Non-enzymatic glycation of antithrombin III in vitro

Non-enzymatic glycation of antithrombin III (AT-III) has been proposed as a significant contributor to the increased incidence of thrombo-occlusive events in diabetics. AT-III, isolated from normal human plasma by means of heparin affinity and ion-exchange chromatography, was incubated with 0-0.5 M glucose in neutral phosphate buffer at 37 degrees C. The extent of non-enzymatic glycation could be monitored by uptake of radioactivity as well as by binding to a phenylboronate affinity resin, which effectively retards AT-III containing ketoamine-linked glucose. Non-enzymatically glycated AT-III (approx. 1 mol glucose/mol protein) bound heparin nearly as efficiently as non-glycated AT-III. The two AT-III preparations were equally active in inhibiting thrombin cleavage of chromogenic substrate. Following incubation with [14C]glucose, structural analyses of cyanogen-bromide-cleaved peptides of enzymatically glycated AT-III showed that the [14C]glucose adducts were distributed over many sites on the molecule. This lack of specificity contrasts with the restricted sites of modification on hemoglobin, albumin and ribonuclease A, and explains why non-enzymatic glycation of AT-III has little if any effect on its function.     

Isolation and characterization of an antithrombin III variant with reduced carbohydrate content and enhanced heparin binding

Two distinct forms of antithrombin III were isolated by chromatography of normal human plasma on heparin-Sepharose. The predominant antithrombin species present (AT-III alpha), which eluted from the affinity column in 1 M NaCl, was identified as the antithrombin III form which has been previously characterized. Ionic strength of the buffer was increased to elute a variant form of antithrombin III, designated as AT-III beta. The molecular weight of AT-III beta is less than that of AT-III alpha, but physicochemical studies do not indicate measureable differences in the polypeptide portion of the proteins. Carbohydrate determination revealed the sole detectable structural difference in the two antithrombins: levels of hexosamine, neutral sugars, and sialic acid in AT-III beta were all 25-30% less than in AT-III alpha. Kinetic studies of thrombin inactivation by both antithrombins, in the presence of nonsaturating amounts of heparin, indicated that AT-III beta inhibited thrombin more rapidly. AT-III beta is also distinguishable from AT-III alpha on the basis of heparin-binding affinity estimated from titration of protein fluorescence with heparin. Thus, antithrombin III exists as two molecular entities in human plasma which differ both structurally, in carbohydrate content, and functionally, in their heparin-binding behavior.     

Further studies on the contribution of electrostatic and hydrophobic interactions to protein adsorption on dye-ligand adsorbents.

The adsorption equilibria of bovine serum albumin (BSA), gamma-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB coupling density, and the net negative charges of proteins (or aqueous - phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4 micromol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 mM Tris-HCl buffer, pH 7.5). However, because CB-6AS and CB-4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2 M. The electrostatic exclusion effect is even found for CB-6AS with a CB density as low as 2.38 micromol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For gamma-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption.     

Purification of a catalase from Thermus thermophilus via IMAC chromatography: effect of the support

A hexameric Mn-catalase was purified from crude extracts of Thermus thermophilus using ammonium sulfate precipitation and ion metal-chelate affinity chromatography (IMAC). Eupergit 250 and Sepabeads FP-EP3 epoxy supports derivatized with iminodiacetic acid (IDA) and copper were used, at similar micromole/packed milliliter of support. Although Eupergit 250-IDA-Cu support adsorbed 80% of the total proteins in the extract, it exhibited a minimum affinity for the catalase. On the other hand, Sepabeads FP-EP3-IDA-Cu allowed the full adsorption of the catalase activity, which could be desorbed in fractions of different purity. This was attributed to a different geometrical congruence of the support surfaces with the enzyme surface, resulting in a different ability to form multipoint interactions with the proteins. Thus, by a cleanup step, followed by a negative chromatographic step using Eupergit 250-IDA-Cu2+ and by the adsorption of the catalase on Sepabeads-IDA-Cu2+ support, a pure enzyme fraction was obtained and its N-terminal end was sequenced.     

Production of human antithrombin-III in a serum-free culture of CHO cells.

A simple method was developed to establish serum-independent Chinese hamster ovary (CHO) cells that grew and secreted high level of human antithrombin-III (AT-III). First, human AT-III and mouse dihydrofolate reductase (DHFR) cDNAs were transfected into DHFR-deficient CHO cells. Transfected cells were treated with increasing concentrations of methotrexate (MTX) and clones secreting high levels of AT-III (10-20 micrograms/ml/3 day) in a serum-containing medium were obtained. Serum-independent clones were derived from the serum-dependent clones by simply culturing the cells for a few weeks in a serum-free medium. In a serum-free medium the established serum-independent clones grew at normal rate and produced almost equivalent amount of AT-III to that of the serum-dependent, parent clones. In addition, AT-III from the serum-independent clones has specific activity similar to that of plasma-derived AT-III.     

Production of Human Antithrombin-III in a Serum-free Culture of CHO Cells

A simple method was developed to establish serum-independent Chinese hamster ovary (CHO) cells that grew and secreted high level of human antithrombin-III (AT-III). First, human AT-III and mouse dihydrofolate reductase (DHFR) cDNAs were transfected into DHFR-deficient CHO cells. Transfected cells were treated with increasing concentrations of methotrexate (MTX) and clones secreting high levels of AT-III (10–20 µg/ml/3 day) in a serum-containing medium were obtained. Serum-independent clones were derived from the serum-dependent clones by simply culturing the cells for a few weeks in a serum-free medium. In a serum-free medium the established serum-independent clones grew at normal rate and produced almost equivalent amount of AT-III to that of the serum-dependent, parent clones. In addition, AT-III from the serum-independent clones has specific activity similar to that of plasma-derived AT-III.     

Physiological variant of antithrombin-III lacks carbohydrate sidechain at Asn 135

Both normal antithrombin-III (AT-III alpha) and the high heparin affinity form (AT-III beta) were isolated from pooled human plasma. AT-III beta had a lower negative charge and lower molecular mass than AT-III alpha. Sialidase and endo-F digestion indicated that the inherent difference resided in the oligosaccharide component of the molecule. CNBr fragmentation showed there was an oligosaccharide sidechain missing between residues 104 and 251, subdigestion with trypsin indicated that Asn 135 was not glycosylated in AT-III beta. Chromatography of total tryptic digests on concanavalin A-Sepharose confirmed that the high heparin affinity form of antithrombin lacked an oligosaccharide moiety at Asn 135.     

Essential role of the concentration of immobilized ligands in affinity chromatography:: Purification of guanidinobenzoatase on an ionized ligand

Guanidinobenzoatase, a plasma protein with possible application as a ‘tumor marker’, has been fully purified by one-step affinity chromatography. The affinity matrix was prepared by ‘controlled’ immobilization of an enzyme inhibitor (agmatine) onto commercial agarose gels containing carboxyl moieties activated as N-hydroxysuccinimide esters. In this way, agmatine becomes immobilized through an amido bond and preserves an ionized guanidino moiety. Different matrices with different concentration of ligands were prepared in order to evaluate their properties as affinity supports. Interestingly, matrices with a very low concentration of immobilized ligands (2 μmol/ml, corresponding to the modification of only 5% of active groups in the commercial resins) exhibited a low capacity for unspecific adsorption of proteins (as anion-exchange resins) and displayed also a high capacity for specific adsorption of our target protein. On the other hand, when affinity matrices possessed a moderate concentration of agmatine (10 μmol/ml of gel or higher), two undesirable phenomena were observed: (a) the matrix behaves as a very good anionic exchange support able to non-specifically adsorb most of plasma proteins and (b) the specific adsorption of our target protein becomes much lower. The latter phenomenon could be due to steric hindrances promoted by the interaction between each individual immobilized ligand and the corresponding binding pocket in the target protein. These hindrances could also be promoted by the presence of a fairly dense layer of immobilized ligands covering the support surface, thus preventing interactions between immobilized ligands and partially buried protein-binding pockets. In this way, a successful affinity purification (23.5% yield, ×220 purification factor, a unique electrophoretic band) could be achieved by combination of three approaches: (i) the use of affinity matrices possessing a very low density of immobilized ligands, (ii) performing affinity adsorption at high ionic strength and (iii) performing specific desorption with substrates or substrate analogues.     

Heparin binding domain of human antithrombin III inferred from the sequential reduction of its three disulfide linkages. An efficient method for structural analysis of partially reduced proteins

Human antithrombin III (AT-III) was partially reduced under mild conditions in the absence or presence of low molecular weight heparin. Quantitation of reduced disulfide bonds was facilitated by the application of a water-soluble color reagent, 4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid (S-DABIA). The study shows that the three disulfide linkages of AT-III can be sequentially reduced, with Cys8-Cys128 being the most sensitive, followed by Cys21-Cys95, while Cys247-Cys430 is the most resistant to the mild reduction conditions. The rate of reduction of Cys8-Cys128 and Cys21-Cys95 was significantly decreased in the presence of heparin. The reduction of Cys8-Cys128 was also found to correlate quantitatively with the loss of heparin-accelerated antithrombin activity, heparin binding affinity, and heparin-induced fluorescence enhancement. These results suggest that Cys8-Cys128 is required for the integrity of the heparin binding domain of AT-III and support previous findings that lysyl residues surrounding Cys128 (Lys107, Lys114, Lys125, and Lys136) constitute an important part of the heparin binding site in AT-III.     

Affinity chromatography in the separation of human alpha 1-antitrypsin (alpha 1-AT) and antithrombin-III (AT-III).

The two antiproteases alpha 1-antitrypsin (alpha 1-AT) and antithrombin-III (AT-III) have been purified simultaneously from human plasma. Purification procedure consisted of gel filtration on Sephadex G-200 after initial processing of plasma, followed by ion exchange chromatography on DEAE-Sephadex A50 and DEAE-Cellulose, at a pH of 9.0 and pH 8.3 respectively. The two proteins could not be separated by any of these procedures including a lower pH (7.4) in ion exchange chromatography. Affinity chromatography on heparin-Sepharose separated the proteins since alpha 1-AT did not bind to the matrix. Alpha 1-AT unbound to the heparin-Sepharose was subsequently purified through con A-Sepharose affinity column. The final yield of both the proteins was about 20%. The molecular weight estimated on SDS electrophoresis for AT-III and alpha 1-AT was 63,000 and 50,000, respectively.     

Immunolabeling efficiency of protein A-gold complexes

A systematic study of the adsorption of protein A on colloidal gold particles varying in size from 5-16 nm was performed at different protein concentrations. The number of protein A molecules bound per colloidal particle was evaluated and the Scatchard analysis of the adsorption parameters was applied for each size of the colloid. The binding of protein A to the colloidal gold surface exhibited the same affinity pattern for all of the particle sizes. At low concentrations of stabilizing protein, adsorption took place with high affinity (Kd 1.96-3.3 nM) and the maximum number of protein A molecules attached with this affinity correlated well with the surface of the particle. At higher concentrations of protein A, adsorption exhibited a significantly lower affinity (Kd 530-800 nM), and no saturation was recorded. Competition by albumin did not reveal a preferential removal of the "low-affinity" bound protein A molecules, contradicting the model of successive shells of stabilizing protein around the colloidal particle. The immunolabeling efficiency of conjugates having the same size of gold nucleus but carrying different numbers of protein A molecules was comparatively investigated by quantitative post-embedding immunocytochemistry. Protein A-gold formed with 5-10-nm colloids gave the highest intensity of labeling when carrying the maximum number of protein A molecules that could be adsorbed with high affinity. Overloading as well as underloading these complexes resulted in a significant decrease of their immunoreactivity. The most efficient conjugates were obtained when stabilization was performed with 6 micrograms protein A/ml gold sol of 5 and 10 nm particle diameter, and 15 micrograms protein/ml of 15-nm colloid.     

Site-directed mutagenesis of alanine-382 of human antithrombin III

Antithrombin III Hamilton is a structural variant of antithrombin III (AT-III) with normal heparin affinity but impaired serine protease inhibitory activity. The molecular defect of AT-III-Hamilton is a substitution of threonine for alanine at amino acid residue 382. Recently it has been shown that both plasma-derived and cell-free-derived AT-III-Hamilton polypeptides act as substrates rather than inhibitors of thrombin and factor Xa. In the present study, the cell-free expression phagemid vector pGEM-3Zf(+)-AT-III1-432 was mutated at amino acid residue 382 of AT-III to generate 7 cell-free-derived variants. All these cell-free-derived AT-III variants were able to bind heparin as effectively as cell-free-derived normal AT-III. In terms of alpha-thrombin inhibitory activity each variant reacted differently. Variants could be grouped into 3 categories with respect to thrombin-AT-III complex formation: (1) near normal activity (glycine, isoleucine, leucine, valine); (2) low activity (threonine, glutamine); (3) no detectable activity (lysine). These data suggest that mutations at position 382 of AT-III may have a variable effect on protease inhibitory activity, depending on either the stability of the P12-P9 region of the exposed loop of AT-III, or the inability of the amino acid residue at position 382 to interact with a conserved hydrophobic pocket consisting of phenylalanine (at positions 77, 221 and 422) and isoleucine (position 412) residues.     

Selection of ceramic fluorapatite‐binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography

Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA‐specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N‐terminal sequence was found in two selected peptides: F4‐2 (KPRSMLH) and F5‐4 (KPRSVSG). The peptide F5‐4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5‐4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage‐derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Protein interactions with nanosized hydrotalcites of different composition

Nanosized hydrotalcite-like compounds (HTlc) with different chemical composition were prepared and used to study protein adsorption. Two soft proteins, myoglobin (Mb) and bovine serum albumin (BSA), were chosen to investigate the nature of the forces controlling the adsorption and how these depend on the chemical composition of the support. Both proteins strongly interact with HTlc exhibiting in most cases a Langmuir-type adsorption. Mb showed a higher affinity for Nickel Chromium (NiCr-HTlc) than for Nickel Aluminum (NiAl-HTlc), while for BSA no significant differences between supports were found. Adsorption experiments in the presence of additives showed that proteins exhibited different types of interactions onto the same HTlc surface and that the adsorption was strongly suppressed by the addition of disodium hydrogen phosphate (Na₂HPO₄). Atomic force microscopy images showed that the adsorption of both proteins onto nanoparticles was followed by the aggregation of biocomposites, with a more disordered structure for BSA. Fluorescence measurements for adsorbed Mb showed that the inorganic nanoparticles induced conformational changes in the biomolecules; in particular, the interactions with HTlc surface quenched the tryptophan fluorescence and this process was particularly efficient for NiCr-HTlc. The adsorption of BSA onto the HTlc nanoparticles induced a selective quenching of the exposed fluorescent residues, as indicated by the blue-shift of the emission spectra of tryptophan residues and by the shortening of the fluorescence decay times.     

Adsorption Behavior of Multicomponent Protein Mixtures Containing α1−Proteinase Inhibitor with the Anion Exchanger, 2-(Diethylamino) ethyl-Spherodex

The equilibrium binding behavior of α₁-proteinase inhibitor (α₁-PI) in the presence of human serum albumin (HSA) has been determined in packed bed systems with the anion exchanger, 2-(diethylamino) ethyl (DEAE) -Spherodex. Experimental data derived for the individual proteins were compared with the corresponding data obtained from batch adsorption studies as well as studies in which mixtures of these two proteins were loaded at different concentration ratios onto columns of the same anion exchange adsorbent. The results confirm that α₁-PI has a greater affinity for the anion exchanger, although competitive adsorption was observed as the inlet concentration of HSA was increased. Under these conditions, decreased binding capacities and lower dynamic adsorption rates were observed for α₁-PI with the DEAE-Spherodex anion exchange adsorbent. The results are discussed in terms of the influence which various contaminants that occur in multicomponent mixtures of proteins from human plasma can have on the equilibrium binding characteristics of a target protein with weak or strong ion exchange adsorbents under conditions approaching concentration overload in preparative chromatographic systems. These investigations have also addressed, as the first part of an iterative approach for the simulation of the adsorption behavior of multicomponent mixtures of human plasma proteins with ion exchange and affinity chromatographic adsorbents, the ability of noncompetitive and competitive Langmuirean models to simulate the adsorption of α₁-PI in the presence of different concentrations of HSA to DEAE-Spherodex.     

Thiophilic adsorption of immunoglobulins--analysis of conditions optimal for selective immobilization and purification

Immunoglobulins have been selected by their general affinity for adjacent sulfone-thioether sulfur groups as a useful model system for the characterization of thiophilic interaction chromatography. Mercaptoethanol coupled to divinylsulfone-activated agarose (thiophilic or T-gel) provided an affinity matrix for the efficient and reversible immobilization of the immunoglobulins. The adsorption/desorption process was investigated as a function of protein concentration, temperature, flow rate, and pH in different concentrations of ammonium sulfate. Immobilization of these proteins was (as a function of pH) found to be both dependent and independent of the adsorption-promoting effects of water-structure-forming salts. Buffer conditions are recommended for the selective adsorption of immunoglobulins from unfractionated human serum. These results indicate that thiophilic interaction chromatography provides a new and effective alternative for the immobilization and purification of immunoglobulins and other proteins under conditions known to preserve structure and biological activity.     

Examination of protein adsorption in fluidized bed and packed bed columns at different temperatures using frontal chromatographic methods

The influences of various experimental parameters on the dynamic adsorption capacity (DAC) and the dynamic adsorption rate (DAR) of a biomimetic affinity silica-based adsorbent in fluidized and packed bed columns operated under plug flow conditions and at different temperatures have been investigated with different inlet concentrations of hen egg white lysozyme (HEWL) and human serum albumin (HSA). The DACs as well as the DARs of both the fluidized and packed beds were examined at 10% saturation (i.e., at the QB value) and the experimental data compared with the corresponding data obtained from batch equilibrium adsorption procedures. Parameters examined included the fluid superficial velocity and protein concentration and their effect on the binding capacity and column efficiency. Consistent with various results reported from this and other laboratories on the behavior of biospecific affinity adsorbents derived from porous silica and zirconia particles, adsorbents prepared from Fractosil 1000 were found to exhibit appropriate rheological characteristics in fluidized bed systems under the experimental conditions. Moreover, changes in temperature resulted in a more significant effect on the breakthrough profiles of HSA compared to HEWL with the immobilized Cibacron Blue F3G-A with Fractosil 1000 adsorbent. This result suggests that temperature effects can possibly be employed profitably in some processes as part of a strategy to enhance column performance with fluidized bed systems for selective recovery of target proteins. At relatively low superficial velocities of the feed, the DARs with HEWL and HSA were similar for both the fluidized and packed bed column systems, whereas, at high superficial velocities, the DARs for these proteins were larger with the packed bed columns.     

Dye-ligand poly(GMA–TAIC–DVB) affinity adsorbent for protein adsorption

Macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) was prepared by a radical suspension copolymerization. Reaction of the copolymer with 2-hydroxyethyl amine was employed to obtain a hydrophilic matrix. An affinity dye, Cibacron blue 3GA, was then coupled covalently to prepare a novel macroporous affinity adsorbent. The surface and pore structure of the affinity adsorbent were examined by scanning electron micrography (SEM). SEM observations showed that the affinity adsorbent abounded in macropores. Bovine serum albumin (BSA) and lysozyme (Lys) were used as samples to examine the adsorption properties of the adsorbent. Under appropriate conditions, the affinity adsorbent had a capacity of 15.5 mg BSA/g and 22.3 mg Lys/g (wet adsorbent weight). The adsorbed proteins could be desorbed by increasing liquid phase ionic strength or by using a NaOH solution, and the adsorbent could be recycled for protein adsorption.     

Affinity chromatography of plasma proteins (guanidinobenzoatase): use of mimetic matrices and mimetic soluble ligands to prevent the binding of albumin on target affinity matrices

Serum albumin is the most abundant protein in plasma and it has a high capacity to bind many small compounds and macromolecules. In this way, albumin may promote important interferences during affinity chromatography of plasma proteins. Guanidinobenzoatase (GB) is a very relevant plasma protease that seems to be related to tumoral processes. This enzyme may be adsorbed on tailor-made agmatine-amide-agarose (CH-A) supports (e.g., the ones having 2 μmol of guanidino groups per ml of agarose attached to the support, through a 6 C aliphatic chain). Such tailor-made supports containing a very low concentration of ionized groups are hardly able to adsorb any protein by anion-exchange. However, they are able to strongly adsorb albumin. In order to solve this problem new mimetic affinity matrices have been designed: (i) by using the same ligand immobilized through a different chemical linkage [guanidino groups attached via secondary amino bonds, (AEA)] or (ii) by using slightly different ligands (e.g., 1,8-octanediamine containing a primary amino group instead of a guanidino one) also attached to the support via amido bonds (CH-DAO). Albumin adsorbs on the target and on the two mimetic matrices while GB is mainly adsorbed on the target one. Moreover, the adsorption of albumin on the affinity matrix (CH-A) is very strongly inhibited by the presence of low concentrations of soluble ligands (e.g., 1,8-octanediamine containing two ionized primary amino groups). On the contrary, the adsorption of GB on CH-A is hardly inhibited by the presence of such mimetic soluble ligand. In this way, the former offering of crude GB samples to AEA plus the use of mimetic inhibitors during adsorption of the extract on CH-A completely prevent the undesirable adsorption of albumin. In a such way, an extremely selective adsorption of GB can be performed. Such an improved chromatography procedure allows a very easy affinity purification and detection of GB.     

Affinity adsorption of lysozyme on a macroligand prepared with Cibacron Blue 3GA attached to yeast cells

The objective of this study was the development of affinity adsorbent particles with the appropriate characteristics to be applied in protein purification using the affinity ultrafiltration method. To prepare affinity macroligands Cibacron Blue 3GA, as a ligand molecule, was immobilized by covalent bonding onto yeast cell walls, the support material or matrix. The maximum attachment of the ligand to the matrix was 212 μmol/g (ligand dry weight/yeast dry weight). Lysozyme was selected as the protein model for the adsorption studies. Its adsorption onto the matrix without ligand and matrix with attached ligand were investigated batch-wise. The adsorption equilibrium isotherms appeared to follow a typical Langmuir isotherm. The maximum adsorption capacity (q(m)) of the Cell-Cibacron macroligand for lysozyme was 110 mg/ml of wet macroligand. The adsorbent was also employed for the separation of lysozyme from hen egg white. High purity lysozyme was obtained.