Affinity chromatography of porcine pepsin and pepsinogen using immobilized ligands derived from the specific substrate for this enzyme

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.     

Binding mitochondria to cryogel monoliths allows detection of proteins specifically released following permeability transition

Following proapoptotic signals such as calcium-induced mitochondrial permeability transition or translocation of proapoptotic proteins, mitochondria induce cell death through release of apoptogenic proteins. The mechanism of release and the identity of the released proteins are currently debated. Earlier attempts at identification of the apoptogenic proteins have been hampered by a high nonspecific background. Our aim was to develop a novel method where background release was eliminated, allowing proteins specifically released from mitochondria following proapoptotic stimulation to be identified. Liver mitochondria were immobilized and washed on cryogel monoliths prior to induction of protein release (calcium or Bid/Bax). Immobilized mitochondria exhibited normal morphology and swelling response and retained respiratory activity. The released proteins were collected, concentrated, separated on polyacrylamide gels which were cut into pieces, trypsin-digested, and analyzed using liquid chromatography-tandem mass spectrometry. Control samples contained no protein, and stimulation with calcium and Bid/Bax resulted in identification of 68 and 82 proteins, respectively. We conclude that, in combination with the robust proteomic approach, immobilization on cryogel monoliths is a fruitful approach for studying specific protein release from isolated mitochondria. We propose that this method is a powerful tool to further characterize the role of mitochondria in cell death induction.     

Anomalous behavior of bacteriophage lambda polypeptides in polyacrylamide gels: resolution, identification, and control of the lambda rex gene product.

The resolution of lambia proteins was compared on the two types of sodium dodecyl sulfate-polyacrylamide gels commonly in use. The two kinds of gel differ essentially in the ratio of the cross-linker, N'-N-bismethylene-acrylamide (bisacrylamide), to acrylamide monomer. Several lambda proteins migrate relatively more slowly in gels with high bisacrylamide/acrylamide ratios (HB gels) than in gels with low ratios, although the two types of gel are of roughly equivalent porosity. This effect is illustrated by a change in relative position of both the Rex and Int proteins, with apparent increases in molecular weight of about 8 and 15%, respectively, in the HB gels. This work confirms that like repressor and Int, the 28.5-kilodalton protein, identified as Rex on HB gels, is postively regulated by the lambdacII and cIII products and negatively controlled Cro. An intact y site is required for Rex and repressor expression after infection, whereas their synthesis in a lysogen is dependent upon a functional maintenance promoter, Prm.     

Polyacrylamide gels with modified cross-linkages

The retention of low molecular weight proteins during electrophoresis through gradient polyacrylamide gels was improved when a gradient of N,N′,N″-triallyl citric triamide (TACT) was superimposed on the gradient of acrylamide and N,N′-methylenebisacrylamide (MBA). Gels cross-linked only with N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) are soluble in dilute periodic acid or dilute aqueous solutions of bases. DHEBA cross-linked gradient gels have a smaller pore structure at high acrylamide concentrations and a more open structure at low acrylamide concentrations than gels cross-linked with MBA. Proteins labeled with tritium and carbon-14 were fractionated through DHEBA cross-linked gradient gels and the isotopes measured after solution of the gel with periodic acid. The mild solubilizing conditions enhanced isotope resolution. The characteristics of several cross-linking molecules are discussed and reasons advanced for the superiority of those with acrylamido end groups.     

New methods for the preparation of biospecific adsorbents and immobilized enzymes utilizing trichloro-s-triazine

Methods for preparing biospecific adsorbents and immobilized enzymes utilizing Sepharose CL as a support and trichloro-s-triazine as the linking agent are described. The difficulties encountered during conventional aqueous and mixed aqueous-phase reactions of trichloro-s-triazine with insoluble polyols, particularly reagent hydrolysis, are avoided by performing the activation reactions in anhydrous organic phase and replacing the second chlorine on the triazine ring by an aromatic amine. Ligands can be coupled to the activated support in either aqueous or organic phase. The methods have been applied to the attachment of a number of different enzymes, proteins, and small-molecule ligands to Sepharose. The superiority of the triazine linkage to the cyanogen bromide linkage is demonstrated.     

A simple method for the preparation of polyacrylamide gels containing thioglycoside ligands.

A simple method was developed for the preparation of acrylamide derivatives containing thioglycosides. The synthetic scheme involves the preparation of an O-acetylated 1-thiosugar via a pseudothiourea derivative, the controlled addition of the thiosugar to N,N′-bismethyleneacrylamide to form a monoaddition product in which one of the acrylamide groups remains unchanged, and finally de-O-acetylation. Similar reaction schemes using bisacrylamido derivatives of 1,2-diaminoethane and 1,6-diaminohexane lead to analogous compounds with longer aglycon chain lengths. The thioglycoside derivatives of acrylamide thus prepared could be copolymerized with acrylamide to form polymers or gels containing thioglycosides as ligands. These gels were successfully used as affinity materials for the purification of peanut lectin and in cell-adhesion studies.     

A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described. The slab get consists of two porous layers of acrylamide of the following composition: 4% acrylamide, 0.04% bisacrylamide for the stacking gel, and 10% acrylamide, 0.1% bisacrylamide for the separating gel, both layers having different buffers. The separating gel mixture (final pH 9.0) and the buffers of the electrode chamber (pH 8.45) consist of Tris and glycine in such a ratio that no acid or base is necessary to adjust the pH. The resulting gel system has the following advantages: (a) it is able to resolve the components from large-volume samples (up to 200 microliter) after an overnight electrophoresis run while still maintaining the capacity to produce very sharp bands; (b) it has a high and broad resolution, allowing the separation on the same gel of proteins with apparent molecular masses between 10,000 and 450,000 Da; (c) it is very easy to prepare and shows excellent reproducibility in the electrophoretic patterns; (d) when used as a second dimension in tandem with isoelectric focusing, it improves the resolving power of two-dimensional gel electrophoresis; and finally, (e) its low crosslinker-to-acrylamide ratio allows the effective and rapid transfer of proteins to nitrocellulose membrane, thus improving the usefulness of protein blotting. In all cases, adrenal medullary chromaffin cell proteins were used as test samples.     

Protein-blotting on Polybrene-coated glass-fiber sheets. A basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/polyacrylamide gel

A procedure has been developed which allows the immobilization on glass-fiber sheets coated with the polyquaternary amine, Polybrene, of proteins and protein fragments previously separated on sodium-dodecylsulfate-containing polyacrylamide gels. The transfer is carried out essentially as has been used for protein blotting on nitrocellulose membranes [Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl Acad. Sci. USA 76, 4350-4354], but is now used to determine the amino acid composition and partial sequence of the immobilized proteins. Protein transfer could be carried out after staining the proteins in the gels with Coomassie blue, by which immobilized proteins are visible as blue spots, or without previous staining, after which transferred proteins are detected as fluorescent spots following reaction with fluorescamine. The latter procedure was found to be more efficient and yielded binding capacities of +/- 20 micrograms/cm2. Fluorescamine detection was of equal or higher sensitivity than the classical Coomassie staining of proteins in the gel. Immobilized proteins could be hydrolyzed when still present on the glass fiber and reliable amino acid compositions were obtained for various reference proteins immobilized in less than 100 pmol quantities. In addition, and more importantly, glass-fiber-bound proteins could be subjected to the Edman degradation procedure by simply cutting out the area of the sheet carrying the immobilized protein and mounting the disc in the reaction chamber of the gas-phase sequenator. Results of this immobilization-sequencing technique are shown for immobilized myoglobin (1 nmol) and two proteolytic fragments of actin (+/- 80 pmol each) previously separated on a sodium-dodecylsulfate-containing gel.     

Polyphenoloxidases immobilized in organic gels: Properties and applications in the detoxification of aromatic compounds

Gelatine gels originate from water in oil microemulsions in which the ternary system consists of isooctane/ sulfosuccinic acid bis [2-ethyl hexyl] ester/water; the solubilization of gelatin in the water pool of these microemulsions transforms them into viscous gels in which it is possible to cosolubilize various reactive molecules. These gels were used to immobilize two phenoloxidases, a laccase from Trametes versicolor and a tyrosinase from mushroom. The best balance between gel retention and catalytic activity was reached at a gelatine concentration of 2.5% (w/v) in the case of tyrosinase, while laccase immobilization was independent of gelatine concentration. Both enzymes kept the same optimum pH as the corresponding soluble controls, while a partial loss of activity was observed when they were immobilized. Immobilized enzymes showed an increased stability when incubated for several days at 4 degrees C with a very low release from the gels in the incubation solutions. The immobilization of tyrosinase and of laccase enhanced stability to thermal inactivation. Furthermore, gel-entrapped tyrosinase was almost completely preserved from proteolysis: more than 80% of the activity was maintained, while only 25% of the soluble control activity was detected after the same proteolytic treatments. A column packed with gel-immobilized tyrosinase was used to demonstrate that enzymes immobilized with this technique may be reused several times in the same reaction without loosing their efficiency. Finally, gel-entrapped tyrosinase and laccase were capable of removing naturally occurring and xeno-biotic aromatic compounds from aqueous suspensions with different degrees of efficiency. (c) 1995 John Wiley & Sons, Inc.     

Preparation and characterization of Rhizopus amyloglucosidase immobilized on poly(o-toluidine)

The starch hydrolyzing enzyme amyloglucosidase (AMG) from Rhizopus was immobilized onto the protonated salt (TS) and basic (TB) forms of chemically synthesized poly(o-toluidine) (POT) using adsorption and covalent binding. The polymers were activated with glutaraldehyde prior to covalent bonding. The immobilization efficiency was affected by the pH of the immobilization medium, contact time and amount of enzyme. After immobilization, the pH and temperature were changed to conditions under which the enzyme is most active. Immobilized AMG was more stable with respect to changes in pH and increases in temperature compared to free AMG. The immobilized enzyme retained high catalytic activity after multiple uses and showed enhanced stability with storage compared to free enzyme.     

Preparation and Some Properties of Chitosan Bound Enzymes

An immobilization method using chitosan prepared from chitin as an insoluble carrier was investigated. Glucose isomerase, urease, glucamylase, trypsin and glucose oxidase were attached to chitosan by the aid of water soluble carbodiimide. Their activity yields were as follows; glucose isomerase 32%, urease 44%, glucamylase 8%, trypsin 10%, glucose oxidase 37%.

Immobilized glucose isomerase showed no significant changes in optimal temperature and heat stability. But pH optimum of reaction and pH stability range were somewhat lowered. The inhibitory effects of bivalent metal ions were considerably reduced by immobilization and similar tendency was observed for buffer reagents such as Tris or veronal. Immobilized glucose isomerase was inhibited by 8 m urea or 6 m guanidine hydrochloride in nearly the same way as free enzyme. With SDS, cysteine or mercaptoethanol free glucose isomerase was scarcely affected by these reagents, while immobilized enzyme considerably suffered to a loss of its activity.     

Coimmobilization of lactate dehydrogenase and low molecular weight thiols on sepharose

Lactate dehydrogenase (EC 1.1.1.27) and dithiothreitol (DTT) were coimmobilized on Sepharose activated with cyanogen bromide. It was demonstrated that addition of 10 mM DTT (but not 2-mercaptoethanol) during immobilization increased the enzyme specific activity 1.5-5-fold, depending on the initial extent of Sepharose activation by cyanogen bromide. The total activity increased two- to threefold. The lactate dehydrogenase preparations were rich in matrix-immobilized sulfhydryl groups (1.8-13.0 nmol per ml gel). The presence of DTT increased the stability of immobilized lactate dehydrogenase.     

Production of acrylamide using immobilized cells of<Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> M33

The cellsof Rhodococcus rhodochrous M33, which produce a nitrile hydratase enzyme, were immobilized in acrylamide-based polymer gels. The optimum pH and temperature for the activity of nitrile hydratase in both the free and immobilized cells were 7.4 and 45°C, respectively, yet the optinum temperature for acrylamide production by the immobilized cells was 20°C. The nitrile hydratase of the immobilized cells was more stable with acrylamide than that of the free cells. Under optimal conditions, the final acrylamide concentration reached about 400 g/L with a conversion yield of almost 100% after 8 h of reaction when using 150 g/L of immobilized cells corresponding to a 1.91 g-dry cell weight/L. The enzyme activity of the immobilized cells rapidly decreased with repeated use. However, the quality of the acrylamide produced by the immobilized cells was much better than that produced by the free cells in terms of color, salt content, turbidity, and foam formation. The quality of the aqueous acrylamide solution obtained was found to be of commercial use without further purification.     

Laccase stabilization by covalent binding immobilization on activated polyvinyl alcohol carrier

AIMS: Attempts were made to immobilize laccase from Panus conchatus. METHODS AND RESULTS: The laccase was immobilized on carboxylated polyvinyl alcohol (PVA) activated by N-hydroxysuccinimide (N-HSI) in aqueous solution at different pHs, temperatures, and with different reaction times. An optimum condition for laccase immobilization is at pH 3.2, 40 degrees C and 12 h, respectively. Immobilization of laccase increased optimal pH for reaction with 2, 2'-azinobis (3-ethylbenzthiazoline-6-solfonate) (ABTS) and pH stability. Immobilized laccase proved to be reacted consecutively 17 times with only a 50% decrease on activity and be used in removal of 2,4,6-trichlorophenol (TCP). CONCLUSIONS: It was possible to immobilize the laccase on carboxylated polyvinyl alcohol by activation with N-hydroxysuccinimide in HAc-NaAc buffer. The immobilized laccase is both stable and reusable. SIGNIFICANCE IMPACT OF THE STUDY: The results indicate that this immobilized laccase can be used in the removal of poisonous effluent from pulp bleaching mills.     

A new immobilization technique of whole cells and enzymes with colloidal silica and alginate

A mixed gel composed of colloidal silica and alginate (As gel) was prepared for the immobilization of enzymes or microorganisms. The physical strength of AS gel increased with the amount of colloidal silica. The ethanol production rate of Saccharomyces cerevisiae (IFO 0224) immobilized in AS gel was higher than in alginate gel (Al gel) in the early phase of growth. At a concentration of glucose of more than 10%, the ethanol production of immobilized yeast in AS gel was higher than in Al gel. Any difference was not recognized in the diffusion coefficient of glucose between AS and Al gels. The AS gel had an ability to retain proteins such as bovine serum albumin and gamma-globulin. The alkaline protease and beta-galactosidase in AS gel continued their function for a long time, but those immobilized in Al gel did not. Immobilized beta-galactosidase in AS gel had a higher thermal stability than in Al gel or free enzymes.     

A mechanically strong matrix for protein electrophoresis with enhanced silver staining properties.

Duracryl is a mechanically strong and elastic acrylamide-based matrix, useful for a wide variety of electrophoretic applications. The matrix is stable as a refrigerated solution for one year. Upon addition of appropriate catalysts, Duracryl forms a polymer-reinforced polyacrylamide gel matrix suitable for electrophoresis. The polymer-reinforced gel is superior to conventional polyacrylamide gels in terms of mechanical strength, elasticity and protein silver staining properties. Protein detection sensitivity by silver staining, as well as the linear response of silver deposition versus protein load, is equivalent to standard acrylamide/N,N'-methylene bisacrylamide gels. Additionally, the silver staining properties of the Duracryl matrix result in proteins appearing as monochromatic shades of grey instead of red, brown and yellow, as is the case of conventional polyacrylamide matrices. Monochromatic shades of grey are more suitable for image analysis and densitometry. The matrix is compatible with standard electroblotting and protein N-terminal sequencing procedures. Low acrylic acid content and conductivity allow incorporation of the matrix into isoelectric focusing gels. The matrix was found not to alter polypeptide migration relative to the standard acrylamide/N,N'-methylene bisacrylamide matrix.     

Coimmobilization of Lactate Dehydrogenase and Low-Molecular-Weight Thiols on Sepharose

Lactate dehydrogenase (EC 1.1.1.27) and dithiothreitol (DTT) were coimmobilized on Sepharose activated with cyanogen bromide. It was demonstrated that the addition of 10 mM DTT (but not 2-mercaptoethanol) during immobilization increased the enzyme specific activity 1.5–5-fold depending on the initial extent of Sepharose activation by cyanogen bromide. The total activity increased two- to threefold. The lactate dehydrogenase preparations were rich in matrix-immobilized sulfhydryl groups (1.8–13.0 nmol per ml gel). The presence of DTT increased the stability of immobilized lactate dehydrogenase.     

The immobilization of enzymes on nylon structures and their use in automated analysis

1. Glucose oxidase (EC 1.1.3.4) and urease (EC 3.5.1.5) were covalently attached through glutaraldehyde to low-molecular-weight nylon powder. 2. Immobilized derivatives of glucose oxidase and urease were prepared by cross-linking the respective enzymes within the matrix of a nylon membrane. 3. An improved process is described for the immobilization of glucose oxidase and urease on the inside surface of partially hydrolysed nylon tube. 4. Automated analytical procedures are described for the determination of glucose with each of the three immobilized glucose oxidase derivatives and for the determination of urea with each of the three immobilized urease derivatives. 5. The efficiencies of the three immobilized enzyme structures as reagents for the automated determination of their substrates were compared.     

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.     

A micromethod for the quantitation of sialoglycoconjugate immobilization on insoluble supports: Its use in the preparation of supports with varying ligand concentration

A micromethod is described for the evaluation of immobilization of sialoglycoconjugates on insoluble supports. Ligands were radioactively labeled in their sialic acid moieties after mild periodate oxidation and borotritide reduction, or in the glycosylamino residue after borotritide reduction of the Schiff's base formed between reducing sialooligosaccharides and β-(p-aminophenyl)-ethylamine. Sephadex G-25, Sepharose 4B, and Cellulose MN 2100 were activated by CNBr or periodate oxidation. The hydrazido derivatives of these supports were prepared using both activation methods, and activated to azido-supports using nitrous acid. Controlled Pore Glass-glycophase activated by periodate oxidation was also studied. The investigation of conditions for the binding of the radioactive ligands was carried out in the microassay using 0.5-ml aliquots of the activated supports. The stability of the bound ligands in dependence on various parameters was investigated using the immobilized radioactive ligands. Multivalent linkages formed between ligand and support gave increased stability to release compared to monovalent attachment, for cyanogen bromide activation. The use of periodate activation yielded ligands with much greater stability even for monovalent linkages.The microassay was used successfully to predict conditions for the batchwise preparation of immobilized ligands.