Antigenic properties of nitro aromatic derivatives of polyethylene glycol

The immunogenic properties of the dihapten and monohapten derivatives of polyethylene glycol with different nitroaromatic groupings were studied. 2,4-dinitrophenyl, 2, 4, 6-trinitrophenyl and trinitrophenyl-ethyl groupings were used as hapten groups. The injection of monohapten compounds was found to induce the accumulation of antibody-forming cells secreting antibodies to trinitrophenyl in the spleen of normal and athymic nude mice. As early as on day 3 the number of antibody-forming cells considerably exceeded their background level, the process of B-cell activation being, to a certain extent, thymus-independent. Dihapten compounds were not immunologically active. The effect rendered by the nitroaromatic derivatives of polyethylene glycol, revealed in this study, is linked with the known capacity of polyethylene glycol to adsorb on the surface of cell membranes.     

Zeolites as new chromatographic carriers for proteins--easy recovery of proteins adsorbed on zeolites by polyethylene glycol

Zeolites are able to adsorb proteins on their surface and might be suitable as a new type of chromatographic carrier material for proteins and for their conjugates (Matsui et al., Chem. Eur. J. 7 (2001) 1555-1560). Interestingly, maximum adsorption was observed at the isoelectric point (pI) of each protein. The current study was performed to investigate the desorption of proteins from the zeolites at pI. Proteins adsorbed to zeolites could be desorbed at pI by polyethylene glycol (PEG), but not by conventional eluents. The eluted proteins still retained their activities. The zeolite Na-BEA was an especially good composite for desorption by PEG. Using this method for the adsorption and desorption of proteins at pI, we succeeded in separating various proteins. The application of zeolites to biochemistry and biotechnology is also discussed.     

Isolation and purification of bacterial ribosomes from endotoxin using polyethylene glycol

The difficulties arising in the study of the immunogenicity of bacterial ribosomes and in their possible use as vaccines are due to the fact that preparative ultracentrifugation, constituting a necessary stage in most of the methods used for the isolation of ribosomes, has a low productive capacity. To develop a more effective method for obtaining Shigella ribosomal vaccines, an attempt to use the method of precipitation with 10% polyethylene glycol (PEG), proposed by Expert-Bezan?on et al., has been made. The serological determination of O antigen has shown that nearly contained in the supernatant fluid S-30 can be detected in precipitated ribosomes. Taking into account the wide spectrum of the biological activity of bacterial endotoxin, it must be removed from the vaccine. The study has revealed that precipitation by means of ethanol (15-35%), low pH (4,2-4,7) and PEG (4-8%) can be used for this purpose. In accordance with the chosen method, the clarified material obtained by precipitation with 10% PEG is fractionated by means of 5% PEG which causes the complete precipitation of ribosomes, thus leaving endotoxin in the solution. Centrifugation in the density gradient of saccharose and electron microscopy have demonstrated that ribosomes isolated by this method possess typical sedimentation properties and structure. The yield of ribosomes is 3 times greater than that obtained by ultracentrifugation. Fractionation with PEG may be used as the method of the mass production of ribosomal vaccines.     

New PEG2 type polyethylene glycol derivatives for protein modification

Although proteins with 2,4-bis (o-methoxypolyethylene glycol)-6-chloro-s-triazine (PEG2-Cl) as a divalent PEG modification have some advantages compared to proteins with the linear PEG modification, PEG2Cl cannot react with amino groups at neutral pH. Therefore, we have prepared new PEG2 derivatives that have an activated ester as the functional group. We confirmed that these derivatives are useful for the divalent modification of proteins, such as bSOD and rhG-CSF. © Rapid Science Ltd. 1998     

Enhanced activity of Candida rugosa lipase modified by polyethylene glycol derivatives

The derivatives of polyethylene glycol (PEG) were prepared by reacting PEG with propylene oxide to enhance its hydrophobicity and introduce a branched structure. The PEG derivatives were activated with cyanuric chloride and used to modify the lipase fromCandida rugosa. The maximum specific activity of lipase modified with the PEG derivatives was about 2-fold of that modified with PEG for the esterification of oleic acid and lauryl alcohol in hexane.     

Synthesis of polyethylene glycol (PEG) derivatives and PEGylated-peptide biopolymer conjugates

We synthesized a library of 50 poly(ethylene glycol) (PEG) derivatives to expand the extent of conjugation with biologically active molecules (biopolymers, peptides, drugs, etc.) and biomaterial substrates. The formation of PEG derivatives was confirmed with HPLC, (1)H and (13)C NMR. PEG derivatives were polymerized into networks in order to study the role of PEG and terminal functional groups in modulating the hydrophilicity of biomaterials and cell-biomaterial interaction. The resulting surface hydrophilicity and the number of adhered fibroblasts were primarily dependent on the PEG concentration with the molecular weight and the terminal functional group of PEG derivatives being less important. One of PEG derivatives, PEG-bis-glutarate, was utilized to link peptide sequences to gelatin backbone in the formation of novel biomedical hydrogels. PEG-peptide conjugates were characterized by mass spectroscopy. PEG-peptide modified gelatins were characterized by gel permeation chromatography.     

Arginine-specific modification of proteins with polyethylene glycol

In this study, the residue-selective modification of proteins with polymers at arginine residues is reported. The difficulty in modifying arginine residues lies in the fact that they are less reactive than lysine residues. Consequently, typical chemo-selective reactions which employ "kinetic" selectivity (active esters, Michael addition, etc.) cannot be used to target these residues. The chemistry exploited herein relies on "thermodynamic" selectivity to achieve selective modification of arginine residues. ω-Methoxy poly(ethylene glycol) bearing an α-oxo-aldehyde group was synthesized and used to demonstrate the selective modification of lysozyme at arginine residues. In addition, the optimization of reaction conditions for coupling as well as the stability of the formed adduct toward dilution, toward a nucleophilic buffer, and toward acidification are reported. It was concluded that this approach is a convenient, mild, selective, and catalyst-free method for protein modification.     

Time and cell systems as variables in fusion experiments with polyethylene glycol

Summary Cell hybridization was done between a monolayer of B14-150 Chinese hamster cells and a suspension of either mouse leukemia cells or normal human lymphocytes. Cell contact was obtained by centrifugation of the suspension cells onto the monolayer cells in a culture plate. Cell fusion was done by means of polyethylene glycol (PEG). The optimum time for PEG exposure as well as the yield of hybrid cells differed markedly with the different combinations.     

The use of polyethylene glycol in concentration and purification of several bovine viruses.

The polyethylene glycol (PEG) precipitation method was used for the concentration and purification of eight bovine viruses. Good results were obtained from four viruses, parainfluenza--3 virus, bovine enterovirus, bovine adenovirus, and bovine parvovirus. No satisfactory results of concentration were obtained from bovine reovirus, bovine viral diarrhea virus, and infectious bovine rhinotracheitis virus. The failure of concentration of the four viruses seems to be ascribed rather to the resuspending of virus from the virus--PEG precipitate than to the precipitation of virus from infective culture fluid. This method can be applied as the initial step to the concentration of parainfluenza--3 virus, bovine enterovirus, bovine adenovirus, and bovine parvovirus from a large volume of material, since it is simple, rapid, and inexpensive.     

Protein refolding is improved by adding nonionic polyethylene glycol monooleyl ethers with various polyethylene glycol lengths

Protein refolding from bacterial inclusion bodies is a crucial step for the production of recombinant proteins, but the refolding step often results in significantly lower yields due to aggregation. To prevent aggregation, chemical additives are often used. However, the ability of additives to effectively increase refolding yields are protein dependent, and therefore, it is important to understand the manner in which the substructures of additives confer suitable properties on protein refolding. We focused attention on nonionic detergents, the polyethylene glycol monooleyl ether (PGME) series, and systematically studied the influence of two to 90 polyethylene glycol (PEG) lengths of PGMEs on the refolding of pig muscle lactate dehydrogenase (LDH), hen egg white lysozyme, and yeast α‐glucosidase. PGMEs with longer PEG lengths such as PGME20, 50, and 90 suppressed aggregation, and increased refolding yields. Notably, PGME20 increased the LDH yield to 56.7% from 2.5% without additives. According to the refolding kinetic analysis of LDH, compared with PGME50 and 90, the refolding rate constant in PGME20 solutions remained relatively high at a broad range of concentrations because of its weaker steric hindrance of intramolecular interactions involved in folding, leading to a preference for refolding over aggregation. These findings should provide basic guidelines to identify appropriate PEG‐based nonionic detergents for protein refolding.     

Preparative purification of pig red blood cell hexokinase type III using a new efficient chromatographic support.

In this paper we report the purification of pig erythrocyte hexokinase type III, at preparative level, using 52 liters of starting material (hemolysate). This was possible using a new efficient anion exchanger support, the Toyopearl DEAE 650 M which allows completely to change the strategy of removing hemoglobin from hemolysates, permitting to handle large amounts of starting material and reducing work would have required months using conventional anion exchanger supports, to only 2-3 days. Furthermore, we have tested the binding of other red blood cell enzymes to the Toyopearl DEAE 650 M, showing a wider potential use of this chromatographic support for their purification at a preparative level.     

Lysozyme purification with dye-affinity beads under magnetic field

Magnetic poly(2-hydroxyethyl methacrylate) mPHEMA beads carrying Cibacron Blue F3GA were prepared by suspension polymerization of HEMA in the presence of Fe3O4 nano-powder. Average size of spherical beads was 80-120 microm. The beads had a specific surface area of 56.0m(2)/g. The characteristic functional groups of dye-attached mPHEMA beads were analyzed by Fourier transform infrared spectrometer (FTIR) and Raman spectrometer. mPHEMA with a swelling ratio of 68% and carrying 28.5 micromol CibacronBlueF3GA/g were used for the purification of lysozyme. Adsorption studies were performed under different conditions in a magnetically stabilized fluidized bed (i.e., pH, protein concentration, flow-rate, temperature, and ionic strength). Lysozyme adsorption capacity of mPHEMA and mPHEMA/Cibacron Blue F3GA beads were 0.8 mg/g and 342 mg/g, respectively. It was observed that after 20 adsorption-desorption cycle, mPHEMA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 87.4% with recovery about 79.6%. The specific activity of the desorbed lysozyme was high as 41.586 U/mg.     

Preparative chromatographic purification of diastereomers of silybin and their quantification in human plasma by liquid chromatography-tandem mass spectrometry

A novel preparative HPLC method separating silybin has been developed to meet the need for both silybin A and silybin B standard. After the preparation of silybin A and silybin B standard, a simple, sensitive, selective and reproducible liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with negative electrospray ionization (ESI) was developed for the quantification of silybin A and silybin B in human plasma. Following rapid sample preparation, silybin A, silybin B and naringin (internal standard, ISTD) were separated on a Zorbax Eclipse XDB-C18 column, using methanol-water containing 0.1% formic acid (48:52, v/v) as the mobile phase. The mass spectrometer was operated in selected reaction monitoring (SRM) mode using the transition m/z 481.1-->300.9 for both silybin A and silybin B and m/z 579.2-->271.1 for naringin, respectively. Linear calibration curves were obtained in the concentration range of 2-5000ng/ml with a lower limit of quantitation (LLOQ) of 2ng/ml for both silybin A and silybin B, respectively. The intra- and inter-day precision values were below 7.5% and accuracy was within +/-4.9% at all three quality control (QC) levels, for both silybin A and silybin B, respectively. This method was successfully applied to the stereospecific analysis of silybin in plasma samples from a pharmacokinetic study of silybin A and silybin B in 22 healthy male Chinese volunteers after a single oral dose of silybin-phosphatidylcholine complex (equivalent to 280mg silybin, including 133mg silybin A and 147mg silybin B).     

Radioprotective effect of polyethylene glycol

Polyethylene glycol of molecular weight 400 (PEG-400) had a radioprotective effect of about 20% against lethality when given ip 20 min prior to single or fractionated X-ray doses to the head and neck. Dose modification factors (DMF) based on LD50/15 values ranged from 1.14 to 1.24. A similar DMF of 1.12 based on LD50/30 values was obtained using single doses of whole-body X irradiation. Mice given head and neck irradiation had significantly reduced rectal temperatures (31.3 +/- 3.0 degrees C) 9 days post irradiation compared with unirradiated controls (35.4 +/- 0.6 degrees C). No such reduction was observed when PEG-400 was given with radiation (36.3 +/- 0.9 degrees C). PEG-400 also lessened, but not significantly, the frequency of shivering in irradiated animals. Histopathologic examination of the oral structures demonstrated only marginal protection by PEG-400. Estimation of the alpha/beta ratio from LD50 data on head and neck-irradiated mice yielded values of 4.4 +/- 1.9 (95% confidence limits) Gy without PEG-400 and 7.9 +/- 1.4 Gy with PEG-400. Since it is a non-thiol radioprotector, PEG-400 may be more useful when combined with more conventional thiol-containing radioprotectors.     

Preparative purification of Escherichia coli heat-stable enterotoxin

Heat-stable enterotoxin (STa) isolated from bovine Escherichia coli strains was purified to homogeneity by growing the bacterial strains in a chemically defined medium, desalting, and concentrating the culture filtrate by batch adsorption chromatography on Amberlite XAD-2 resin, batch adsorption chromatography on reversed-phase silica, and preparative reversed-phase high-performance liquid chromatography. This rapid preparative purification scheme gave high recovery yields of pure STa which exhibited biochemical homology to STa purified by more complicated procedures.