Galina Tishchenko

Purification of the specific immunoglobulin G1 by immobilized metal ion affinity chromatography using nickel complexes of chelating porous and nonporous polymeric sorbents based on poly(methacrylic esters). Effect of polymer structure

Ni2+ complexes of the chelating nonporous and porous bead sorbents based on methacrylic esters crosslinked with ethylene dimethacrylate were used in isolation of the horseradish peroxidase-specific immunoglobulin IgG1 from the crude mouse ascitic fluid by immobilized metal ion affinity chromatography (IMAC). Iminodiacetic and aspartic acids were attached to porous poly(glycidyl methacrylate) beads differing in size, morphology and chemical composition. Ethylenediaminetriacetic acid and quinolin-8-ol chelating groups were attached mainly to the surface hydroxyl groups in nonporous poly(diethylene glycol methacrylate) beads through spacers. The latter sorbents exhibited better kinetic characteristics than the former but a very low IgG1 sorption capacity. In a single-step IMAC procedure, the best efficiency in the specific IgG1 purification was obtained with porous sorbents (recovery 92%, purity 73%). Differences in IMAC separations are discussed from the point of view of morphology of polymer beads as well as of the type and concentration of chelating ligands.

Effect of salt concentration gradient on separation of different types of specific immunoglobulins by ion-exchange chromatography on DEAE cellulose

A three-stage process, consisting of an ammonium sulfate precipitation step, dialysis desalination with microporous anion-exchange Neosepta membranes and anion-exchange chromatography on DEAE-cellulose DE-52 was used for the isolation of mouse monoclonal antibodies specific against different antigens. The ascites fluids contained monoclonal antibodies against human IgG, against horseradish peroxidase and against the heavy chain of human IgM. The effect of the salt concentration gradient in the elution buffer was examined with the aim of optimizing chromatographic conditions. The quality of separation of protein zones was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. The immunoreactivity of purified monoclonal antibodies was determined by enzyme-linked immunosorbent assay using a solid-phase adsorbed antigens against which each monoclonal antibody type was directed.

Effect of chitosan on the growth of human colonic bacteria

Growth of 6 bacterial strains representing dominant members of the human colonic microflora was measured in the presence of 0.025, 0.05 and 0.5 % chitosan (from shrimp shells, with a 97 % final degree of deacetylation). The effect of chitosan was variable and dependent on bacterial species. The most susceptible to chitosan were bacteria belonging to generaBacteroides andClostridium (91–97% growth inhibition). On the other hand,Roseburia sp.,Eubacterium sp. andFaecalibacterium sp. were more resistant (63–83 % inhibition of growth). Chitosan can thus be considered as one of the means for influencing the bacterial population in the human colon.

Immobilized-metal affinity sorbents based on hydrophilic methacrylate polymers and their interaction with immunoglobulins

Abstract Hydrophilic homogeneous poly[2-(2-hydroxyethoxy)ethyl methacrylate- co -ethylene dimethacrylate] poly(DEGMA- co -EDMA) beads have been prepared as precursors for introducing the chelating groupings of ethylenediaminetriacetic acid, quinolin-8-ol, or N -(2-pyridylmethyl)glycine. The effect of the chelating ligand structure on coordination with Ni 2+ ions and that of the metal complexes on binding the model protein (horseradish-peroxidase-specific immunoglobulin IgG 1 ) was analyzed. It was found that the protein binding affinity and selectivity was the highest for IMA sorbents with quinolin-8-ol groups.

Electrical resistance and diffusion permeability of microporous polyethylene membranes modified with polypyrrole and polyaniline in solutions of electrolytes

Composite systems based on a microporous polyethylene (PE) membrane modified with conducting polymers such as polypyrrole (PPy) and polyaniline (PANI) have been elaborated. Conducting polymers were deposited onto PE membranes in situ during the oxidative polymerization of pyrrole from gas phase or by the polymerization of aniline in aqueous medium. Composite membranes have a low resistance in electrolyte solutions owing to the coating with conducting polymers inside the pores. The dependence of electrical resistance of modified membranes on concentration of hydrochloric acid or sodium hydroxide solutions, in which the conducting polymers are in protonated or non-protonated states, respectively, was studied using alternating and direct electric currents. Diffusion permeability of composite membranes to acid, salt and alkali solutions has been also studied.

The antimicrobial action of low-molar-mass chitosan, chitosan derivatives and chitooligosaccharides on bifidobacteria

The crude fractions of chitooligosaccharides (COS) and low-molar-mass chitosans (LMWC) were prepared by enzyme hydrolysis of chitosan (CS). Specific growth rate of B. adolescentis, B. bifidum, B. breve, B. catenulatum, B. infantis and B. longum ssp. longum was determined in the presence of 0.025 and 0.5 % COS (<5 kDa), LMWC (5–10 kDa), and 0.025, 0.1 and 0.5 % of CS, chitosan succinate and chitosan glutamate in vitro. Minimum inhibitory concentrations (MIC; assayed by colony counting on TPY agar plates) of COS-LMWC and CS ranged from 0.025 % to 0.75 % of CS-LMWC. The growth of all bifidobacterial strains in the presence of chitosan, its derivatives and LMWC decreased at a concentration of 0.025 %; the bacterial growth was completely inhibited at a concentration of 0.5 %. COS did not show any inhibitory effect, an increased growth rate was even observed in the case of B. bifidum, B. catenulatum and B. infantis.

Chitinolytic enzymes from Clostridium aminovalericum: activity screening and purification.

A strain isolated from the feces of takin was identified asClostridium aminovalericum. In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase (‘endochitinase’), exochitinase, β-N-acetylglucosaminidase, chitosanase and chitin deacetylase. The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin. Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively). Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL). The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27. At pH 4.5, some isoforms of the chitinolytic enzymes (30 % of total enzyme activity) did not bind to Polygran 27. The enzymes were eluted under a stepwise pH gradient (pH 5–8) in 0.1 mol/L phosphate buffer. At merely acidic pH (4.5–5.5), the adsorbed enzymes were co-eluted. However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated. The protein and enzyme recovery reached 90 %.

Chitosan–Oligo(silsesquioxane) Blend Membranes: Preparation, Morphology, and Diffusion Permeability

Preparation of the blend chitosan (CHI) membranes containing polyhedral oligomeric silsesquioxane (POSS) derivatives was investigated. POSS derivatives such as (3-aminopropyl)isobutyl-POSS (amino-POSS), [2-(3,4-epoxycyclohexyl)ethyl]isobutyl-POSS (epoxy-POSS), and octa(tetramethylammonium)-POSS were used. The blend CHI–amino-POSS membranes were predicted to be the most porous due to having the weakest interactions between the components in the blends. The CHI–epoxy-POSS blend membranes were assumed to be more dense owing to chemical binding of the chitosan amino groups with the epoxy groups of POSS. Studies of membrane morphology and diffusion permeability support these predictions.

Theoretical analysis of neutralization dialysis in the three-compartment membrane cell

Abstract A theoretical model of the kinetics of neutralization dialysis across ideally selective ion exchange membranes is suggested. The model is based on the use of the Nernst-Planck equations describing ion transport through both the membranes and adjacent unstirred layers (ULs) of solution. Special emphasis is made on the kinetics of change in pH of the desalinated solution. The character of this change with time is shown to depend essentially on the solution concentration. At sufficiently high concentration of the solutions in the three compartments, the presence of the ULs can be neglected in the model, and the pH value in the desalination compartment is determined only by the relationship between the intrinsic permeabilities of the membranes. In this case, the model predicts the pH to be in either the acidic or the alkaline range of values during most of the desalination experiment, so that the passage of the pH through neutrality, if it occurs at all, is “leaplike”. In contrast, in solutions of sufficiently low concentration,, the exchange of counterions between the compartments is controlled by mass transfer through the ULs. For this limiting case, a rigorous relationship linking the concentrations in the three compartments is derived under which the pH of the desalinated solution assumes the neutral value. Finally, at intermediate concentration of the solutions, the model is found to describe satisfactorily the data of auxiliary experiments with appropriate choice of parameters.

Chitinolytic activities of Clostridium sp. JM2 isolated from stool of human administered per orally by chitosan

The novel chitinolytic bacterium Clostridium beijerinckii strain JM2 was isolated from the stool of healthy volunteers supplied daily per orally with 3 g of chitosan. The bacterium grown on colloidal chitin produced a complete array of chitinolytic enzymes. Significant activities of endochitinase, exochitinase and chitosanase were excreted into the medium (301, 282 and 268 nkat/μg protein, respectively). The high cellular activity of N-acetyl-β-glucosaminidase (NAGase) and chitosanase were detected (732.4 and 154 nkat/μg protein, respectively). NAGase activity represented the main activity associated with the cellular fraction. The activities of both enzymes tested increased from 20 to 50 °C; the optimum reaction temperature estimated being 50 °C. Endochitinase as well as NAGase showed an activity in the pH interval of 4.0–8.0; the optimum pH values were 6.5 and 6.0, respectively. The extracellular endochitinase complex consisted of six isoenzymes with molar mass of 32–76 kDa; in the cellular fraction five bands with molar mass of 45–86 kDa were detected. Exochitinase activity was demonstrated in the form of three bands (with molar mass of 30–57 kDa), NAGase activity displayed one band of 45 kDa.