Selective removal of chymotrypsin using diphenyl alpha-aminoalkylphosphonate immobilized on sepharose gel

A diphenyl alpha-aminoalkylphosphonate derivative, which is an irreversible inhibitor of chymotrypsin-like serine proteases, was immobilized on cyanogen bromide-activated Sepharose, and the selective binding of chymotrypsin to the obtained inhibitor-gel was evaluated using batch and column methods. Complete removal of chymotrypsin in an aqueous solution was done using the column method, while partial removal was done using the batch method.     

Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B.

A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier. This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells. This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin.     

Fractionation of Escherichia coli isoaccepting tRNA species by sepharose 4B column chromatography.

We have determined the elution profile on Sepharose 4B chromatographic column ofthe tRNA isoaccepting species of all 20 amino acids from Escherichia coli MRE 600. Further chromatography on a reversed phase column (RPC-5) is sufficient, in some cases, for a complete purification.     

The rapid preparation of peroxisomes from rat liver by using a vertical rotor.

A method is described for the rapid preparation of peroxisomes from rat liver by using sucrose-density-gradient centrifugation in a vertical rotor. The preparation, shown to be virtually free of mitochondrial and microsomal contamination, can be used to study fatty acid metabolism by isolated peroxisomes.     

Fractionation of Neisseria allergenic preparations by a gel chromatographic method on sepharose 4B

The fraction composition of allergens obtained by different methods from the microbial mass of N. meningitidis, N. gonorrhoeae and N. perflava has been studied. Each method produced its characteristic number of fractions, irrespective of the Neisseria species used. Their molecular weights: more than 2,000,000 daltons, 160,000 daltons, 31,000 daltons and 14,000 daltons. All these fractions are biologically active.     

A novel procedure for the preparation and characterization of catalytically active fatty acid synthetase immobilized on sepharose beads

A novel procedure for immobilization of enzymatically active fatty acid synthetase is presented. The enzyme is coupled to a Sepharose 4B matrix containing covalently attached antibodies which recognize, and bind specifically to, the thioesterase domain of this polyfunctional enzyme. A continuous flow system is described for assay of the immobilized enzyme. Fatty acid synthetase activity apparently is not limited by movement of substrates through the Nernst diffusion layer surrounding the matrix particles, since normal Michaelis-Menten kinetics are observed and reaction rates are independent of flow rate. The Km values for acetyl-CoA and malonyl-CoA, the pH/activity profile, and the reaction products are essentially the same as for the freely soluble enzyme, although the specific activity is lower by about 55%. The preparation and characterization of immobilized subunits of the enzyme could provide a valuable approach for studying the role of structural and functional subunit interactions in the enzyme. In addition, the immobilized enzyme offers a model for studying the properties of this enzyme in a highly structured environment such as might exist in vivo, permitting study of both physical and functional interactions of fatty acid synthetase with other lipogenic enzymes.     

Properties of human hemoglobin immobilized on Sepharose 4B.

This paper reports the properties of human hemoglobin covalently bound to Sepharose 4B both in 'high-affinity' and 'low-affinity' conformations. The results suggest that the coupling reaction is strongly affected by the conformational changes linked to oxygenation of the protein. The rate and the extent of the reaction are different for the oxy and deoxyderivatives, probably due to the change in reactivity of the amino groups in the liganded and unliganded tetramer. The data on the equilibrium which is established between matrix-bound and soluble subunits, measured by the 'subunit-exchange chromatography', indicate that the system displays a minimal heterogeneity when hemoglobin is coupled to the gel in the deoxy state at intermediate protein concentration and pH 8. Maxtrix-bound hemoglobin is characterized by a higher oxygen affinity and by decreased homotropic and heterotropic interactions with respect to hemoglobin in solution, but the changes depend strongly on the conditions used in the coupling procedure.     

Formation of α-keto acids from amino acids using immobilized bacteria and algae

Summary Both free and immobilized cells of the algae Chlorella vulgaris and Anacystis nidulans contain aminoacid oxidase (AAO) activity which is increased by illumination with red light. Both immobilized species are photosynthetically active. By co-immobilizing Chlorella with bacterial cells (Providencia sp. PCM 1298) containing high AAO activity an increased production of keto acid (up to tenfold) is observed due to improved oxygen supply.     

Purification of multiple pea ferredoxins by chromatography at high ionic strength on unsubstituted sepharose 4B

At high concentrations of ammonium sulfate (3–4 M) pea ferredoxin (which is soluble under these conditions) can be adsorbed to Sepharose 4B, either by column chromatography or by batchwise treatment. A reverse (3 M to 1 M) ammonium sulfate gradient results in the elution of three peaks of ferredoxin. The spectral ratio A₄₂₀/A₂₈₀ of 0.47–0.54 indicates that each peak of ferredoxin is highly purified by this single step. A further gel filtration removes residual high molecular weight contaminants from the ferredoxin. The spectrum of the purified pea ferredoxin is typical of other plant ferredoxins in having absorbance peaks at 276 nm, 330 nm, 422 nm and 465 nm. Other chromatographic matrices are capable of adsorbing ferredoxin. Sepharose and Sephacryl were the best adsorbents while Sephadex and cellulose adsorbed ferredoxin less tenaciously. The polyacrylamide-based resins Biogel P-4 and P-200 did not adsorb ferredoxin at high ionic strength.     

Large-scale preparation of phosphoglycerate kinase from Saccharomyces cerevisiae using Cibacron Blue-Sepharose 4B pseudoaffinity chromatography.

A reproducible procedure for the large-scale preparation of phosphoglycerate kinase frombaker's yeast is described. This method includes autolysis of dried yeast in 0.75 m ammonia, heat treatment, ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose, Cibacron blue 3 G-A-Sepharose 4B pseudoaffinity chromatography, and Sephadex G-100 gel filtration. Approximately 1.7 g of homogeneous phosphoglycerate kinase can be obtained from 1 kg of air-dried bakers' yeast (yield 52%, specific activity 890 units/mg at 25°C). In a few cases further purification was achieved by reversible salting out on Sepharose CL-4B, hydroxylapatite chromatography, or ATP-Sepharose 4B affinity chromatography. Differences in the preparation of phosphoglycerate kinase from yeast with those from pig liver and pig muscle are discussed, especially concerning the interaction of the three enzymes with the chromophores of Cibacron blue- and dextran blue-Sepharose.     

Affinity chromatography of galactose containing biopolymers using covalently coupled Ricinus communis lectin to sepharose 4B

A galactose-specific lectin isolated from Ricinus communis beans has been covalently coupled to Sepharose 4B activated with cyanogen bromide. The immonolized lectin retains its polysaccharide-binding property. The Sepharoselectin can be used for the purification of polysaccharides containing terminal nonreducing galactose.Only a small fraction of “native fetuin’ and ‘native ceruloplasmin’ are retarded on Sepharose-lectin. On analysis it was observed that hey had a lower content of sialic acid as compared to the native and unbound glycoproteins (sialated fractions). However, on desialation, fetuin and ceruloplasmin were completely adsorbed to Sepharose-lectin. The asialoglycoproteins interact strongly with Sepharose-lectin as compared to ‘partially sialated glycoproteins’. This has been attributed to the exposure of galactose residues of these glycoproteins on enzymatic desialation. These experiments demonstrated that Sepharose-lectin interacts with glycoproteins through their terminal, nonreducing galactose. On the basis of these experiments it is suggested that Sepharose-lectin can be used as an analytical tool for separation of ‘fully sialated glycoproteins’ from the ‘partially sialated glycoproteins’.     

固定化酵母连续发酵生产酒精工业应用的研究

本实验以海藻酸钠为基本载体,选择性地加入适量化学物质,制备新型复合载体。该载体能较好地避免单纯海藻酸钠载体易降解、易软化［１］以及颗粒上浮的问题。经反复使用不发粘、弹性好,无毒并能保持较高的发酵水平。     

Purification of human milk bile salt-activated lipase by cholic acid-coupled sepharose 4B affinity chromatography

Sequential chromatography of human milk whey on concanavalin A—Sepharose 4B followed by cholate—Sepharose 4B yielded a bile salt-activated lipase with 150-fold purification. The lipase was not retained by concanavalin A—Sepharose 4B but was retained by the cholate—Sepharose 4B, from which it was eluted with 2% sodium cholate. The affinity chromatography procedure on cholate—Sepharose 4B was based on the specific structural requirement of the enzyme for a 7-hydroxyl group of bile salt. Sodium deoxycholate, which lacks the 7-hydroxyl group, was effective in removing the nonspecifically bound proteins without affecting the binding of the enzyme. Bile salt-activated lipase showed a single band on urea-sodium dodecyl sulfate—polyacrylamide gel electrophoresis with an apparent molecular weight of 125,000, and based on densitometric measurement accounted for 0.5–1.0% of the protein mass of human whole milk. A rabbit antiserum to the purified bile salt-activated lipase caused no inhibition of human milk lipoprotein lipase activity but completely inhibited bile salt-activated lipase activity.     

Affinity chromatography of yeast alpha-glucosidase using ligand-mediated chromatography on immobilized phenylboronic acids.

The synthesis of 3-nitro-4-(6-aminohexylamido)phenylboronic acid is described. The properties of two novel forms of immobilized phenylboronate agarose adsorbents [m-aminophenylboronic acid-Matrex Gel and 3-nitro-4-(6-aminohexylamido)phenylboronic acid-Sepharose CL-6B] were investigated. Both gels bind and selectively retard the glycoprotein alpha-glucosidase from yeast. The retardation is affected by following parameters: (i) pH, (ii) presence of sugar, (iii) concentration of sugar and (iv) buffer species (especially triethanolamine). Five sugars were studied, namely sorbitol, fructose, ribose, glucose and maltose. The concentration of sugar required to produce significant retardation increased in the above order, whereas the ability of sugar to form a complex with boron decreases in the same order. These effects were observed with crude as well as pure enzyme. Since alpha-glucosidase is a glycoprotein, it is proposed that this protein is mainly bound to these immobilized phenylboronates via sugar (glyco) residues. Displacement of the enzyme from the column is effected by the sugar in the buffer (or in a preincubation mixture). However, the marked pH-dependence (this retardation effect could only be observed at pH 7.4) suggests that these results are not due solely to hydrophobic or ionic mechanisms and are more complex than simple sugar-phenylboronic acid interactions.