilute buffer solution at pH 89, with a

.bacteriostat-containing solution e.g., 25% ethanol and 0.02% sodium azide .Five to ten 10 times higher substitution of triazinyl dyes have been observed onto the

matrices bearing sulfhydryl or amino groups in much shorter times few minutes to.hours even with monochlorotriazinyl dyes. For example epichlorohydrin-activated

agarose can be treated with ammonia to create amino groups on agarose surfaces. Usingsodium sulfide instead of ammonia gives a thiol matrix. The N-linked dyes are morestable than the conventional ones. However, it is often more difficult to elute theproteins. It should also be noted that S-linked dye cannot clean with NaOH.

As mentioned before, in order to reduce the steric hindrance in the interaction withthe immobilized ligand and the target protein molecules, spacer arms are introducedbetween the matrix surface functional groups and the ligand at the immobilization step.

.Poly ethyleneimine , dextran and diaminoalkane spacers have been introduced betweenthe hydroxyl groups of the matrix and the carbon atoms in which chlorine atoms areattached on the triazinyl dyes. These studies have shown some improvement in

w xselectivity 6769 .Alternatively, triazinyl dyes have been immobilized onto agarose beads via the

primary anthraquinonoid amino groups by using different activating agents and spacer .molecules. In most cases with Cibacron Blue F3G-A , protein binding was either

w xreduced or eliminated 70 , except in one case, in which much better purification ofhorse liver alcohol dehydrogenase from a crude extract with Procion Blue H-B attached . w xvia a spacer arm Sepharose 28 .

One important consideration in dye-ligand immobilization is the purity of the dye.Textile dyes do often contain a variety of minor components, such as stabilizing agents . . e.g. phosphate buffer , diluents e.g. NaCl , and anti-dusting agent e.g., dodecylben-


.zene to improve the dyes handling properties, and also isomers of the main component.All these contaminants affect adversely the dye properties in their use as affinity ligands,

w xand therefore, they should be removed before use by applying several techniques 71 .

2.6. Adsorption and elution

2.6.1. AdsorptionDye-ligand adsorbents are often supplied as slurries in solutions containing anti-

.bacterial agents e.g., sodium azide , which are needed for storage. After packing withinthe chromatography columns, they should be flushed with equilibration buffers whichshould be pre-filtered, therefore should contain no particle contamination, which maycause in rapid fouling of the column. The sample to be treated in the column should alsobe filtered to remove contaminating particles before affinity separation.

The pH and ionic strength of the equilibration buffer should be the same of thesample solution containing the target protein, and are adjusted to maximize proteinbinding. The composition of buffers should be selected correctly in each specific case.Phosphate buffers are often used because they reduce nonselective proteindye-ligandinteraction. Ionic strength may be adjusted by adding sodium or potassium chloride tothe buffer. When low conductivity buffers are required, MOPS, MES, HEPES, or Trisare often chosen.

2q 2q 2q 3q 3q .Metal ions Mg , Ca , Zn , Fe , Al , etc. may be added into the buffers .about 0.110 mM to increase the affinity of the dye-ligand to the target protein,andror to stabilize the protein molecules in the aqueous media. However, precipitationof the metal ions, which is pH dependent, may cause problems, therefore necessaryprecautions should be taken into consideration.

The affinity interaction between the immobilized dye-ligands and target proteins arealso effected by temperature. Therefore, it should be kept constant at the optimum valueboth at the sorption and elution steps.

The particle size of the adsorbent is an important parameter in optimizing adsorptionprocess. Smaller particles exhibited larger surface to volume ratio, which in turn allowshigher adsorption capacities and faster adsorption rates. But, it should be noted thatsmaller particles yield higher pressure drops in the column, which means that higherpressure differences should be applied to allow the liquid phase through the column,which may cause mechanical deformation especially of soft adsorbent particles.

.Porous particles should be preferred, in which much higher surface area internalwould be available for both the ligand immobilization, therefore for adsorption of thetarget protein molecules. Proteins are large molecules, therefore the pore size should belarge enough to allow easy trafficking of the protein molecules in and out.

Linear flow rates of buffer solution through the chromatography columns should alsobe optimized. High flow rates may minimize the film-diffusion resistance, and led theprotein molecules reach to the adsorbent active sites faster. However, high flow ratesreduce the protein adsorption in single pass units, simply due to shorter residence times

.in the columns. In addition, some mechanical deformation or even disintegration of theadsorbent matrix may occur at high flow rates. Flow rates in a range of 1030 cmrhmay be suitable in many applications.


. . .Fig. 5. Alternative strategies for adsorption: A Apositive bindingB; B Anegative bindingB; and C two-step .negative and positive .

.Selecting suitable column dimensions, especially column length L and diameter . D ratio is important. Higher LrD ratios may result higher linear velocities when the

.volumetric rate is constant , which may be beneficial from the mass transfer point ofview, but higher pressure drops are yielded in the column, which may cause mechanicaldeformation of the adsorbent particles as mentioned above.

.There are two alternative protocols can be followed in the adsorption step Fig. 5 . Inthe first one, which is the so-called Apositive bindingB, adsorbent carries a suitabledye-ligand which selectively binds the target protein. Ideally only few contaminatingproteins should bind to the column at the optimized adsorption conditions. Thesenon-specifically adsorbed contaminating proteins should be removed from the columnby using flushing equilibrating buffer before the elution step. Alternatively, a Anegative

bindingB protocol can be applied, in which contaminating proteins other than the target.protein molecules or others are adsorbed in the column preferentially. Higher recoveries

and purities can be achieved by a two-step process, in which the protein mixture is firstpassed through a negative binding column, and the effluent of this column is thendirected to the second ApositiveB binding column.

Affinity adsorption is a monolayer adsorption process, which means that adsorptionequilibrium is reached when all the ligand molecules are combined with the complemen-tary target molecules. This phenomenon may be described by simple adsorptionequilibrium expressions, namely Langmuir and Fruendlich equations given below. Theseequations can be used to predict the adsorption capacities of the affinity sorbents.

Langmuir equationC)sQ C)r K qC) . 1 . .s m d


Fruendlich equation1rn) )C sk C . 2 . .s

) . )Here, C is adsorbed solute concentration at equilibrium mgrg solid ; C is solutes .concentration in bulk liquid at equilibrium mgrml ; Q is maximum binding capacitym

. .in Langmuir isotherm model mgrg solid ; K is dissociation constant mgrml ; k isd .capacity parameter in Freundlich isotherm model mgrg solid ; n is exponential

parameter in Freundlich isotherm model.Adsorption should be followed by using an on-line dedector UV, pH, conductivity,

.refractive index, etc. . Thus, it would be possible to follow the movement of adsorptionzone during the process. When the zone reaches to the exit, as depicted in Fig. 6A, therewill be no more adsorption, because this means that all the ligands immobilized on thecarrier matrix are occupied by the target molecules. The elution step should be appliedafter this point. For continuous affinity separation, it is recommended to use a two

.columns in series Fig. 6B , in which the second column are used for adsorption, whileelution is applied to the first one.

2.6.2. ElutionA washing step is often applied to remove unselectively adsorbed contaminants from

the column before the target molecules are eluted. The composition, volume and flowrate of the washing buffer should be optimized. Elution should be performed with a highrecovery and preferably in a small volume.

Elution of the bound proteins may be achieved by nonselective or selective processes.The objective, whether they are specific or non-specific, is to be completely elute thedesired protein, at the same time, minimizing the amount of contaminating proteinwhich may be co-eluted. As mentioned before, interactions between dye-ligands andproteins may be as a result of hydrophobic, electrostatic, and hydrogen bonding. In the

. .Fig. 6. A Movement of adsorption zone in the column; and B two column system for continuous affinityseparation.


nonselective elution, pH, ionic strength, or polarity of the elution buffer is changed.These methods have been widely employed for protein elution from dye-ligand adsor-bents in both step-wise and gradient techniques. If the electrostatic interactions aredominant, increasing of the pH may be sufficient to elute the bound protein molecules.If the cation exchange is important, a very sharp effect of ionic strength would beobserved on protein elution. If hydrophobic interactions are predominating, polarity ofthe elution buffer can be reduced to promote elution by using ethylene glycol or glycerol .about 1050% . The amount of buffer to be used should be minimized. Minimum ionic

.strength low salt concentration and pH should be used for sharp elution. .Chaotropic agents urea, guanidine hydrochloride, sodium thiocyanate, etc. are

highly effective on elution of proteins. However, chaotropes may cause significantdegree of protein denaturation, and therefore they are not recommended to be used at the

.elution step. Chaotropic solutions 28 M concentration are effective to removeresidual proteins for regenerating dye-ligand sorbents for repeated use.

In cases where metal ions are involved in binding, it has been sometimes found thatsimply omitting the metal ion from the elution buffer can cause desorption. However, incases where the ternary proteinmetal-dye complexes is stronger, chelating agents suchas EDTA must be added to the elution buffer in order to disrupt the complex.

Since the interaction of many proteins with immobilized dyes occurs at the proteinsactive site, affinity elution techniques have proven useful in a great number of casesw x72 . This technique can be very effective because the affinity eluant competes with thedye ligand for the same ligand-binding site on the adsorbed target protein. Nucleotidecofactors such as NADPq, NADH, ATP, and AMP have been used to elute dehydroge-nases and other nucleotide-dependent enzymes from immobilized Cibacron Blue F3GAand other reactive dyes. Substrates, products, cofactors, inhibitors, etc. are all potentialcandidates for specific elution.

2.7. Some selected applications of dye-ligand affinity systems

Cibacron Blue F3GA which has a specific binding for nicotinamide adenine q. . dinucleotide NAD -dependent enzymes , and Procion Red HE3B which has a

q.specific binding for nicotinamide adenine dinucleotide phosphate NADP -dependent.enzymes were widely used for the purification of various hydrogenases and kinases

w x7376 . Dyes were also successfully employed for plasma fractionation and purificationw x w x77 . Hanford et al. 78 used a Cibacron Blue F3GA-Sepharose column to recover the

w xhuman serum albumin from the Cohn fraction IV precipitate. Anspach et al. 79modified monodisperse silica with different silanes for immobilization of varioustriazine dyes including Procion Red HE3B, Procion Red MX5B, and Cibacron BlueF3GA. Lactate dehydrogenase and malate dehydrogenase from different species andaldehyde reductase from rat brain were purified by affinity elution using the substrate ofthe enzyme and NADH. They showed that Cibacron Blue F3GA is more selective for

w xNADH-dependent enzymes than with the two Procion dyes. Koch et al. 80 studiedaffinity chromatography of serine proteases on the Cibacron Blue F3GA-carryingSepharose CL-4B and they showed that C2, factor II, factor IX, trypsin, chymotrypsin

w xand proteinase 3 serine proteases bound to Blue Sepharose. Rehberg et al. 81 purified


human cholesteryl ester transfer protein from lipoprotein-depleted serum and plasma in athree step procedure utilizing commercially available triazine dyes e.g., Procion Red

.H-E3B, Cibacron, Brillant Red 4G-E, Procion Yellow M-8G immobilized on agarose.The activity is approximately 50.000 and 100.000 fold purified relative to the Owen et

w xal. 82 described the use of a Procion Red HE-7B derivatized perfluorocarbon supportin the affinity extraction of malate dehydrogenase from a homogenized Saccharomycescerevisiae feed. The equipment was used to purify the enzyme on a continuous basis injust under 80% yield, and purification factor of at least 10 was achieved. Sherwood et al.w x 83 have used Procion Red H-8BN which was coupled Sepharose 6B 1.96 mol dyerg

.moist weight gel in order to purified carboxypeptidase G on a large scale from2Pseudomonas sp. strain RS-16 by a three-step procedure involving the dye affinity step.

.They have shown that in the presence of Zn II ions, the enzyme is quantitatively boundto the dye-Sepharose, whilst in the absence of metal ions binding occurred only at verylow levels. Elution of the enzyme from the column was achieved by using chelating

.agents e.g., EDTA in conjuction with a step change in pH. A methylotropic hydrox-ypyruvate reductase was partially purified from a crude bacterial extract using Cibacron

. w xBlue F3GA-attached poly EGDMA-HEMA microspheres 84 . It was reported thatthese dye-affinity microspheres revealed good adsorption properties as an affinitysupport and will be effective in processing large volumes of crude extract or liquidculture medium containing target protein.

w xTravis et al. 85 reported the use of Cibacron Blue F3GA-Sepharose beads forplasma separation; a highly pure human plasma albumin and other albumin-free plasmaproteins were obtained by using either a linear NaCl gradient elution or a 0.5 N NaSCN

w xelution. Harris and Byfield 86 employed Procion Red HE-3B-Sepharose 4B beads toexctract plasminogen from human serum. Several researchers investigated the binding

w xmechanism of serum albumin to Cibacron Blue F3GA-agarose beads 87,88 . Boyer andw xHsu 89 studied the effects of ligand concentration, pH and ionic strength on protein

w xadsorption on Cibacron Blue F3GA-Sepharose CL-6B beads. Muller-Schulte et al. 90 .prepared radiation-grafted polyamide and poly vinylalcohol in microparticulate form

and they used these microparticles for the affinity chromatographic separation of humanserum albumin using Cibacron Blue F3GA as affinity ligand. They also tested adsorp-tion characteristics of the commercial media including Sepharose, Biograft, Fractogeland VA-epoxy. Camli et.al developed Cibacron Blue F3GA-carrying uniform macrop-

. w xorous poly styrene-co-divinyl-benzene particles for specific albumin adsorption 91 .w xMcCreath et al. 92,93 developed a perfluorocarbon affinity emulsion derivatised with

the triazine dye Reactive Blue 4 for the purification of human serum albumin fromblood plasma. They reported that these liquid affinity supports present an excitingopportunity to develop a range of unit operations for the continuous purification of

w x .proteins. Nash et al. 9496 produced poly styrene-divinylbenzene chromatography .matrices and then they modified these materials with poly vinylalcohol . The adsorption

capacities of lysozyme and human serum albumin on these Procion Yellow HE-3G,w xProcion Blue MX-R-coupled matrix were investigated. Yu et al. 97 developed a

.concept of polymer-shielded dye-affinity chromatography. Poly N-vinyl pyrrolidonetreatment of a Blue-Sepharose column resulted in the binding of the polymer due tomulti-point interaction with dye-ligands. The bound polymer molecules significantly


decreased both the adsorption of foreign proteins and non-specific binding of the targetenyzmes without seriously impairing enzyme interactions with the dye ligands viaspecific nucleotide binding sites. The realization of only specific interactions improvedrecoveries and elution efficiency. In other words, the bound polymer served as a lid,opening the ligand for strong specific interactions but preventing more weak non-specific

w xinteractions. Tuncel et al. 98 produced polystyrene microspheres by phase inversionpolymerization of styrene in ethanol-methoxyethanol medium. These microspheres werethen coated with polyvinylalcohol to decrease non-specific protein adsorption. ThenCibacron Blue F3GA was attached for specific albumin adsorption. They achieved high

w xalbumin adsorption capacities. Alderton et al. 99 immobilised Procion Red H-3B,Procion Red HE-3B, Procion Red HE-7B and Procion Yellow HE-4R for purification ofimmunotoxins from an Escherichia coli fermentation extract. They reported that thesematerials show promise for the isolation of immunotoxins from immunoconjugationmixtures. Triazine dyes, such as Cibacron Blue F3GA were found to have group

specificity for albumin, dehydrogenase and lysozyme. Macroporous poly glycidyl-trially.isocynaurate-divinylbenzene carrying Cibacron Blue F3GA have been recently used for

w x w xaffinity separation of bovine serum albumin and lysozyme 100 . Denizli et al. 101,102prepared a series of dye affinity sorbents which based on polyvinylalcohol and poly 2-

.hydroxyethylmethacrylate carrying reactive dyes. They studied albumin separation bothin batch and column system from different media inluding human plasma and obtainedhigh albumin adsorption capacities.

G-DNA structures formed by a 27-mer guanosine-rich oligodeoxyribo-nucleotidewere isolated by dye-ligand chromatography, using a Reactive Green 19-agarose resin.The experiments were performed in the presence of Liq, Naq and Kq, which are able to

w xstabilize G structures to different extents 103 .Reactive chlorotriazine dyes have also been used as affinity labels for variety of

enzymes and other biological molecules dehydrogenases, kinases, aspartate transcar-. w xbamoylase, ricin A, etc. 104 .

So far, only a few dye-affinity sorbents were reported for blood detoxificationw x105109 . Bilirubin, a bile pigment, is formed as a result of the catabolism ofhemoglobin from aged red blood cells in all mammals. Although its physiologicalfunctions in the human body are not fully understood, it has been suggested that itprobably serves as a chain breaking antioxidant. It deposits in tissue, especially in thebrain and it is toxic. Disorders in the metabolism of bilirubin, especially common amongnewborn infants, may cause jaundice, a yellow discoloration of the skin and other

w x tissues. Denizli et al. 104,105 attemped to utilize dye-carrying i.e., Alkali Blue 6B,.Congo Red and Cibacron Blue F3GA pHEMA based microspheres as specific sorbent

for removal of bilirubin from human plasma in a batch and packed-bed column system.They showed that these dye-affinity microspheres are promising adsorbents for bilirubinremoval from human plasma.

Iron is an essential element for a broad spectrum of biological processes whichinclude electron transfer, transport, storage and activation of oxygen, nitrogen fixationand DNA synthesis. However, it has also a potential toxicity, and the toxic effects ofiron overload are well known. Chronic iron overload may occur in a variety of diseases

.where the administration of parental iron is necessary e.g. thalassemia, aplastic anemia .


Acute iron intoxication is also a frequent, sometimes life-threating form of poisoning,w xespecially among young children. Denizli et al. 106 immobilized Cibacron Blue F3GA,

.Alkali Blue 6B and Congo Red onto the poly EGDMA-HEMA microspheres, and thenthey were used for iron removal from aqueous solutions and human plasma. Theyshowed that these dye-affinity microspheres are suitable for repeated use for more thansix cycles without noticable loss of adsorption capacity.

The chronic toxicity of cadmium compounds includes kidney damage with protein-uria of low-molecular-weight molecules. An epidemic of Japanese itai-itai disease is

.believed to be the result of chronic ingestion of Cd II , with altered renal tubularfunction, impaired regulation of calcium and phosphorus, manifesting bone demineral-ization, osteomalacia, and pathological fractures. No specific treatments for acute orchronic cadmium poisoning are available. However, in addition to supportive therapyand hemodialysis, heavy metal poisoning is often treated with a chelating agent. Ibrahim

w xet al. 107 prepared Cibacron Blue F3GArthionein carrying pHEMA microspheres wasbound to these dye-affinity sorbents. Then they were used for cadmium removal from

.human plasma poisoned with Cd II . It was reported that obtained results made thesedyerthionein carrying pHEMA microspheres potential candidates for future detoxifica-tion studies.

Aluminum has recently been considered as a causative agent in dialysis encephalopa-thy, osteodystrophy, and microytic anemia occuring in patients with chronic renal failure

.who undergo long-term hemodialysis. Only a small amount of Al III ions in dialysissolutions may cause these disorders. Encephalopathy has also occured in childrenconsuming aluminum hydroxide as a phosphate binder for renal disorders. Aluminumhas also been implicated in neurotoxicity associated with amyotrophic lateral sclerosis, aform of parkinsonism and in Alzheimers disease. Denizli et al used Congo Red,

.Cibacron Blue F3GA and Alkali Blue 6B immobilized poly EGDMA-HEMA micro-spheres for aluminum removal from aqueous solutions, drinking water and reverse

w x .osmos water 108 . The researchers showed that affinity of Al III ions for Congo Redmolecules is significantly higher than for Cibacron Blue F3GAand Alkali Blue 6B.

Several examples where metal ions have been observed to play an important role arelisted in Table 2. In this system, the exposed electron-donating amino acid residues on

Table 2Examples of metal ion-promoted protein adsorption dextran on dye-affinity sorbentsProtein Promoting metal ion Reactive dye References

2q w xCarboxypeptidase G Zn Procion Red HE-8BN 882q w xAlkaline phosphatase Zn Procion Yellow H-A 1102q w xHexokinase Mg Procion Green H-4G 25

2q w xTyrosinase Cu Procion Blue HE-RD 1113q w xOvalbumin Al Cibacron Blue F3GA 1103q w xCatalase Fe Cibacron Blue F3GA 1123q w xFe Congo Red 1132q w xAlbumin Cu Congo Red 1142q w xZn Cibacron Blue F3GA 1153q w xGlucose oxidase Fe Cibacron Blue F3GA 1132q w xLysozyme Cu Cibacron Blue F3GA 116


the protein surface, such as the imidazole group of histidine, thiol group of cysteine andw xindoyl group of tryptophan, contribute to the binding of proteins to metal ions 110116 .

2.8. Membrane dye-affinity chromatography

In recent years, separation units consist of affinity membranes have been consideredw xas an important alternative to the adsorption columns containing sorbents 117 . Microp-

orous membranes have the advantages of large surface area, short diffusion path and lowpressure drop. As a result of the convective flow of solution through the pores, the masstransfer resistance is tremendously reduced and the binding kinetics dominates theadsorption process. This results in a rapid processing, which greatly improves theadsorption, washing, desorption and regeneration steps and decreases the probability ofinactivation of biomolecules.

w xWeissenborn et al. 118 have first studied pre-purified human serum albumin andmalate dehydrogenase adsorption onto Cibacron Blue F3GA immobilized nylon mem-branes. In order to decrease non-specific hydrophobic protein adsorption, dextran,

.hydroxyethylcellulose and polyvinyl-alcohol were covalenltly linked to bisoxirane-activated nylon membranes. Covalent immobilization of hydrophilic polymers on mem-branes eliminated non-specific protein binding. However, the dynamic permeability ofthe membranes was reduced due to polymer coating. Slightly better protein recoverieswere observed with dextran and hydroxyethylcellulose-coated membranes. They havealso used these modified membranes for separation of recombinant L-alanine dehydroge-nase from crude fermentation broth. Although enzyme recoveries were up to 90% usingcell-free supernatant, more than 50% of the product was lost, and the dynamic capacitiesdecreased remarkably. The hydrophobic coating was no positive effect on derucing this

w xfouling. Champluvier and Kula 119 have used Cibacron Blue F3G-A and severalProcion dyes as affinity ligands. They have immobilized dyes onto nylon 66 isotropic

.membranes with or without using a spacer polyethyleneimine PEI , via glutaralde-.hyde . The amount of dyes immobilized were in the range of 0.566.65 mgrg

.membrane. Using the spacer PEI it was possible to increase the Cibacron blueattachment up to 21.4 mgrg membrane. They have performed both batch and filtrationmode adsorption experiments, using albumin and lysozyme as model proteins. Theadsorption values for albumin and lysozyme were in the range of 40120 mgrcm2.

w xWashing the membranes with 1 M NaCl restored the initial capacity. Guo et al. 120have studied alkaline phosphatase recovery in a membrane affinity chromatographysystem in which Cibacron Blue F3GA and Active Red K2BP were immobilized asaffinity ligands. It was possible to immobilize up to 90 mg of Active Red K2BP onto 1 g

of membrane matrix a chemically cross-linked cellulose films with large pore size and.high porosity . They have used a membrane cartridge containing 80 sheet of membrane,

and were able to reach recoveries up to 60% of activity and a 40-fold purification withthe red affinity membranes by using 1 M NaCl as the eluent. Cibacron Blue F3GA-graftedpolyethyleneimine-coated titania microporous membranes were used to the affinity

w x . w xseparation of human serum albumin 121 . Poly 2-hydroxyethylmethacrylate 122 , or . .poly vinylalcohol -coated poly propylene hollow-fiber-affinity membranes carrying

w xCibacron Blue F3GA 123 were also prepared for separation of proteins and enzymes


w xincluding albumin and catalase. Langlotz et.al. 124 found the dye-ligand membranes .from Sartorius Gottingen, Germany highly efficient to recover malate dehydrogenase

w xfrom unclarified E. coli homogenate. Champluvier and Kula 125 introduced anadsorbent for the selective binding of enzymes, in the form of microporous Sartobindmembranes carrying Cibacron Blue F3GA in the recovery of glucose-6-phosphate

w xdehydrogenase from Saccharomyces cerevisiae. Ruckenstein and Zeng 126,127 pre- . pared monochloro-Cibacron Blue F3GA anthraquinone type , Procion Red HE-3B azo

. .type , or Procion Blue MX-R dichloro type is attached to macroporous chitosan andw xchitin and used to the adsorption of albumin. Suen et al. 128,129 prepared cellulose

membrane disc and polsulfone hollow fibers carrying different spacer arms and CibacronBlue F3GA and they used these dye-affinity membranes for lysozyme adsorption. Suen

w x .and Tsai 130 also studied commercially available Immobilon AV polyvinylidenemembrane of Millipore as the solid support for a plate-and-frame adsorptive filter. Theyused Cibacron Blue 3GA as dye-affinity ligand and used these materials for lysozymeadsorption. Kassab et al immobilized Cibacron Blue F3GA onto commercially availablemicroporous polyamide hollow fiber membranes for human serum albumin isolation

w xfrom human plasma and they reported very high protein adsorption capacity 131 .w xDenizli et al. 132 investigated lysozyme adsorption onto Cibacron Blue F3GA and

. .Cu II incorporated microporous poly hydroxyethylmethacrylate membrane and theyshowed that metal incorporation significantly increased the protein adsorption.

Affinity membranes are also employed in direct competition to chromatographicw xmatrices 133,134 . Application of selective membranes in the cross-flow mode was

described to allow adsorption of the target protein on the membrane and separation ofw xcell debris at the same time 135 .

2.9. Affinity extractionAffinity extraction based on aqueous two-phase systems has many advantages for

w xlarge-scale bioseparation processes 136,137 . Introduction of affinity ligands into thesesystems has a profound and selective influence on the partitioning efficiency of the

w xtarget proteins. Dye affinity ligands have been used in free 138 or bound to a waterw xsoluble carrier polymer 139 , usually polyethylene glycol, which is also the two-phase

forming component in these systems. However, this procedure still has some limitationsin the recovery and reduce of the ligands and polymers.

Recently, reversed micellar extraction systems by using dye ligands have beenattracted as an alternative affinity separation technique due to its simplicity and

w xscalability 140 . Cibacron Blue F3GA has also been considered as an effective ligand inw xthese systems for lysozyme and albumin extraction 141 .

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Dye-ligand Affinity Systems