sepharose and divinyl-sulfone

The immobilization of ficin (a cysteinyl proteases) on vinyl sulfone agarose produced its almost full inactivation. It was observed that the incubation of the free and immobilized enzyme in β-mercaptoethanol produced a 20 % of enzyme activity recovery, suggesting that the inactivation due to the immobilization could be a consequence of the modification of the catalytic Cys. To prevent the enzyme inactivation during the immobilization, switching off of ficin via Cys reaction with dipyridyl-disulfide was implemented, giving a reversible disulfide bond that produced a fully inactive enzyme. The switch on of ficin activity was implemented by incubation in 1 M β-mercaptoethanol. Using this strategy to immobilize the enzyme on vinyl sulfone agarose beads, the expressed activity of the immobilized ficin could be boosted up to 80 %. The immobilized enzyme presented a thermal stabilization similar to that obtained using ficin-glyoxyl-agarose beads. This procedure may be extended to many enzymes containing critical Cys, to permit their immobilization or chemical modification.

Topics: Disulfides; Endopeptidases; Enzyme Stability; Enzymes, Immobilized; Ficain; Hydrogen-Ion Concentration; Mercaptoethanol; Peptide Hydrolases; Sepharose; Sulfhydryl Compounds; Sulfones

Penicillin G acylase (PGA) from

Topics: Enzyme Stability; Enzymes, Immobilized; Escherichia coli; Hydrogen-Ion Concentration; Osmolar Concentration; Penicillin Amidase; Sepharose

Lipase B from

Topics: Butyrates; Enzymes, Immobilized; Ethylenediamines; Lipase; Sepharose

Lipase from Thermomyces lanuginosus (TLL) has been covalently immobilized on heterofunctional octyl-vinyl agarose. That way, the covalently immobilized enzymes will have identical orientation. Then, it has blocked using hexyl amine (HEX), ethylenediamine (EDA), Gly and Asp. The initial activity/stability of the different biocatalysts was very different, being the most stable the biocatalyst blocked with Gly. These biocatalysts had been utilized to analyze if the enzyme activity could decrease differently along thermal inactivation courses depending on the utilized substrate (that is, if the enzyme specificity was altered during its inactivation using 4 different substrates to determine the activity), and if this can be altered by the nature of the blocking agent and the inactivation conditions (we use pH 5, 7 and 9). Results show great changes in the enzyme specificity during inactivation (e.g., activity versus triacetin was much more quickly lost than versus the other substrates), and how this was modulated by the immobilization protocol and inactivation conditions. The difference in the changes induced by immobilization and inactivation were confirmed by fluorescence studies. That is, the functional and structural analysis of partially inactivated immobilized enzyme showed that their inactivation pathway is strongly depended on the support features and inactivation conditions.

Topics: Aspartic Acid; Enzymes, Immobilized; Ethylenediamines; Eurotiales; Fungal Proteins; Glycine; Lipase; Microspheres; Sepharose; Substrate Specificity; Sulfones; Triacetin

Lipase from Thermomyces lanuginosus (TLL) and lipase B from Candida antarctica (CALB) have been immobilized on divinylsulfone (DVS) activated agarose beads at pH 10 for 72 h. Then, as a reaction end point, very different nucleophiles have been used to block the support and the effect of the nature of the blocking reagent has been analyzed on the features of the immobilized preparations. The blocking has generally positive effects on enzyme stability in both thermal and organic solvent inactivations. For example, CALB improved 7.5-fold the thermal stability after blocking with imidazole. The effect on enzyme activity was more variable, strongly depending on the substrate and the experimental conditions. Referring to CALB; using p-nitrophenyl butyrate (p-NPB) and methyl phenylacetate, activity always improved by the blocking step, whatever the blocking reagent, while with methyl mandelate or ethyl hexanoate not always the blocking presented a positive effect. Other example is TLL-DVS biocatalyst blocked with Cys. This was more than 8 times more active than the non-blocked preparation and become the most active versus p-NPB at pH 7, the least active versus methyl phenylacetate at pH 5 but the third one most active at pH 9, versus methyl mandelate presented lower activity than the unblocked preparation at pH 5 and versus ethyl hexanoate was the most active at all pH values. That way, enzyme specificity could be strongly altered by this blocking step.

Topics: Ascomycota; Candida; Catalysis; Enzyme Stability; Enzymes, Immobilized; Fungal Proteins; Hydrogen-Ion Concentration; Kinetics; Lipase; Nanotechnology; Sepharose; Substrate Specificity; Sulfones

A variety of applications in glycobiology exploit affinity chromatography through the immobilization of glycans to a solid support. Although several strategies are known, they may provide certain advantages or disadvantages in how the sugar is attached to the affinity matrix. Additionally, the products of some methods may be hard to characterize chemically due to non-specific reactions. The lack of specificity in standard immobilization reactions makes affinity chromatography with expensive oligosaccharides challenging. As a result, methods for specific and efficient immobilization of oligosaccharides remain of interest. Herein, we present a method for the immobilization of saccharides using N'-glycosylsulfonohydrazide (GSH) carbohydrate donors. We have compared GSH immobilization to known strategies, including the use of divinyl sulfone (DVS) and cyanuric chloride (CC), for the generation of affinity matrices. We compared immobilization methods by determining their immobilization efficiency, based on a comparison of the mass of immobilized carbohydrate and the concentration of active binding sites (determined using lectins). Our results indicate that immobilization using GSH donors can provide comparable amounts of carbohydrate epitopes on solid support while consuming almost half of the material required for DVS immobilization. The lectin binding capacity observed for these two methods suggests that GSH immobilization is more efficient. We propose that this method of oligosaccharide immobilization will be an important tool for glycobiologists working with precious glycan samples purified from biological sources.

Topics: Binding Sites; Chromatography, Affinity; Glycomics; Hydrazines; Oligosaccharides; Plant Lectins; Sepharose; Sulfones; Triazines

Heat shock proteins (HSPs) hsp70/hsc70, hsp90 and hsp96 were separated from mammalian cells and tissues on a gel obtained by the reaction of beta-mercaptoethanol with divinyl sulfone-activated Sepharose CL-6B (thiophilic gel or T-gel). Hsp90 revealed a much higher affinity towards the T-gel than the other HSPs. One-step thiophilic interaction chromatography of proteins resulted in a more than 80% purity and 85% yield of hsp90. Based on this observation, a simple and efficient method for the purification of hsp90 and a procedure for the simultaneous purification of several HSPs (hsp70/hsc70, hsp90 and hsp96) using thiophilic interaction chromatography was developed. All the HSPs were recovered with a high yield and purity (90-99%). The results indicated that the thiophilic gel is a highly efficient affinity matrix for the purification of hsp90 and can be used in the protocols of purification of different HSPs from cells and tissues of various animal species.

Topics: Animals; Antigens, Neoplasm; Cells; Chromatography, Affinity; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Mammals; Mercaptoethanol; Molecular Weight; Organ Specificity; Sepharose; Sulfhydryl Compounds; Sulfones

The interaction of porcine pepsin A with immobilized derivatives of aromatic amino acids was investigated. Divinyl sulfone-activated Sepharose was used to immobilize N-acetyl-l-phenylalanine and 3,5-diiodo-l-tyrosine via their free carboxyl groups and l-tyrosine via its amino group. Immobilized l-tyrosine was iodinated after coupling. The optimum conditions for the separation of porcine pepsin A using the prepared affinity carriers were studied and the following parameters were established: enzyme recovery, reproducibility of analyses, capacity and dependence of the elution peak area on the concentration of the loaded enzyme. The ability of the prepared affinity carriers to retain various types of proteins was compared under optimum conditions for porcine pepsin A separation. While immobilized 3,5-diiodo-l-tyrosine and iodinated l-tyrosine-Sepharose adsorbed relatively high amounts of bovine serum albumin and ovalbumin, only negligible amounts of these proteins were adsorbed to immobilized N-acetyl-l-phenylalanine. The behavior of porcine pepsin A was the same as its complex with pepstatin A on the prepared affinity carriers, indicating that the enzyme active site is not involved in the studied interaction.

Topics: Amino Acids, Aromatic; Animals; Chromatography, Affinity; Ethylenediamines; Pepsin A; Phenylalanine; Sepharose; Sulfones; Swine; Tyrosine

Affinity chromatography of porcine protease and its zymogen was carried out on immobilized components of specific substrate used for the pepsin determination. For the immobilization of N-acetyl-L-phenylalanine and iodinated derivative of L-tyrosine, divinyl sulfone activated Sepharose was used. Ligands with blocked amino group and free carboxyl one were linked to Sepharose via ethylene diamine spacer using carbodiimide reaction. Conditions of affinity chromatography of porcine pepsin and pepsinogen on the prepared carriers were optimized: the effect of pH, ionic strength and a nature of the buffers used on adsorption of the enzyme and zymogen to an affinity carrier, as well as their elution was studied. The following parameters were taken into consideration: capacity of the prepared affinity matrices, reproducibility of experiments and the enzyme stability. Pepsin was adsorbed to both immobilized ligands at pH 3.5-4.0; for the elution of the enzyme it was necessary to increase ionic strength (up to 0.5 M). For the adsorption of pepsinogen pH 5.2 was found to be optimum, for its desorption, an increase of ionic strength was used.

Topics: Amino Acids; Animals; Chromatography, Affinity; Indicators and Reagents; Ligands; Pepsin A; Pepsinogen A; Sepharose; Substrate Specificity; Sulfones; Swine

By coupling 3-(2-mercaptoethyl)quinazoline-2,4(1H,3H)dione (MECH) to divinyl sulfone activated agarose, a novel thiophilic matrix was obtained which allows the binding of immunoglobulins from different sources. In contrast to other thiophilic gels, antibodies are bound at low ionic strength and can easily be desorbed in intact form by elution with dilute alkali. The potential of using the MECH-gel was demonstrated by the purification of antibodies from human and animal (goat, rabbit, mouse) sera. The functional integrity of the purified antibodies was established with cytoplasmic islet cell antibodies from the sera of patients with type I diabetes and autoantibodies against thyroid peroxidase from patients with Graves' and Hashimoto's disease.

Topics: Animals; Autoantibodies; Chromatography, Affinity; Diabetes Mellitus, Type 1; Fluorescent Antibody Technique, Indirect; Graves Disease; Humans; Immunoglobulins; Iodide Peroxidase; Islets of Langerhans; Osmolar Concentration; Protein Binding; Quinazolines; Radioimmunoassay; Sepharose; Sulfones; Thyroiditis, Autoimmune

The requirements for divinylsulfone (DVS)-based gels to act as thiophilic adsorbents, binding immunoglobulins in a salt-dependent manner have been examined. No differences in protein binding were observed for a DVS-activated gel reacted with mercaptoethanol (the T-gel), or for the same gel treated at high pH to hydrolyse the active groups and/or allow the formation of cross-links within the matrix, indicating that an O atom may be substituted for the thioether without affecting the thiophilic interactions. Extending the time of the activation reaction between DVS and the matrix results in increased amounts of sulfone attached to the gel, but decreased levels of active vinyl groups. When coupled to mercaptoethanol, these adsorbents bound more IgG than gels activated for shorter periods. This provides a convenient method to prepare thiophilic adsorbents of high capacity while minimising the amount of DVS used. The immobilised vinylsulfone must be linked to an electron donating atom for IgG to bind. When the vinyl was instead reduced with sodium borohydride, protein binding was decreased. No IgG bound to amine-coupled DVS-activated adsorbents, perhaps due to an overall positive charge on these gels at pH 7.4. The binding of human IgG to the adsorbents is dependent on ligand density, with little protein binding to gels having less than 16 mumol sulfone per ml. The binding increased with the ligand density above this level, with more than 25 mg IgG binding per ml to an adsorbent having 114 mumol sulfone per ml. The lack of binding at low ligand densities would be expected if the IgG must interact with two or more sulfone ligands to be retained on the adsorbent.

Topics: Borohydrides; Cross-Linking Reagents; Gels; Hydrolysis; Immunoglobulin M; Ligands; Mercaptoethanol; Oxidation-Reduction; Protein Binding; Sepharose; Structure-Activity Relationship; Sulfones

During the use of chromatographic supports for the purification of proteins or the selective removal of substances by immunoaffinity, leakage of the antibodies immobilized on the matrix is systematically observed. When the cleansing of blood plasma by extracorporeal circulation is concerned, it is of prime importance that the immunoadsorbents exhibit an extensive chemical stability over the whole range of experimental conditions. To study and minimize this leakage, a matrix, Sepharose CL-4B, was activated by various chemical reagents and coupled to goat anti-apolipoprotein B polyclonal antibodies. Immunoadsorbents thus prepared were compared with those obtained earlier by cyanogen bromide activation. It turns out that divinyl sulphone- and tresyl chloride-activated supports lead to similar results in terms of coupling yield and adsorption capacity, but to a significant reduction in released antibodies.

Topics: Animals; Antibodies; Antibody Specificity; Apolipoproteins B; Butylene Glycols; Chromatography, Affinity; Cyanogen Bromide; Goats; Humans; Hydrogen-Ion Concentration; Immunosorbent Techniques; Immunosorbents; Sepharose; Sulfones

As a model for the isolation of a labile or trace protein, the purification of uteroglobin (UGL) by immunoaffinity chromatography is described. Antibody was isolated from sheep antiserum by immunoprecipitation, and coupled to divinylsulphone-activated agarose (Mini Leak). For the immunoabsorption stage rabbit uterine mucosal scrapings were defatted and incubated directly with the immunosorbent. After washing and desorption, the UGL preparation contained relatively few high molecular weight impurities and these were removed by gel chromatography. Purification was monitored at each step by two-dimensional SDS polyacrylamide gel electrophoresis and immunoelectrophoresis. Furthermore, affinity-purified UGL was tritiated with N-succinimidyl[2,3-3H]propionate and assayed by fluorography. In order to determine absolute UGL concentrations a competitive ELISA was developed.

Topics: Animals; Antibody Specificity; Chromatography, Affinity; Cross-Linking Reagents; Enzyme-Linked Immunosorbent Assay; Glycoproteins; Immune Sera; Immunoelectrophoresis; Immunoglobulin G; Mucous Membrane; Precipitin Tests; Rabbits; Sepharose; Sheep; Sulfones; Uteroglobin

Divinylsulphone-activated agarose is an attractive alternative to several of the activated supports usually used. Unlike CNBr-activated gels, it does not leak the immobilized protein at high pH. It reacts readily with proteins at near-neutral pH (unlike the epoxy-activated supports). Generally, divinylsulphone-activated agarose reacts with amino, hydroxyl, and sulphydryl groups, thus allowing immobilization of a wide spectrum of ligands. Moreover, it is available in an aqueous suspension free of organic solvents and neither requires time-consuming swelling nor washing.

Topics: Animals; Cattle; Chemical Phenomena; Chemistry, Physical; Chromatography, Affinity; Drug Stability; Goats; Hydrogen-Ion Concentration; Immunoglobulins; Immunosorbent Techniques; Proteins; Rabbits; Sepharose; Sulfones