Structural organization of glutamate dehydrogenase hexamer. 1. Immobilization on sepharose as a model for analysis of structural-functional characteristics of the enzyme

Bovine liver glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3) and its radioactive phosphopyridoxyl derivative were covalently immobilized on Sepharose CL-4B with different degrees of cyanogen bromide activation. The catalytical and regulatory properties of the immobilized samples of the enzymes were studied. It was shown that the enzymes were immobilized through a single subunit of hexamer when sepharose was activated by small amounts of cyanogen bromide (less than 5 mg per 1 ml of gel). In this case, the immobilization did not alter the catalytical and regulatory properties of glutamate dehydrogenase. The immobilized radioactive phosphopyridoxyl derivative of glutamate dehydrogenase completely imitated the immobilized native enzyme and can be used as a convenient model for structural and functional investigation of catalytically active hexamer of glutamate dehydrogenase.     

Biospecific fractionation matrices for sequence specific endonucleases

Fractionation of several type II specific restriction endonucleases was achieved by separation on two novel biospecific matrices. The matrices are pyran, a copolymer of divinyl ether of maleic anhydride, and Cibacron Blue F3GA, a blue dye commonly used for the calibration of molecular sieves. Both compounds are insolubilized by coupling to sepharose through a cyanogen bromide linkage and in their soluble form inhibit the restriction endonucleases which we have tested. These affinity matrices can be used to obtain restriction endonucleases from crude extracts after removal of nucleic acids. They have also proven to have a high capacity when used as subsequent steps in enzyme purification. Their additional advantage is the rapid development time and reusability of columns packed with the two matrices.     

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.     

Cellulase immobilized on a soluble polymer

Summary Aspergillus niger cellulase was imobilized on cyanogen bromide activated dextran of varying molecular weights. The effect of different concentrations of cyanogen bromide used for the activation process was also studied. About 50% conjugation and 70% retention activity was achieved in the immobilized cellulase. The pH activity of immobilized enzyme was unchanged, but exhibited more stable activity at acidic pH than the free enzyme. Higher resistance to heat inactivation was also observed.     

Immobilized Human Plasmins: Preparation and Enzymatic Properties

Plasminogen was immobilized on agarose using either a commercially available substituted gel, or the cyanogen bromide (CNBr) activation procedure as a means of coupling the protein to agarose. Coupling the zymogen to the gel followed by its activation with urokinase yielded an immobilized plasmin. The immobilized enzymes have esterase, amidase and protease activity towards lysine and arginine esters, lysine anilide and casein, respectively. They activate plasminogen by a linear non-autocatalytic process. Both enzyme preparations are stable for extended periods of time in the absence of any stabilizing agents, and are not denatured by high salt concentrations or detergents.     

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.     

硫氧还蛋白抗体柱的制备

目的:探索硫氧还蛋白(Trx)抗体柱对Trx融合蛋白纯化的可行性。方法与结果:对含有Trx基因的质粒表达载体pTrxFus进行改造,在Trx读框之后加入6×His序列,并在大肠杆菌中表达C端带有6×His标签的Trx,经Ni2+柱亲和纯化后制备多克隆抗体;把经蛋白A纯化后的抗体偶联在溴化氰活化的琼脂糖凝胶上,制成Trx抗体柱;用此抗体柱纯化与Trx融合表达的豇豆胰蛋白酶抑制剂(CpTI),SDS-PAGE结果显示获得了纯度较高的Trx-CpTI。结论:用Trx抗体制成的免疫亲和层析柱可以有效纯化Trx融合蛋白。     

Enzymatically active subunits of Bacillus stearothermophilus enolase bound to Sepharose

The octameric enolase from Bacillus stearothermophilus was immobilized onto Sepharose 4B activated by the cyanogen bromide reaction under conditions for achieving essentially a single-point attachment. The immobilized enzyme was dissociated with guanidine hydrochloride to yield bound monomeric enolase. The Sepharose-bound subunit regained activity upon removal of the denaturant. It was also possible to rehydribize immobilized monomers to native octamers. Of note, the thermal stability of the immobilized enolase subunit does not appreciably differ from that of the parent soluble octameric enzyme. Thus, these results indicate that single subunits of thermophilic enolase are active and that oligomerization is not a prerequisite for the enzymic activity as well as for thermal stability.     

Continuous production of oligoglucuronans by immobilized glucuronan lyase

A glucuronan lyase was incubated with sepharose matrices pre-activated with N-hydroxysuccinimide (NHS), cyanogen bromide (CNBr) or epoxy. The CNBr- and NHS-activated gels showed satisfactory immobilization yields whereas no enzyme could be immobilized using the epoxy coupling group. Glucuronan lyase immobilized on CNBr-gel ensured rapid conversion of several glucuronans into oligomers with an enzymatic activity identical to that of the free enzyme. As classically observed when using free enzymes, the acetylation degree of glucuronan limited enzyme activity. Nevertheless, this immobilized system made it easier to obtain accurately oligomers with different polymerization degrees, notably in modifying the glucuronans residence times in column. Thus oligoglucuronan pools with polymerization degrees between 2 and 25 could be obtained with a productivity ranging from 120 mg h^?1 to 1.2 g h^?1 using 0.9 ml of chromatographic gel with immobilized glucuronan lyase. This methodology opens the way to continuous and large oligoglucuronan productions.     

Immobilization of invertase on sepharose-linked enzyme glycosyl recognizing polyclonal antibodies

Polyclonal antibodies directed against the yeast invertase glycosyls were raised by immunizing rabbits with neoglycoprotein-I and neoglycoprotein-II. The neoglycoproteins were prepared by separately coupling the N-linked large and small molecular weight yeast invertase oligosaccharides respectively to bovine serum albumin with the help of glutaraldehyde. Antibodies specifically recognizing the invertase oligosaccharides were purified from the sera of rabbits immunized with either neoglycoprotein using an affinity column of sepharose 4B-linked yeast invertase. Specific immunoaffinity supports for the immobilization of invertase were constructed by coupling the affinity-purified antineoglycoprotein-I or antineoglycoprotein-II antibodies to cyanogen bromide activated sepharose-4B. Both the affinity adsorbants were effective in binding and improving the thermal stability of invertase. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 605-609, 1997.     

Inactivation and reactivation of horseradish peroxidase immobilized by various procedures.

Horseradish peroxidase (HRP) immobilized by coupling the amino acid side chain amino groups or carbohydrate spikes to the matrix has been studied for its resistance to heat, urea-induced inactivation and ability to regain activity after denaturation in order to understand the influence of the nature of immobilization procedure on these processes. The various immobilized preparations were obtained and their properties studied: Sp-HRP was obtained by direct coupling of HRP to cyanogen bromide-activated Sepharose, Sp-NHHRP by coupling periodate oxidized and diamine-treated enzyme to the cyanogen bromide activated Sepharose, SpNH-COHRP by coupling periodate-treated enzyme to amino-Sepharose and SpCon A-HRP by binding of the enzyme on Con A-Sepharose. All the immobilized preparations exhibited higher stability against heat-induced inactivation as compared to the native HRP. Sp-NHHRP was most stable followed by Sp-HRP, SpNH-COHRP and SpCon A-HRP. Sp-NHHRP was also superior in its ability to regain enzyme activity after thermal denaturation, although Sp-HRP regained maximum activity after urea denaturation. Inclusion of Ca2+ was essential for the reactivation of all preparations subsequent to denaturation by urea.     

Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.     

The synthesis of 8-(6-aminohexyl)-amino-GTP and -GDP and their application as ligands in affinity chromatography

Two guanine nucleotide analogs, 8-(6-aminohexyl)-amino-guanosine-5′-triphosphate and 8-(6-aminohexyl)-amino-guanosine-5′-diphosphate, were synthesized from the monophosphate by phosphorylation with pyrophosphate or orthophosphate. Structural assignments were made according to nuclear magnetic resonance spectra. The ability of these nucleotides to act as enzyme substrates has been determined. Both guanosine nucleotides were used as ligands for affinity chromatography by attaching them to Sepharose 4B by the cyanogen bromide method. The effectiveness of these new affinity columns in enzyme purification was investigated with polypeptide elongation factor II from rat and pig liver.     

Enzymatic hydrolysis of racemic ibuprofen esters using Rhizomucor miehei lipase immobilized on different supports

This research describes the immobilization of Rhizomucor miehei lipase (RML) and chemically aminated RML (NH₂-RML) on different supports including octyl-sepharose (octyl-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH₂-RML), glyoxyl sepharose (Gx-RML and Gx-NH₂-RML) and glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH₂-RML). The highest immobilization yield was achieved for octyl-RML (>98%) followed by CNBr-RML (88%). Octyl-RML had the most specific activity (13.6) among all derivatives. The other preparations had moderate activities likely because of chemical reaction during covalent attachment of the enzyme. The catalytic behavior of lipase immobilized in hydrolysis reactions was investigated using methyl, ethyl, propyl, butyl and isobutyl-ibuprofen esters and the influence of the alkyl chain and the alcoholic residue of the ester were studied. Butyl ester was the most interesting ester for carrying out hydrolysis. The highest enantioselectivity of enzyme (E = 8.8) was obtained with isooctane/sodium phosphate buffer pH 7.0 at temperature of 40 °C. Increasing temperature from 40 to 50 °C caused decreasing in enantioselectivities and conversions. Also esterification of ibuprofen was carried out in solvent systems containing isooctane and two ionic liquids (ILs); [BMIM][PF₆] and [BMIM][BF₄]. Poor conversions and enantioselectivities were observed during esterification in all solvents.     

Stability of antibody attachment in immunosorbent chromatography

Detachment of immobilized antibody from its support matrix in an immunosorbent system prepared by the cyanogen bromide activation route was demonstrated. The immunosorbent system, however, was stable under slightly basic conditions. Detachment of antibody from the support material occurred mainly during the elution of the antigen complexed with the immobilized antibody. The antibody was detached from the matrix by different elution buffers. The detachment pattern of antibody was independent of the number of cycles used and also independent of the support materials. A change in the molecular structure of the detached antibody occurred as revealed by an alteration in the ultraviolet absorption spectra of the released antibody. The antibody detachment from the support matrix occurred in more than one antigen-antibody system suggesting that the leakage phenomenon may be a widespread disadvantage associated with the cyanogen bromide activation procedure. Detachment of the antibody could be reduced to < 10 ng ml⁻¹ by immobilizing antibody on the properly oxidized polysaccharide support material or on the N-hydroxysuccinimide activated ester gel. Antibody dissociation from the matrix did not occur when antibodies were immobilized by either amine or amide attachment, thus, immunosorbents prepared by such strategies are suitable for the immunochromatographic purification of proteins from complex mixtures.     

A new method for measuring sulfotransferase

Agarose can be activated by adding cyanogen bromide, dissolved in acetonitrile, to beads suspended in a solution of sodium carbonate. The necessity for manual titration and the use of a pH meter are thus eliminated. Activation for 1 min results in coupling capacities comparable to those reported for the titration method. The coupling capacity is influenced by the initial carbonate concentration and by the duration and temperature of the activation reaction. The coupling capacities of the activated gels are very reproducible.     

Regulation of particulate guanylate cyclase by Escherichia coli heat-stable enterotoxin: receptor binding and enzyme kinetics

1. Escherichia coli heat-stable enterotoxin (ST) induces a secretory diarrhea by binding to receptors on brush borders of intestinal villus cells, activating particulate guanylate cyclase and increasing intracellular concentrations of guanosine 3',5'-cyclic monophosphate (cyclic GMP). 2. However, little is known concerning coupling of receptor-ligand interaction to enzyme activation. 3. This study compares the kinetics of toxin-receptor binding and enzyme activation to better understand this transmembrane signal cascade. 4. Toxin receptor binding was linear and saturable with 50% of maximum displacement of [125I]ST by unlabeled toxin observed at 1.1 x 10(-7) M. ST increased the maximum velocity (Vmax) of guanylate cyclase with magnesium or manganese as the cation substrate without altering the affinity of the enzyme for its substrate or its positive cooperativity. 5. The concentration of toxin yielding half-maximum stimulation of guanylate cyclase was 1.2 x 10(-6) M, 10-fold higher than the affinity of the ligand for its receptor. 6. These data are consistent with the suggestion that ST-receptor interaction is coupled to activation of particulate guanylate cyclase. 7. However, the discrepancy between the affinity of ST for its receptor and its efficacy in activating the enzyme suggests that this coupling is complex. 8. Possible mechanisms underlying this coupling are discussed.     

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 effect of various coupling methods on the adsorption of serum proteins by immobilized concanavalin A

Concanavalin A was coupled to Sepharose 6B after activation by cyanogen bromide, divinyl sulfone, or glutaraldehyde and its adsorption behavior toward human serum proteins was investigated. The capacity and selectivity of the lectin were influenced markedly by the method used for its immobilization. When coupled to agarose via CNBr, the resulting absorbent showed the highest capacity and the lowest selectivity relative to the other two derivatives. When coupled to agarose via divinyl sulfone, the lectin exhibited high selectivity but its adsorption capacity was significantly reduced. Coupling to agarose via glutaraldehyde gave an absorbent that behaved, in some respects, differently from the other two. The variability in the adsorption behavior of the immobilized concanavalin A is attributed in part to variations in the degree of multipoint attachment of the lectin or its subunits to the agarose matrix. The selectivity increases also with increasing sample load, irrespective of the coupling method used, apparently due to protein-protein displacement.     

The use of BrCN for assembling modified DNA duplexes and DNA-RNA hybrids; comparison with water-soluble carbodiimide.

Both cyanogen bromide (BrCN) and 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide may be used as coupling reagents for the template-directed assembly of DNA duplexes containing the sugar-phosphate backbone modification. Both reagents show similar ligation site structure-specific trend. Practical recommendations are given for selection of the condensing reagent depending on the properties of the duplex. Based on 31P NMR spectroscopy data, a scheme is suggested for BrCN activation of the nucleotide phosphomonoester group. Using both condensing reagents, we studied the condensation of oligonucleotides containing ribo-segments (from mononucleotide residue to full sequence) on the DNA template. Efficiency of the chemical ligation of RNA oligomers was shown to be much lower than that of DNA analogues. The coupling yield depends on the position of the RNA segment in the hybrid duplexes and on the position of the phosphate group in the nick.