Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports

The stabilization achieved by different immobilization protocols have been compared using three different enzymes (glutaryl acylase (GAC), D-aminoacid oxidase (DAAO), and glucose oxidase (GOX)): adsorption on aminated supports, treatment of this adsorbed enzymes with glutaraldehyde, and immobilization on glutaraldehyde pre-activated supports. In all cases, the treatment of adsorbed enzymes on amino-supports with glutaraldehyde yielded the higher stabilizations: in the case of GOX, a stabilization over 400-fold was achieved. After this treatment, the enzymes could no longer be desorbed from the supports using high ionic strength (suggesting the support-protein reaction). Modification of the enzymes immobilized on supports that did not offer the possibility of react with glutaraldehyde showed the same stability that the non modified preparations demonstrating that the mere chemical modification did not have effect on the enzyme stability. This simple strategy seems to permit very good results in terms of immobilization rate and stability, offering some advantages when compared to the immobilization on glutaraldehyde pre-activated supports.     

Method of purifying glutaraldehyde for microscopic research on biological objects

A simple and reliable method of purification and determination of glutaraldehyde concentration for histochemical fixation is proposed. Purification of glutaraldehyde is provided by vacuum distillation with a rotational-filmy evaporator, and its concentration is determined using refractometer.     

Effect of preservation with metaperiodate and glutaraldehyde on the immunogenic properties of vessels]

Experiments were conducted on guinea pigs and rabbits. It was shown that treatment of human femoral vessels with metaperiodate and glutaraldehyde solutions led to a sharp (10-fold) reduction of the amount of water-soluble proteins in the tissue homogenates, to a decrease of the sensitising and anaphylactic activity of the extracts, to inertia in the precipitation test with homologous antiserum to crude tissue.     

Immobilization and stabilization of cephalosporin C acylase on aminated support by crosslinking with glutaraldehyde and further modifying with aminated macromolecules

In this work, cephalosporin C acylase (CA), a heterodimeric enzyme of industrial potential in direct hydrolysis of cephalosporin C (CPC) to 7‐aminocephalosporanic acid (7‐ACA), was covalently immobilized on the aminated support LX1000‐HA (HA) with two different protocols. The stability of CA adsorbed onto the HA support followed by crosslinking with glutaraldehyde (HA–CA–glut) was better than that of the CA covalently immobilized on the glutaraldehyde preactivated HA support (HA–glut–CA). The thermostabilization factors (compared with the free enzyme) of these two immobilized enzymes were 11.2‐fold and 2.2‐fold, respectively. In order to improve the stability of HA–CA–glut, a novel strategy based on postimmobilization modifying with aminated molecules was developed to take advantage of the glutaraldehyde moieties left on the enzyme and support. The macromolecules, such as polyethyleneimine (PEI) and chitosan, had larger effects than small molecules on the thermal stability of the immobilized enzyme perhaps due to crosslinking of the enzymes and support with each other. The quaternary structure of the CA could be much stabilized by this novel approach including physical adsorption on aminated support, glutaraldehyde treatment, and macromolecule modification. The HA–CA–glut–PEI20000 (the HA–CA–glut postmodified with PEI M_w = 20,000) had a thermostabilization factor of 20‐fold, and its substrate affinity (K_m = 14.3 mM) was better than that of HA–CA–glut (K_m = 33.4 mM). The half‐life of the immobilized enzymes HA–CA–glut–PEI20000 under the CPC‐catalyzing conditions could reach 28 cycles, a higher value than that of HA–CA–glut (21 cycles). © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:387–395, 2015     

Ultrastructure of human E-rosettes: a re-examination

In contrast with previous published studies, it has been found that T-lymphocytes interact through broad-base contacts with sheep erythrocytes (SRBC) in E-rosettes. An electron dense material was spontaneously observed between the lymphocyte and erythrocyte plasma membranes. These findings fit more closely with the known metabolic effects of SRBC upon lymphocytes and the SRBC-capping phenomenon.     

Immobilization of thylakoids in porous particles and stabilization of the photochemical processes by glutaraldehyde action at subzero temperature

Summary Lettuce thylakoids were immobilized by the action of glutaraldehyde at sub-zero temperature in presence of albumin. Particles with sponge structure and good mechanical properties were obtained. The activity yield after immobilization was found equal to 70% for the oxygen production. Photosystems I and II were both active after immobilization. The yield for the ATP production was 27%. An increase of stability of the immobilized thylakoids was observed when stored under illumination.     

Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate

Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.     

Crosslinking of concanavalin A with glutaraldehyde

Crosslinking of Concanavalin A with low concentrations of glutaraldehyde gives a mixture of products. A specific product having about 66% of the biological activity of the native molecule was characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed the presence of monomers, dimers, trimers, tetramers, and a small amount of pentamers as products. The presence of alpha-methyl mannoside during crosslinking changed the nature of the products, yielding a product retaining 80% of the biological activity. The crosslinked products showed greater stability than the native molecule at alkaline pH. However, the greatest stability under alkaline conditions was shown by the native molecule itself where alpha-methyl mannoside was present.     

The production of specific immunoenzyme conjugates to human immunoglobulins by the glutaraldehyde method]

The results of studies aimed at obtaining class-specific conjugates to human immunoglobulins to be used in the enzyme immunoassay (EIA) are presented. At the first stage of the studies purified IgA, IgM and IgG preparations were obtained. These preparations were used for obtaining immunologically active immunosorbents on the basis of bromocyanic Sepharose. Specific antibodies to human IgA, IgM and IgG were isolated from animal sera by the method of affinity chromatography. These antibodies were conjugated with peroxidase by the glutaraldehyde method. The specific activity of the conjugates were determined in EIA. The results thus obtained revealed that all preparations exhibited high specific activity and gave no cross reactions with immunoglobulins of other classes.     

Reexamination of the polymerization of pyridoxylated hemoglobin with glutaraldehyde

Pyridoxylated adult human hemoglobin (HbAo) was prepared using a one molar equivalent of pyridoxal 5-phosphate (PLP) per heme and reduced with either NaCNBH3 or NaBH4. A separate sample was pyridoxylated and passed through a mixed-bed ion exchange column without reduction. All three preparations had a P50 of 29 +/- 2 torr and a cooperativity of n = 2.4 +/- 0.1. These preparations, in both the oxy and deoxy forms, were then treated with 7 equivalents of glutaraldehyde per tetramer at pH 6.8 at 4 degrees C and at room temperature. The polymerization invariably reduced the P50 to 18 +/- 2 torr with Hill coefficients of less than 2. These solutions, with or without further reduction using NaCNBH3, all retained the PLP in differing amounts (2-3 moles/tetramer). Methemoglobin concentrations were increased during the polymerization reaction. The normal pyridoxylation procedure, using sodium borohydride reduction, resulted in a number of different molecular species. Polymerization with glutaraldehyde caused a further proliferation of molecular species that could not be separated by anion exchange chromatography or by isoelectric focusing. The extent of polymerization, estimated by gel exclusion chromatography and SDS polyacrylamide gel electrophoresis, was from 40 to 50%. Analysis of the reverse phase chromatograms, which separate the heme and the alpha- and beta-chains, showed extensive polymerization and distribution of the radioactively labeled PLP on the protein for all preparations. All of the polymerized and pyridoxylated samples were unstable, and showed different chromatographic patterns after storage at 4 degrees C for 1 month. Attempts to stabilize these preparations by further reduction with NaCNBH3 gave products with a lower P50 and lower cooperativity. When the reactions were conducted with a purified HbAo, heterogeneity was somewhat decreased compared to the normally used stroma-free hemoglobin, but a large number of molecular species were still formed.     

About mechanism of chitosan cross-linking with glutaraldehyde

The regularities of the reaction of aminopolysaccharide chitosan with glutaraldehyde (GA) have been considered. The equilibrium forms of GA in water have been thoroughly studied by NMR spectroscopy. It has been established that at pH 5.6, the exchange of the protons of O=CHCH₂ groups for deuterium occurs, indicating the presence of an anion, a product of the first stage of the aldol reaction; at pH > 7.2, the formation of the products of an aldol reaction and aldol condensation takes place. The kinetics of the reaction between the amino groups of chitosan and GA, the kinetics of gel formation in chitosan solutions in the presence of GA, and the kinetics of changes in the rigidity of gels formed have been studied by UV spectroscopy. IR spectra of cross-linked chitosan have been obtained. It has been shown that chitosan catalyzes the polymerization of GA to form irregular products; in this process, the length of oligomeric chains in modified or cross-linked chitosan and the concentration of conjugated bonds increase with the GA concentration and pH of the reaction medium.