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At least 55 records · Page 3Linked to original sources
PubMed1976

Studies on the Treponema pallidum immobilizing activity in normal human serum. 3. The kinetics of immobilization reaction of normal and immune sera.

The influence of immobilizing antibody, complement and lysozyme, on the T. pallidum immobilization reactions of normal and immune sera was studied. Lysozyme shortened the lag periods and increased the reaction rates of the reactions of normal and immune sera. At high concentrations of added lysozyme, variations in the concentrations of immobilizing antibody and complement, within a wide range, did not further influence the kinetics of the two reactions. Preincubation with lysozyme did not influence the treponemes in the following immune serum immobiliation reaction provided the lysozyme was removed before the addition of antibody and complement. Normal serum was found to immobilize T. pallidum more rapidly than immune serum. This was seen also if the reaction mixtures were almost identical, the only differences being the immobilizing IgM antibody involved in the normal and the IgG antibody involved in the immune serum reaction.

Blood↗
PubMed1984

Specific immobilization of an enzyme by monoclonal antibody: immobilization of guinea pig liver transglutaminase.

A simple method for enzyme immobilization using a monoclonal antibody raised against the enzyme was studied, taking guinea pig liver transglutaminase as an example. This method consists of two steps; a conjugation of monoclonal antibodies, which bind with the enzyme without inhibitory effect on the activity, with agarose gel bead supports, and immobilization of enzyme proteins onto the conjugated monoclonal antibodies through the antigen-antibody immunoreaction. The following are the advantages of this method. (i) Activity recovery after the immobilization was very high, (ii) enzyme proteins in crude enzyme preparation could be immobilized specifically, and (iii) immobilized enzymes could be replaced easily by fresh enzyme proteins through a urea-buffer treatment.

Acyltransferases↗
PubMedDate not supplied

[Immobilized luciferase of Luciola mingrelica fireflies. The kinetic properties and thermostability of luciferase immobilized on cellulose films].

Luciferase of fireflies Luciola mingrelica was immobilized on cellulose films activated by cyanuric chloride or sodium periodate. Kinetic properties and the contribution of diffusional obstacles to the kinetics of the immobilized enzyme were examined. External and internal diffusion were found to influence the kinetic parameters. The stability of the enzyme was investigated at 25 degrees C and pH 7.8. Thermoactivation of the immobilized enzyme was shown to proceed in two stages: fast and slow. Dithiotreitol and cystein stabilized the enzyme at the fast stage while salt supplements at both stages. The fast thermoinactivation stage was apparently associated with the oxidation of luciferase SH-groups. It is demonstrated that the immobilized enzyme of Luciola mingrelica can be employed to measure ATP traces with the detection limit 0.1 mM. The enzyme immobilized on cellulose films can be used repeatedly.

Adenosine Triphosphate↗
PubMed1994

A general method for immobilization of glycoproteins on regenerable immobilized metal-ion carriers: application to glucose oxidase from Penicillium chrysogenum and horseradish peroxidase.

A general method for immobilization of glycoproteins on immobilized metal-ion carriers is described. The method includes oxidation of the carbohydrate moiety of the glycoprotein with potassium periodate, covalent modification with histidine and immobilization on bivalent metal ion-iminodiacetic acid-agarose. The method is exemplified with horseradish peroxidase and glucose oxidase from Penicillium chrysogenum. The modified and immobilized enzymes are stable for at least 2 months at 4 degrees C. The immobilized enzymes were used as enzyme electrodes for the determination of the glucose concentration.

Biosensing Techniques↗
PubMed2001

Expression microarray hybridization kinetics depend on length of the immobilized DNA but are independent of immobilization substrate.

Expression microarrays are often constructed by the immobilization of PCR products on two-dimensional modified glass slides or on three-dimensional microporous substrates. In this study we investigate whether the length of the immobilized species and the substrate choice influence hybridization dynamics. Using a simple bimolecular mass action controlled model to describe hybridization, we observed that the extent of hybridization and the initial velocities were directly dependent on the length of the immobilized species. An inflection point was noted at a length of 712 bases, above which the influence of length on hybridization rate decreased. Interestingly, we observed no differences in these parameters whether hybridization occurred on a two- or three-dimensional surface. Furthermore, the affinity of the solution phase labeled species for the immobilized species was identical for all arrayed lengths on both surfaces. These data indicate a similar interaction of the noncovalently immobilized species with either surface. Finally, we have determined that competitive hybridization on expression microarrays is nonlinear with respect to time and concentration of competitor. This observation is critical for analysis of expression array data.

Base Pairing↗
PubMed1981

Covalent immobilization of proteins to N-hydroxysuccinimide ester derivatives of agarose. Effect of protein charge on immobilization.

An uncharged N-hydroxysuccinimide ester derivative of agarose, Affi-Gel 10, exhibited excellent capacity for immobilization, at pH 7.5, of proteins having isoelectric points of 6.5--11.0. Under identical conditions, acidic proteins with isoelectric points of 3.3--5.9 did not couple well to this activated gel. Immobilization of acidic proteins increased in the presence of 80 mM CaCl2, or at a pH equal to or less than the isoelectric point. Affi-Gel 15, a new N-hydroxysuccinimide ester derivative of agarose containing a tertiary amine in the spacer arm, coupled acidic proteins efficiently at pH 7.5 but basic proteins coupled poorly. The immobilization of basic proteins to Affi-Gel 15 was increased to useful levels by increasing the ionic strength, or the pH, of the reaction medium. The lectin concanavalin A was efficiently immobilized using either activated gel, and the concanavalin A-agarose derivatives bound 3.9--4.1 mg ovalbumin/ml gel. These studies demonstrate that the charge of the protein relative to the charge of the gel is an important factor affecting the level of protein immobilization to active ester gels.

Animals↗
PubMed1992

Cell growth on immobilized cell-growth factor. II. Adhesion and growth of fibroblast cells on poly(methyl methacrylate) membrane immobilized with proteins of various kinds.

The surface of poly(methyl methacrylate) membrane was partially hydrolysed and the carboxyl groups produced were coupled with various protein molecules with water-soluble carbodiimide. The immobilized proteins were a cell-growth factor insulin, cell adhesion factors fibrinogen and fibronectin, and serum proteins albumin and gamma-globulin. The insulin-immobilized poly(methyl methacrylate) membrane strongly accelerated the growth and slightly accelerated the adhesion of fibroblast cells. The immobilized fibronectin and fibrinogen enhanced the cell adhesion, and the former also accelerated the cell growth. The immobilized albumin and gamma-globulin influenced the adhesion and growth of cells very little. It was found that various proteins specifically influence the adhesion and growth of cells in an immobilized state.

Animals↗
PubMed1994

Immobilization of human thrombomodulin on biomaterials: evaluation of the activity of immobilized human thrombomodulin.

Thrombomodulin (TM) is a newly described endothelial cell associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. In this study, the immobilization of hTM was investigated in detail using surface modified polymers. As the basis of immobilization, poly(acrylic acid) (PAAc) surface-grafted poly(ethylene) (PAAc-g-PE) film was used with the expectation of increasing the immobilization amount of hTM. The effect of the immobilization reaction on the hTM activities, and the comparison of the activities of the immobilized hTM with the free hTM, were studied.

Acrylic Resins↗
PubMed1982

Comparative biochemical studies of the callus matrix in immobilized and non-immobilized fractures.

Fracture healing in both non-immobilized and immobilized rat fracture callus, though different radiographically and histologically; is characterized by similar biochemical changes in the ground substance. Although the callus formed in the immobilized model is appreciably smaller than that formed when the fracture is not immobilized, the chemical composition of the organic matrix of the existing callus undergoes similar modifications regardless of fixation and histological response. Previous studies of endochondral fracture repair have demonstrated an elevation in fracture callus hexosamine content during the early stages of healing of the non-immobilized fracture. This corresponded histologically to a predominance of cartilaginous tissue in the callus matrix. In the present study, the biochemistry, histology, and roentgenographic features of stabilized and non-stabilized rat fractures are compared. Similar elevations in hexosamine are noted at two weeks in both models. These elevations are attributed to changes in proteoglycan rather than glycoprotein concentration of the tissue.

Animals↗
PubMed2001

Modulation of lipase properties in macro-aqueous systems by controlled enzyme immobilization: enantioselective hydrolysis of a chiral ester by immobilized Pseudomonas lipase.

Lipase from Pseudomonas fluorescens (PFL) has been immobilized by using different immobilization protocols. The catalytic behavior of the different PFL derivatives in the hydrolytic resolution of fully soluble (R,S) 2-hydroxy 4-phenyl butanoic acid ethyl ester (HPBE) in aqueous medium was analyzed. The soluble enzyme showed a significant but low enantioselectivity, hydrolyzing the S isomer more rapidly than the R-isomer (E = 7). The enzyme, immobilized via a limited attachment to a long and flexible spacer arm, showed almost identical activity and specificity to the soluble enzyme. However, other derivatives, e.g. PFL adsorbed on supports covered by hydrophobic moieties (octyl, decaoctyl), exhibited significant hyperactivation on immobilization (approximately 7-fold). Simultaneously, the enantioselectivity of the PFL-immobilized enzyme was significantly improved (from E = 7 to E = 80). By using such derivatives, almost pure R ester isomer (e.e. > 99%) has been obtained after 55% hydrolysis of the racemic mixture of a solution of 10% (w/v) (R,S) HPBE. The derivatives could be used for 10 cycles without any significant decrease in the activity of the biocatalyst.

Journal Article↗
PubMed2000

Use of immobilized PCR primers to generate covalently immobilized DNAs for in vitro transcription/translation reactions.

We have developed a novel biochemical method to simultaneously amplify and immobilize a target gene onto insoluble particles using PCR. This method employs the covalent attachment of one of two PCR primers to a particle surface either directly during DNA synthesis of the primer or post-DNA synthesis, through the use of chemical crosslinkers. Immobilization of the target gene can be achieved directly during PCR amplification, with one bead-bound primer and one soluble primer. Alternatively, this can be achieved post-PCR, through covalent attachment of a chemically modified primer incorporated into the amplicon to an activated particle. All of the immobilized DNA templates containing appropriate regulatory regions were fully competent for transcription and translation reactions and several could be re-used in serial reactions. The most successful strategy utilized amino-silanized controlled pore glass beads, which were coupled to phosphorylated primers using carbodiimide chemistry. These bead-bound primers were used during PCR to generate attached DNA templates that could be collected and re-used for at least seven sequential transcription reactions without significant loss in efficiency. This method has also been successfully applied to the amplification, transcription and translation of multiple DNA templates using a single, immobilized primer. The combined PCR-based amplification/immobilization method was shown to be more durable than post-PCR chemical immobilization and affords the convenience of performing sequential PCR amplification, transcription and translation reactions in a single tube.

Animals↗
PubMed1988

[Optimization of methods of immobilization of anti-immunoglobulins. Equilibrium parameters of the interaction of immobilized antibodies with an antigen].

Methods for immobilization of anti-immunoglobulins on insoluble supports were optimized, and the interaction of immunoadsorbents obtained with [125I]-labeled rabbit IgG was investigated. It was shown that this interaction can be adequately described by a rather simple equilibrium model which reflects the interaction of a monovalent antigen with two independent types of binding sites. Within the framework of this model the association constants as well as the concentrations of high affinity binding sites which influence the capacity and efficiency of the separation system were determined. Optimization of the immobilization methods implicated a study on the role of certain functional groups of the antibody involved in the formation of covalent bonds, on the effect of the spacer arm length on the properties of immobilized antibody as well as on the role of the degree of immobilization. It was found that immunoadsorbents obtained after antibody immobilization via lysine or tyrosine residues on matrices with a specific spacer group are the optimal ones.

Animals↗
PubMed2002

On-line synthesis utilizing immobilized enzyme reactors based upon immobilized dopamine beta-hydroxylase.

Immobilized enzyme reactors (IMERs) based upon dopamine beta-hydroxylase (DBH) have been developed. Immobilized artificial membrane (IAM) and glutaraldehyde-P (Glut-P) stationary phases have been used to immobilize DBH. When DBH is immobilized on the Glut-P interphase the enzyme is outside the stationary phase whereas with the IAM interphase the enzyme is embedded within the interphase surroundings. The activity of each IMER and their ability for on-line hydroxylation has been investigated. The resulting IMERs are enzymatically active and reproducible. The IMERs can be utilized through the use of coupled chromatography to characterize the cytosolic (DBH-Glut-P-IMER) and membrane-bound (DBH-IAM-IMER) forms of the enzyme. The substrate is injected onto the individual IMERs and the reactants and products are eluted onto a phenylboronic acid column for on-line extraction. The substrates and products are then transported via a switching valve to coupled analytical columns. The results demonstrate that enzyme-substrate and enzyme-inhibitor interactions can be investigated with the on-line system. These IMERs can be utilized for the discovery and characterization of new drug candidates specific for the soluble form and membrane-bound form of DBH. The effects of flow-rate, contact time, pH and temperature have also been investigated.

Animals↗
PubMedDate not supplied

Highly efficient "tight fit" immobilization of alpha-chymotrypsin in mesoporous MCM-41: a novel approach using precursor immobilization and activation.

The zymogen alpha-chymotrypsinogen A is bound to mesoporous silica MCM-41 with a protein loading of 170 mg/g solid (MCM-Z) by a simple stirring in aqueous tris-HCl buffer (pH 7.2). The bound zymogen is then activated with trypsin to obtain alpha-chymotrypsin immobilized on MCM-41 (MCM-E.I) that displays an effective enzyme activity corresponding to 65 mg protein/g of solid support (3250 BTEE units/g). A direct immobilization of commercially available alpha-chymotrypsin (MCM-E.II) gives lower loading (1250 BTEE units/g). Protein content of the solid support after immobilization is confirmed by thermogravimetric analysis (TGA). The enzyme is tightly bound to the support and can be used over 100 recycles over 1 week in aqueous as well as reverse micellar media. The immobilized enzyme (MCM-E.I) has been used for resolution of N-acetyl-dl-amino acid esters and racemic trans-4-methoxy-3-phenylglycidic acid (PGA) methyl ester.

Chymotrypsin↗
PubMed2006

Electrochemistry of immobilized redox enzymes: kinetic characteristics of NADH oxidation catalysis at diaphorase monolayers affinity immobilized on electrodes.

In the class of NADH:acceptor oxidoreductases, the diaphorase from Bacillus stearothermophilusis a particularly promising enzyme for sensing NADH, and indirectly a great number of analytes, when coupled with a NAD-dependent dehydrogenase as well as for the design of mono- and multienzyme affinity sensors. The design and rational optimization of such systems require devising immobilization procedures that prevent dramatic losses of the enzymatic activity and a full kinetic characterization of the immobilized enzyme system. Two immobilization procedures are described, which involve recognition of the biotinylated diaphorase by a monolayer of neutravidin adsorbed on the electrode surface either directly or through the intermediacy of a monolayer of biotinylated rabbit immunoglobulin. Thorough kinetic characterization of the two systems is derived from cyclic voltammetric responses. A precise estimate of the enzyme coverages is obtained after comparing the enzyme kinetics of the immobilized and the homogeneous system.

Biosensing Techniques↗
PubMed1976

Studies on the Treponema pallidum immobilizing activity in normal human serum. 2. Serum factors participating in the normal immobilization reaction.

The T. pallidum immobilization reaction to be achieved by unheated normal serum was found to be complement dependent and the results presented suggested that complement was activated via the classical pathway. Besides complement, an immobilizing antibody of the IgM class was necessary for the immobilization reaction to occur. Lysozyme exerted an enhancing effect on the normal serum immobilization reaction.

Blood↗
PubMed1983

[Streptokinase immobilization in insoluble carriers, and properties of the immobilized protein].

Immobilization of streptokinase was performed by bromine cyan-activated cellulose and by aminoethyl cellulose using glutaric aldehyde and N-cyclohexyl-N'-[2-(4-morpholinyl)-ethyl-carbodiimide. The specific activator activity of the immobilized streptokinase is 70-100% of that of free streptokinase. In multiple application of the immobilized protein preparations streptokinase obtained by bromine cyan-activated cellulose is the most stable: it retains more than 40% of initial activity after 10 repeated applications. The immobilized streptokinase is shown to be more thermostable as compared to the soluble one.

Cellulose↗
PubMed1993

Cell growth on immobilized cell growth factor: 5. Interaction of immobilized transferrin with fibroblast cells.

Transferrin was immobilized on poly(methyl methacrylate) membrane and the interaction of the immobilized transferrin with mouse fibroblast cells STO was investigated. The immobilized transferrin was stable without decomposition over 48 h and showed higher activities of cell growth acceleration than free transferrin and transported ferric ion as effectively as free transferrin. The specific interaction of the immobilized transferrin with fibroblast cells was confirmed by experiment using the antibody. It was considered that cell growth acceleration and ferric ion transportation occur by different mechanisms.

Animals↗