Isolation of human thrombin using affinity chromatography on gramicidin C-silochrome C 80

Thrombin purification is conducted by biospecific chromatography on gramicidin C-silochrome C 80. Preparations possessing the fibrinogen-coagulating activity of 2500-3200 NIH units per 1 mg of protein and containing 98% of active sites are obtained. Data obtained from electrophoresis in PAAG with the presence of DS-Na show the alpha-thrombin content to be 96%; the admixture of beta-thrombin possessing no coagulating activity does not exceed 4%. The kinetic constants are presented for thrombin hydrolysis of tosyl-L-arginine methyl ester (TAME), benzoyl-L-arginine ethyl ester (BAEE) and chromogenic substrate S-2238. The addition of isopropanol increases sharply the stability of thrombin when storing it in the aqueous-salt solutions.     

Evaluation of cellulose-based biospecific adsorbents as a stationary phase for lectin affinity chromatography

Macroporous cellulose Granocel was evaluated as a matrix for the immobilization of two lectins Concanavalin A (ConA) (108 kDa) and Wheat Germ Agglutinin (WGA) (36 kDa). Two different methods were employed for the immobilization of the lectins via their protein moieties by a Schiff's bases reaction. One of them results in covalent coupling of the lectin directly to the support and the other gives the attachment through a long spacer arm which benefits the immobilization of voluminous ConA molecules. The adsorbents were characterized by the glycoproteins sorption recording adsorption kinetic data and isotherms. The adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg/ml support and a high recovery (up to 93%). The adsorption isotherms of glucose oxidase (GOD) onto ConA adsorbents reveals an adsorption behavior with high and low affinity binding sites. The dissociation constant K(d) of the ligand-sorbate complex is approximately 1 x 10(-6) and 0.4 x 10(-5)M, respectively. It was supposed that the second step is related to the sorption of solvated GOD onto already adsorbed GOD forming sorbate dimers.     

Methacrylamidohistidine in affinity ligands for immobilized metal-ion affinity chromatography of human serum albumin

Different biologands carrying synthetic adsorbents have been reported in the literature for protein separation. We have developed a novel and new approach to obtain high protein adsorption capacity utilizing 2-methacrylamidohistidine (MAH) as a bioligand. MAH was synthesized by reacting methacrylochloride and histidine. Spherical beads with an average size of 150–200 μm were obtained by the radical suspension polymerization of MAH and 2-hydroxyethyl-methacrylate (HEMA) conducted in an aqueous dispersion medium. p(HEMA-co-MAH) beads had a specific surface area of 17.6 m²/g. Synthesized MAH monomer was characterized by NMR. p(HEMA-co-MAH) beads were characterized by swelling test, FTIR and elemental analysis. Then, Cu(II) ions were incorporated onto the beads and Cu(II) loading was found to be 0.96 mmol/g. These affinity beads with a swelling ratio of 65%, and containing 1.6 mmol. MAH/g were used in the adsorption/desorption of human serum albumin (HSA) from both aqueous solutions and human serum. The adsorption of HSA onto p(HEMA-co-MAH) was low (8.8 mg/g). Cu(II) chelation onto the beads significantly increased the HSA adsorption (56.3 mg/g). The maximum HSA adsorption was observed at pH 3.0 Higher HSA adsorption was observed from human plasma (94.6 mg HSA/g). Adsorption of other serum proteins were obtained as 3.7 mg/g for fibrinogen and 8.5 mg/g for γ-globulin. The total protein adsorption was determined as 107.1 mg/g. Desorption of HSA was obtained using 0.1 M Tris/HCl buffer containing 0.5M NaSCN. High desorption ratios (up to 98% of the adsorbed HSA) were observed. It was possible to reuse Cu(II) chelated-p(HEMA-co-MAH) beads without significant decreases in the adsorption capacities.     

Affinity surfactants as reversibly bound ligands for high-performance affinity chromatography

Pyridine was coupled covalently to a nonionic ethoxylated alcohol: octaethylene glycol n-hexadecyl ether. This modified surfactant was found to be a reversible, competitive inhibitor of horse serum cholinesterase. The surfactant bound irreversibly, in aqueous media, to octadecyl-bounded reverse phase silica particles commonly used for high-performance liquid chromatography. The amount of ligand bound was found to be 550 mumol/ml of packing, a concentration that is over 100 times higher than what can be normally bound to agarose affinity chromatography supports. With this packing, a 280-fold purification of cholinesterase from horse serum and a 79-fold purification of human serum cholinesterase were accomplished, with yields greater than 80%, using a 2-cm-long column and a 7-min elution time. The affinity surfactant could be eluted from the column using a 6:4 (v/v) mixture of methanol and isopropanol. This technique should be generally applicable in the development of biospecific supports for high-performance affinity chromatography.     

NMR screening of new carbocyanine dyes as ligands for affinity chromatography

Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N‐carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α‐chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un‐substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α‐chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α‐chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD‐NMR technique was successfully used to screen cyanine–protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography. Copyright © 2014 John Wiley & Sons, Ltd.     

Design and selection of ligands for affinity chromatography

Affinity chromatography is potentially the most selective method for protein purification. The technique has the purification power to eliminate steps, increase yields and thereby improve process economics. However, it suffers from problems regarding ligand stability and cost. Some of the most recent advances in this area have explored the power of rational and combinatorial approaches for designing highly selective and stable synthetic affinity ligands. Rational molecular design techniques, which are based on the ability to combine knowledge of protein structures with defined chemical synthesis and advanced computational tools, have made rational ligand design feasible and faster. Combinatorial approaches based on peptide and nucleic acid libraries have permitted the rapid synthesis of new synthetic affinity ligands of potential use in affinity chromatography. The versatility of these approaches suggests that, in the near future, they will become the dominant methods for designing and selection of novel affinity ligands with scale-up potential.     

Methacryloylamidohistidine in affinity ligands for immobilized metal-ion affinity chromatography of ferritin

A new metal-chelate adsorbent utilizing 2-methacryloylamidohistidine (MAH) was prepared as a metalchelating ligand. MAH was synthesized using methacryloly chloride and histidine. Monosize nanospheres with an average diameter of 450 nm were produced by emulsion polymerization of 2-hydroxyetylmethacrylate (HEMA) and MAH. Then, Fe³⁺ ions were chelated directly onto the monosize nanospheres. Mon-poly(HEMA-MAH) nanospheres were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and elemental analysis. Fe³⁺ chelated monosize nanospheres were used in ferritin adsorption from an aqueous solution. The maximum ferritin adsorption capacity of Fe³⁺-chelated mon-poly(HEMAMAH) nanospheres was 202 mg/g at pH 4.0 in acetate buffer. The non-specific ferritin adsorption on the monpoly( HEMA-MAH) nanospheres was 20 mg/g. The adsorption behavior of ferritin could be modeled using both Langmuir and Freundlich isotherms. The adsorption capacity decreased with increasing ionic strength of the binding buffer. High desorption ratios (> 95% of the adsorbed ferritin) were achieved with 1.0 M NaCl at pH 7.0. Ferritin could be repeatedly adsorbed and desorbed with the Fe³⁺-chelated mon-poly(HEMA-MAH) nanospheres without significant loss of adsorption capacity.     

Synthesis of a biospecific adsorbent for the purification of the three human retinoic acid receptors by affinity chromatography.

The total synthesis of an affinity gel suitable for the purification of retinoic acid receptors (hRARs) is reported. A chalcone derived from a potent retinobenzoic acid (Ch55) was chosen as the ligand and fixed to an immobilized matrix by coupling with the N-hydroxysuccinimide ester of agarose (Affi-Gel 10, Bio-Rad Laboratories). Efficiencies of purification of the different human RARs were tested, using recombinant receptors produced with the baculovirus expression system.     

Magnetic biospecific affinity adsorbents for lysozyme isolation

Summary Magnetic biospecific affinity adsorbents for lysozyme isolation have been prepared. They were obtained by incorporation of fine magnetite particles into the structure of chitin, agar or agarose. Hen egg white lysozyme was obtained in 90% purity in one step.     

Easy assembly of ligands for glycosidase affinity chromatography.

An improved, high-yield synthesis of the corresponding N-carboxypentyl derivatives of three iminoalditol glycosidase inhibitors has been developed for affinity chromatography enzyme purification. Reductive amination of 1-deoxynojirimycin (or its D-manno or D-galacto analogues) with methyl 5-formylvalerate and NaBH3CN at neutral pH afforted an aminoester which upon hydrolysis with aqueous 5% HCl gave the desired aminoacid in 97% overall yield. These amino acids could then be covalently attached using water-soluble carbodi-imide to 6-aminohexyl Sepharose 4B.     

Avidin column as a highly efficient and stable alternative for immobilization of ligands for affinity chromatography

The avidin/biotin system was applied as a general mediator in the adsorption/desorption or immobilization of biologically active macromolecules to solid supports. In this context, model biotinylated proteins (lectins and antibodies) were attached to avidin-coupled Sepharose. As examples for affinity chromatography, peanut agglutinin and anti-transferrin antibody were used to isolate asialofetuin and transferrin, respectively. The capacity and product yields were significantly better than those achieved with conventional affinity chromatography on CNBr-activated Sepharose columns containing the same lectin or antibody. Moreover, the columns were characterized by improved stability properties exhibiting remarkably low levels of leakage.     

Cyclic and linear oligocarbamate ligands for human thrombin.

Several classes of compounds have been tested as potential inhibitors of the serine protease thrombin, an important regulator of blood coagulation cascades. We describe here the discovery of a new class of thrombin inhibitors based on an unnatural carbamate biopolymer. Oligocarbamate thrombin inhibitors were identified through the screening of diverse cyclic trimer, cyclic tetramer, and linear tetramer libraries using the one bead, one peptide method. Whereas the cyclic trimer oligocarbamate ligands bound thrombin with modest affinity, a cyclic tetramer oligocarbamate inhibited thrombin with an apparent Ki of 31 nM. Linear oligocarbamate tetramers bound thrombin with inhibition constants in the 100-nM range. These nonpeptidic, oligomeric molecules may provide the basis for further drug development and studies of thrombin ligand interactions.     

Aptamers as affinity ligands for downstream processing

Aptamers are single‐stranded synthetic oligonucleotides that are able to capture their target molecule with high affinity and specificity. Therefore, they can be thought of as nucleic acid‐based alternative to antibodies, which have several advantages over their amino acid‐based counterparts. Consequently, aptamers can be used in different applications based on molecular recognition including affinity separations. This review will summarize the state‐of‐the‐art in aptamer‐based affinity separations; will discuss the current limitations and will highlight possible future prospects. The first part will point out the advantages of aptamers in downstream processing. Here, the properties of aptamers will be discussed along with their implications on downstream processing from a user's point of view. In the second part, a brief summary of the literature is given with focus on aptamer‐based separation of proteins. Finally, some drawbacks of aptamers will be illustrated and possibilities to overcome these limitations will be suggested. New technologies in the fields of aptamer selection and synthesis are expected to further promote the use of aptamers as affinity ligands in downstream processing.     

Anhydroelastase: enhanced affinity toward product-type ligands revealed by affinity chromatography

Anhydroelastase was effectively isolated by a single operation of affinity chromatography from a complex mixture produced by phenylmethylsulfonylation and alkaline treatment of porcine pancreatic elastase. The adsorbent used for the chromatography was 6-aminohexanoyl-trialanine, which corresponds to a product of elastase action, immobilized on Sepharose 4B. Successful resolution by the operation indicated that this immobilized ligand possesses the highest affinity for anhydroelastase among various proteins including regenerated elastase in the mixture. Comparative affinity chromatography on immobilized anhydroelastase and on immobilized native elastase further confirmed the stronger interaction of anhydroelastase with the product-type peptides. Immobilized anhydroelastase was also found to be useful in the purification and search for naturally occurring proteinase inhibitors.     

New prospects for a method of biospecific chromatography

The polyacrylamide hydrogels with covalently immobilized ovomucoid from the duck's egg white were synthesized by radical copolymerization. These hydrogels can defend the immobilized insulin against the action of proteolytic enzymes. Biospecific interaction of the polysaccharide component of ovomucoid with lectins leads to the targeting transport of the hydrogel particles onto the small intestine wall.     

Sepharose 4B as a matrix for affinity chromatography. A spin-labelling investigation using nitroxides as model ligands

Nitroxide spin labels were attached to CNBr-activated Sepharose 4B directly and through oligoglycines and oo-amino-carboxylic acids of varying length. The homogeneity of the carbohydrate environments of directly attached labels was investigated by measuring dipolar interactions between nitroxides as a function of solvation and of spin dilution with a diamagnetic analogue, as well as by electron exchange between the nitroxides and paramagnetic metal ions in solution. Only the exchange experiment revealed any inhomogeneity, suggesting that a small proportion of sites may be less accessible than the majority. The distances between sites were sufficiently small to allow, in principle, multiple-site interactions between quite small proteins in solution and immobilized ligands. Reorientation of the label at the matrix, characterized by the correlation time t, became more rapid with increasing spacer length n. For n > 12, the decrease in t was less pronounced. The two types of spacer behaved similarly. Thus an ideal spacer length for affinity separations is 12 atoms; this is in good agreement with data from a variety of affinity separations. The results of electron spin resonance studies of the effect of non-aqueous solvent on directly and indirectly labelled Sepharose 4B were used to suggest reasons why enzymes immobilized on Sepharose may be stabilized to denaturing solvents.     

In vitro toxicity assays for dye ligands used in affinity chromatography

Some reactive textile dyes have been used for years as biomimetic ligands in protein purification. There has been reluctance, however, to use these dyes on a large scale for therapeutically applicable proteins for fear of possible dye leakage and consequent contamination. Therefore, toxicological data are necessary to quantify the level of this hazard. This study deals with a series of in vitro toxicity investigations with eukaryotic cells (growth, polyploidy, etc.) and with prokaryotic cells (Escherichia coli) for genotoxic studies. Both approaches demonstrated a lack of or slight toxicity for Reactive Blue 2 and Reactive Red 120 and their derivatives over the range 10–62.5 μg/ml in several assays.