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Thin macroporous poly(vinyl alcohol) (PVA) hydrogels were produced by cross-linking of PVA in a semi-frozen state with glutaraldehyde (GA) on glass slides or in the wells of microtiter plates. The 100-130 μm-thick gels were mechanically transferable, squamous translucent films with a high porosity of 7.2 ± 0.3 mL/g dry PVA i.e. similar to larger cylindrical PVA monoliths of the same composition. Additional treatment of the gels with 1% GA increased the aldehyde group content from 0.7 to 2.4 μmol/mL as estimated using dinitrophenylhydrazine (DNPH) reagent. Translucency of the gels allowed registration of UV-visible spectra of the DNPH-stained films. The catalytic activity of trypsin covalently immobilized on thin gels in the microtiter plates was estimated with chromogenic substrate directly in the wells, and indicated that the amount of protein immobilized was at least 0.34 mg/mL gel. Human immunoglobulin G (IgG) immobilized on thin gels at 0.1-10 mg/mL starting concentrations could be detected in a concentration-dependent manner due to recognition by anti-human rabbit IgG conjugated with peroxidase and photometric registration of the enzymatic activity. The results indicate good permeability of the hydrogel pores for macromolecular biospecific reagents and suggest applications of thin reactive PVA hydrogels in photometric analytical techniques., (© 2019 The Authors.)

Effective technologies are required to remove organic micropollutants from large fluid volumes to overcome present and future challenges in water and effluent treatment. A novel hierarchical composite filter material for rapid and effective removal of polar organic contaminants from water was developed. The composite is fabricated from phenolic resin-derived carbon microbeads with controllable porous structure and specific surface area embedded in a monolithic, flow permeable, poly(vinyl alcohol) cryogel. The bead-embedded monolithic composite filter retains the bulk of the high adsorptive capacity of the carbon microbeads while improving pore diffusion rates of organic pollutants. Water spiked with organic contaminants, both at environmentally relevant concentrations and at high levels of contamination, was used to determine the purification limits of the filter. Flow through tests using water spiked with the pesticides atrazine (32 mg/L) and malathion (16 mg/L) indicated maximum adsorptive capacities of 641 and 591 mg pollutant/g carbon, respectively. Over 400 bed volumes of water contaminated with 32 mg atrazine/L, and over 27,400 bed volumes of water contaminated with 2 μg atrazine/L, were treated before pesticide guideline values of 0.1 μg/L were exceeded. High adsorptive capacity was maintained when using water with high total organic carbon (TOC) levels and high salinity. The toxicity of water filtrates was tested in vitro with human epithelial cells with no evidence of cytotoxicity after initial washing., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Macroporous monolithic composites with embedded divinylbenzene-styrene (DVB-ST) polymeric particles were prepared by cryogelation techniques using poly(vinyl alcohol) or agarose solutions. Scanning electron microscopy images showed multiple interconnected pores with an average diameter in the range of 4 to 180 μm and quite homogeneous distribution of DVB-ST particles in the composites. Biocompatibility of the composites was assessed by estimation of the C5a fragment of complement in the blood serum and concentration of fibrinogen in the blood plasma which contacted the composites. A time-dependent generation of C5a fragment indicated weak activation of the complement system. At the same time, the difference in fibrinogen concentration, one of the most important proteins in the coagulation system of the blood, between the pristine blood plasma and the plasma, circulated through the monolithic columns, was insignificant.

Adsorption of myoglobin (Mb), bovine serum albumin (BSA) and γ-globulin (GG) onto activated carbons (ACs) with different pore size distributions, and poly(vinyl alcohol) (PVA) monolithic cryogels containing AC particles was studied. The highest initial rate of Mb adsorption was observed for AC having the largest specific surface area (1939 m(2) g(-1)) and pore volume (1.82 cm(3) g(-1)). The adsorption kinetics of proteins was characterized by a bimodal shape of the distribution f(D) function of an effective diffusion coefficient. Adsorption isotherms of Mb and GG were of Freundlich type within the studied range of equilibrium concentrations (10-150 μg mL(-1)). The distributions of free energy of protein adsorption were bimodal and reflected both interactions with carbon surfaces and self-association of proteins. Adsorbed amounts of Mb were the highest among the proteins studied (up to 700 mg g(-1) carbon), which was attributed to the higher fraction of pores accessible for Mb. Incorporation of carbon particles into PVA-based cryogel resulted in macroporous monolithic composite materials (AC-PVA) exhibiting good flow-through properties. Scanning electron microscopy of the composites showed macroporous aggregates of carbon particles held together by films and bridges of PVA. The rates of adsorption and adsorbed amounts of proteins on AC-PVA were reduced compared to the pristine carbon and depended on the carbon content in the composites. Nevertheless, adsorption of Mb on AC-PVA took place even in the presence of 500-fold higher concentration of BSA. This indicated a possibility of Mb clearance from blood plasma using the PVA-carbon monoliths.

A set of glutaraldehyde (GA) cross-linked poly(vinyl alcohol)/activated carbon (PVA/GA/AC) composites prepared in the form of monolithic rods using a cryogelation technique and studied using adsorption, mercury porosimetry, scanning electron microscopy (SEM), and quantum chemistry methods display porosity similar to that of PVA/GA cryogel at a high GA content (content ratio GA/AC = 1 and GA/PVA = 0.2). GA cross-linked PVA multilayer coverage is an effective barrier for adsorption on AC particles. Variations in surface chemistry (AC initial and oxidized in air at 300 °C for 12 h) and content (14-62.5%w/w) of ACs in PVA/GA/AC composites relatively weakly affect their textural characteristics at a high GA content (specific surface area S(BET) < 120 m²/g, pore volume V(p) < 0.35 cm³/g). However, PVA/GA/AC composite rods formed with a lower concentration of GA (content ratio GA/AC = 1/6 and GA/PVA = 1/10) have significantly greater S(BET) (∼500 m²/g) and V(p) (>0.55 cm³/g) values because of improved accessibility of the AC surface. This provides better adsorption of methylene blue as a probe compound.

Reversible changes of the height of a polymer brush containing phenylboronic acid were studied. The polymer brush thickness underwent reversible changes of 0.5-1 nm, in response to the changes in composition of the contacting aqueous phase from deionized water to bicarbonate buffer and vice versa, apparently due to the conformational transition of the weak polyelectrolyte to the more extended electrically charged state. Adsorption of mucin glycoprotein to the polymer brush took place due to boronate/sugar interactions between the glycoprotein and the graft copolymer and resulted in further increase of the brush height by ca. 1.5 nm, as observed by means of spectral correlation spectroscopy and ellipsometry., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Boronate-containing thin polyacrylamide gels (B-Gel), polymer brushes (B-Brush) and chemisorbed organosilane layers (B-COSL) were prepared on the surface of glass slides and studied as substrates for carbohydrate-mediated cell adhesion. B-COSL- and B-Brush-modified glass samples exhibited multiple submicron structures densely and irregularly distributed on the glass surface, as found by scanning electron microscopy and atomic force microscopy. B-Gel was ca. 0.1 mm thick and contained pores with effective size of 1-2 microm in the middle and of 5-20 microm on the edges of the gel sample as found by confocal laser scanning microscopy. Evidence for the presence of phenylboronic acid in the samples was given by time-of-flight secondary ion mass-spectrometry (ToF SIMS), contact angle measurements performed in the presence of fructose, and staining with Alizarin Red S dye capable of formation specific, fluorescent complexes with boronic acids. A comparative study of adhesion and cultivation of animal cells on the above substrates was carried out using murine hybridoma M2139 cell line as a model. M2139 cells adhered to the substrates in the culture medium without glucose or sodium pyruvate at pH 8.0, and then were cultivated in the same medium at pH 7.2 for 4 days. It was found that the substrates of B-Brush type were superior both regarding cell adhesion and viability of the adhered cells, among the substrates studied. MTT assay confirmed proliferation of M2139 cells on B-Brush substrates. Some cell adhesion was also registered in the macropores of B-Gel substrate. The effects of surface microstructure of the boronate-containing polymers on cell adhesion are discussed. Transparent glass substrates grafted with boronate-containing copolymers offer good prospects for cell adhesion studies and development of cell-based assays.

Boronate-containing polymer brushes were synthesized by free radical copolymerization of N,N-dimethylacrylamide (DMAA) and N-acryloyl-m-phenylboronic acid (NAAPBA) (9:1) on the surface of 3-mercaptopropyl-silylated glass plates and capillaries. The brushes were characterized with time-of-flight secondary ion mass-spectrometry (ToF SIMS), atomic force microscopy and contact angle measurements. Fructose caused a well-expressed drop spreading on the surface of copolymer-grafted glass, due to the strong interaction with the boronate groups. Sedimentation of murine hybridoma cells M2139 or human myeloid leukemia cells KG1 onto the DMAA-NAAPBA copolymer-grafted glass plates from 10 mM phosphate buffer solution (pH 8.0) resulted in the cell adhesion. The adhered M2139 and KG1 cells could be quantitatively detached from the grafted plates with 0.1 M fructose, which competed with cell surface carbohydrates for binding to the boronates. Evaluation of the binding strength between M2139 cells and the copolymer brush was performed by exposure of the adhered cells to a shear stress. Detachment of a fraction of 18% of the adhered M2139 cells was obtained at a shear force of 1400-2800 pN/cell generated by the running phosphate buffer (pH 8.0), whereas the remaining adhered cells (70%) could be detached with 0.1 M fructose dissolved in the same buffer. Possible applications of the boronate-containing polymer brushes to affinity cell separation can be based upon the facile recovery of the attached cells., (2008 Wiley Periodicals, Inc.)

Copolymers of N-acryloyl-m-aminophenylboronic acid (NAAPBA) with N,N-dimethylacrylamide (DMAA) formed insoluble interpolymer complexes with mucin from porcine stomach at pH 9.0. The complex formation based on boronate-sugar interactions took place between the similarly charged macromolecules and resulted in coacervate particles formation, which depended both on pH and ionic strength of the solution. The coacervation rate displayed a maximum at the intermediate DMAA-NAAPBA copolymer: mucin weight ratio, that is a pattern typical of interpolymer complex formation. The effective hydrodynamic particle diameter of the coacervates monotonously grew from 155+/-20 nm up to 730+/-120 nm in 2 days in 0.1M sodium bicarbonate buffer solution, pH 9.0. Electrophoretic mobility of the resultant nanoparticles was intermediate between those of individual polymers, whereas the particles zeta-potential was -7.5+/-0.4 mV in the above buffer solution. Pre-treatment of the inner mucosal epithelium of excised male pig urethras with 5% (w/v) solutions of acrylamide-NAAPBA or DMAA-NAAPBA copolymers at pH 8.8 allowed for tight occlusion of the lumen by poly(vinyl alcohol)-borax gel injected via a two-way catheter. Leakage of 0.15M NaCl solution through the thus occluded organs could be prevented, while the leakage through the organs occluded by the gel without the pre-treatment was unavoidable. The gel plug could be quickly dissolved on demand after injection of 5% (w/v) aqueous fructose solution into the lumen. The described technique may be useful for temporal occlusion of mucosal lumens in living organisms. In contrast to the conventional mucoadhesive polymers like polyacrylic acid or chitosan, the boronate-containing copolymers display their mucoadhesivity at weakly alkaline pH of 8-9 and physiological ionic strength.

Radical copolymerization of acrylamide (Am) (90 mol%) with N-acryloyl-m-aminophenylboronic acid (NAAPBA) (10 mol%) carried out on the surface of glass slides in aqueous solution and in the absence of chemical cross-linkers, resulted in the formation of thin semitransparent gels. The phenylboronic acid (PBA) ligand density was ca. 160 micromol/ml gel. The gels exhibited a macroporous structure and displayed optical response to sucrose, lactose, glucose and fructose in 50 mM sodium phosphate buffer, in the pH range from 6.5 to 7.5. The response was fairly reversible and linearly depended on glucose concentration in the wide concentration range from 1 to 60 mM at pH 7.3. The character of response was explained by the balance of two competing equilibrium processes: binding of glucose to phenylboronate anions and binary hydrophobic interactions of neutral PBA groups. The apparent diffusion coefficient of glucose in the gels was ca. 2.5 x 10(-7) cm(2)/s. A freshly prepared gel can be used daily for at least 1 month without changes in sensitivity. Autoclaving (121 degrees C, 1.2 bar, 10 min) allows for the gels sterilization, which is important for their use as glucose sensors in fermentation processes., (2008 John Wiley & Sons, Ltd.)

Affinity precipitation of proteins uses polymers capable of reversible soluble-insoluble transitions in response to small environmental changes (temperature, pH or solvent composition). Here we describe protocols for (i) the synthesis of responsive polymers with specific affinity to target proteins and (ii) the purification of proteins using these polymers. The purification is based on precipitation of the affinity complex between the protein and the polymer, which is induced by environmental changes. This separation strategy is simpler and more cost effective than conventional affinity column chromatography. Specifically, we describe the synthesis of thermoresponsive 1-vinylimidazole:N-isopropylacrylamide copolymers. The whole procedure takes 2-3 h when applied to purification of recombinant His-tag proteins or proteins with natural metal binding groups by means of metal chelate affinity precipitation. Optimization of the polymer composition and the type of chelating ions allows for target protein yields of 80% and higher.

Cross-linked agarose particles (Sepharose CL-6B) and baker's yeast cells were found to adhere to siliceous supports end-grafted with boronate-containing copolymers (BCCs) of N,N-dimethylacrylamide at pH> or =7.5, due to boronate interactions with surface carbohydrates of the particles and the cells. These interactions were registered both on macroscopic and on molecular levels: the BCCs spontaneously adsorbed on the agarose gel at pH> or =7.5, with adsorption increasing with pH. Agarose particles and yeast cells stained with Procion Red HE-3B formed stable, monolayer-like structures at pH 8.0, whereas at pH 7.0-7.8 the structures on the copolymer-grafted supports were less stable and more random. At pH 9.0, 50 % saturation of the surface with adhering cells was attained in 2 min. Stained cells formed denser and more stable layers on the copolymer-grafted supports than they did on supports modified with self-assembled organosilane layers derivatized with low-molecular-weight boronate, presumably due to a higher reactivity of the grafted BCCs. Quantitative detachment of adhered particles and cells could be achieved by addition of 20 mM fructose--a strong competitor for binding to boronates--at pH 7.0-9.0. Regeneration of the grafted supports allowed several sequential adhesion and detachment cycles with stained yeast cells. Affinity adhesion of micron-sized carbohydrate particles to boronate-containing polymer brushes fixed on solid supports is discussed as a possible model system suggesting a new approach to isolation and separation of living cells.

Interaction of mono- and disaccharides, polysaccharide particles and yeast cells with boronate-containing copolymers (BCC) of N-acryloyl-m-aminophenylboronic acid (NAAPBA) with N,N-dimethylacrylamide (DMAA) or N-isopropylacrylamide (NIPAM) was studied. The binding of saccharides to BCC of NIPAM resulted in a shift of its phase transition temperature (DeltaTP), which provided a quantitative measure for the complex formation. Among the sugars typical of non-reducing ends of glycoproteins the DeltaTP decreased in the order: N-acetylneuraminic acid > xylose approximately galactose > mannose approximately fucose >> N-acetylglucosamine. Strong specific adsorption of the BCC on the cross-linked agarose gel Sepharose CL-6B (15-30 mg/ml gel at pH 9.2) was registered. The copolymers adsorption was due to boronate-sugar interactions and decreased with pH. Multivalent interaction of the BCC with the agarose gel has been proven by liquid column chromatography exhibiting a weak reversible adsorption of NAAPBA and almost irreversible adsorption of DMAA-NAAPBA copolymer from 0.1 M sodium phosphate buffer, pH 7.9. The two studied BCCs could be completely desorbed from the gel by 0.1 M fructose in aqueous buffered media with pH from 7.5 to 9.2. In turn, the agarose particles and yeast cells were found to adhere to siliceous supports end-grafted with boronate-BCC of N,N-dimethylacrylamide at pH > or = 7.5, due to the actions. Quantitative detachment of adhered particles or cells could be attained by addition of 20 mM or 100 mM fructose, respectively, in the pH range from 7.5 to 9.2. Affinity adhesion of micron-size carbohydrate particles to boronate-containing polymer brushes fixed on solid supports was considered as a model system suggesting a new approach to isolation and separation of living cells., (Copyright 2006 John Wiley & Sons, Ltd.)

Poly(N-isopropylacrylamide), a thermally responsive polymer, was end-grafted to mercaptopropyl derivatives of silica gel, plane glass sheets and glass capillary tubing by free radical polymerization of the monomer in 1,4-dioxane at 100 degrees C. The polymer monolayer attached to the glass carriers provided them with thermally controlled wettability registered by two independent methods: direct measurements of the water contact angle and capillary rise. The water contact angle changed from 54+/-3 degrees to 68+/-3 degrees in the temperature range from 20 to 50 degrees C. The polymer grafting to silica gel (pore diameter 100 A, particle size 5 microm) resulted in 15-30-fold reduction in protein adsorption on the carrier at 35 degrees C. Adsorption isotherms of myoglobin indicate completely different characters of the protein adsorption to silica gel and its polyNIPAM-grafted derivative. Cooling of the grafted carrier containing adsorbed myoglobin to 9 degrees C led to a partial release of the protein to the contacting solution, whereas heating of the system to 35 degrees C resulted in reversible binding of the protein. Adsorption of myoglobin on polyNIPAM-coated silica was ca. 2-fold higher at 35 than at 9 degrees C, most probably due to steric repulsion displayed by the swollen copolymer at the lower temperature.

The copolymer of 3-(acrylamido)phenylboronic acid and N-isopropylacrylamide (82:18, Mn = 47000 g/mol) was prepared by free radical polymerization. The copolymer showed typical thermoprecipitation behavior in aqueous solutions; its phase transition temperature (TP) was 26.5 +/- 0.2 degrees C in 0.1 M glycine-NaOH buffer containing 0.1 M NaCl, pH 9.2. Due to specific complex formation of the pendant boronates with sugars, TP was strongly affected by the type of sugar and its concentration at pH 9.2. Fructose, lactulose, and glucose caused the largest increase in TP (up to 4 degrees C) at 0.56 mM concentration, attributed to the high binding affinity of the sugars to borate and phenylboronate. Among the sugars typical of nonreducing ends of oligosaccharides, N-acetylneuraminic acid had the strongest effect on TP (ca. 2 degrees C at 0.56 mM concentration and pH 9.2), while the effects of other sugars are well expressed at the higher concentrations (16 and 80 mM) and decreased in the order xylose approximately galactose >or= N-acetyllactosamine >or= mannose approximately fucose >> N-acetylglucosamine. The effect exerted on the phase transition by glycoproteins was the strongest with mucin from porcine stomach and decreased in the series mucin > horseradish peroxidase > human gamma-globulin at pH 9.2. As a first approximation, the weight percentage and/or the number of oligosaccharides in glycoproteins determined the character of their interaction with the pendant phenylboronates and, therefore, the effect on the copolymer phase transition.

Copolymers of N-acryloyl-m-aminophenylboronic acid (NAAPBA) with acryamide (AA), N,N-dimethylacrylamide (DMAA), and N-isopropylacrylamide (NIPAM) were found to adsorb on cross-linked agarose gel (Sepharose CL-6B) in the pH range from 7.5-9.2, due to specific boronate-sugar interactions. The molar percentages of phenylboronic acid (PBA) groups in the boronate-containing copolymers (BCCs), as estimated by 1H NMR spectroscopy, were 13, 10, and 16%, respectively, whereas the apparent ionization constants, the pKa values, of the copolymers were similar and equal to 9.0 +/- 0.2 at 20 degrees C. The copolymers adsorption capacities were in the range of 15-30 mg x ml(-1) gel (14-36 micromol pendant PBA ml(-1) gel) at pH 9.2 and decreased with decreasing pH value. The interaction of monomeric NAAPBA with Sepharose CL-6B was characterized by an equilibrium association constant of 53 +/- 17 M(-1), the chromatographic capacity factor k' = 1.8, and a total content of binding sites of 27 +/- 10 micromol x ml(-1) gel at pH 9.2. The weak reversible binding of monomeric NAAPBA and almost irreversible binding of NAAPBA copolymers to the gel at pH 9.2 suggested a multivalent character of the copolymer adsorption. At pH 7.5, the maximal adsorption capacity was displayed by the AA-NAAPBA copolymer (15 mg x ml(-1) gel). All the BCCs could be completely desorbed from the gel by 0.1 M fructose in aqueous buffered media with pH values from 7.5-9.2. The strong adsorption of AA-NAAPBA on agarose gel probably relates to the conformation of the copolymer in aqueous solution and provides opportunities for biomedical applications of the copolymer under physiological conditions. Multivalent, weak-affinity adsorption of BCCs to the agarose gel seems to be a tentative model for the copolymers' binding to oligo- and polysaccharides of cell membranes and mucosal surfaces.

Binding of adenosine to the thermosensitive copolymer of N-isopropylacrylamide and 3-(acrylamido)aminophenylboronic acid (82:18, Mn = 47,000 g . mol(-1)) was studied by equilibrium dialysis at 22 degrees C and 37 degrees C, in a 0.1 M glycine buffer containing 0.1 M NaCl at pH 9.2. The copolymer exhibited a the phase transition temperature (T(p)) of 26.5 degrees C under the above conditions. At 22 degrees C the binding of adenosine to the water-soluble copolymer was well described by a Langmuir model, accounting for preferential ionisation of the boronate-nucleoside complexes and, therefore, restricted reactivity of the rest of boronates. At saturation, the copolymer contained 38% of its phenylboronic acid groups in the form of complexes, whereas the association constant was 1,400 M(-1). At 37 degrees C no binding of adenosine to thermally precipitated copolymer was found, presumably owing to interaction of the phenylboronates with hydrophobic segments of polyNIPAM. At high loading of the copolymer by the reversibly bound adenosine the T(p) steeply increases with increasing fraction of the phenylboronate-adenosine complexes in the chains. The increase of the T(p) observed above the saturating adenosine concentration (>1 x 10(-3) M, 22 degrees C) very probably testifies to competition of the nucleoside with hydrophobic polyNIPAM segments for binding to the pendant phenylboronates.

Conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10-fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4-fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 10(6) and, presumably, cross-linked protein-polymer aggregates of MW > 10(7). Michaelis constant for 5-nitro-3-phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 microM) was found to be slightly higher than that of the free enzyme (12 microM), and evaluation of V(max) testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM-cross-linked PA retained its capacity to synthesize cephalexin from d-phenylglycin amide and 7-aminodeacetoxycephalosporanic acid. The synthesis-hydrolysis ratios of free and polyNIPAM-cross-linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of beta-lactam antibiotics, can be successfully eliminated by cross-linking penicillin acylase to a smart polymer (i.e., polyNIPAM).

Successful immobilized metal affinity chromatography (IMAC) of proteins on Cu2+-iminodiacetic acid Sepharose has been carried out in a displacement mode using a synthetic copolymer of vinyl imidazole and vinyl caprolactam [poly(VI-VCL)] as a displacer. Vinyl caprolactam renders the co-polymer with the thermosensitivity, e.g., property of the co-polymer to precipitate nearly quantitatively from aqueous solution on increase of the temperature to 48 degrees C. A thermostable lactate dehydrogenase from the thermophilic bacterium Bacillus stearothermophilus modified with a (His)6-tag [(His)6-LDH] has been purified using an IMAC column. For the first time it was clearly demonstrated that a polymeric displacer [poly(VI-VCL)] was more efficient compared to a monomeric displacer (imidazole) of the same chemical nature, probably due to the multipoint interaction of imidazole groups within the same macromolecule with one Cu2+ ion. Complete elution of bound (His)6-LDH has been achieved at 3.7 mM concentration of imidazole units of the co-polymer (5 mg/ml), while this concentration of free imidazole was sufficient to elute only weakly bound proteins. Complete elution of (His)6-LDH by the free imidazole was achieved only at concentrations as high as 160 mM. Thus, it was clearly demonstrated, that the efficiency of low-molecular-mass displacer could be improved significantly by converting it into a polymeric displacer having interacting groups of the same chemical nature.

Synthetic copolymers of N-vinylcaprolactam (VCL) and N-vinylimidazole (VI) were studied as thermosensitive, reusable displacers for immobilised metal affinity chromatography (IMAC) of proteins. The copolymer with weight-average molecular mass of 11700 g/mol prepared by free radical polymerisation at a 9:1 monomer molar ratio was separated into several fractions by IMAC and thermal precipitation. The fraction with an average VI content of 8.5% was most efficient as a reusable displacer for IMAC of ovalbumin, lysozyme and other proteins of egg white on Cu2+-IDA-Sepharose. The displacer exhibited a sharp breakthrough curve and binding capacity of 16-20 mg/ml gel, depending on the flow-rate. The recovery of egg white proteins in the course of displacement chromatography was >95%. The displacer could be removed quantitatively from the protein fractions by thermal precipitation at 48 degrees C. Co-precipitation of lysozyme with the displacer was minimal in the presence of 3% (v/v) acetonitrile, while the lysozyme enzymatic activity in the supernatant was completely retained. Addition of free imidazole to the mobile phase increased the rate of protein desorption and allowed better separation of egg white proteins and the displacer in the course of chromatography. The displacement profile of the egg white extract consisted of three zones with different distributions of individual proteins characterised by SDS-PAGE. Regeneration of the column was easily performed with 0.02 M EDTA in 0.15 M sodium chloride, pH 8.0, followed by washing with distilled water and reloading with Cu2+. The displacer could also be regenerated by thermal precipitation at 48 degrees C and subsequent dialysis against dilute hydrochloric acid (pH 2.5).

Composite sorbents based on wide-pore glass and silica coated with N-butyl polyacrylamide (butyl-PA-glass and butyl-PA-silica) were studied. The surface tension of butyl-PA-silica is 50-55 mJ/m2 as evaluated by the sedimentation volume technique. The linear dependences of log k' on ammonium sulphate concentration were determined by isocratic chromatography of the dipetide Trp-Trp and lysozyme on butyl-PA-glass. Both solutes were shown to have a weaker retention on butyl-PA-glass than on butyl-Toyopearl 650C. This weaker retention is beneficial in the purification of sialic acid-binding lectin from Bacillus subtilis.

The applicability of reversed-phase high-performance liquid chromatography (HPLC) to the analysis of the products of recombinant insulin was studied. The influence of several mobile phases in reversed-phase and ion-pair HPLC on selectivity, resolution and sensitivity was investigated. Optimum conditions for the separation of insulin-related proteins on commercial and laboratory-made supports were established by means of three-dimensional optimizations of selectivity and resolution as a function of pH and ionic strength (mu). A mechanism for the separation of proteins with a mobile phase containing a high salt concentration and a pH near the isoelectric point of proteins is proposed. The questions of scaling up are considered. The proposed techniques allow the analysis of the main impurities and ensures a high quality of active insulin production.

Several types of polymeric glycoconjugates, N-substituted polyacrylamides, have been synthesized by the reaction of activated polymers with omega-aminoalkylglycosides: (i) (carbohydrate-spacer)n-polyacrylamide, 'pseudopolysaccharides'; (ii) (carbohydrate-spacer)n-phosphatidylethanolaminem-polyacrylamide, neoglycolipids, derivatives of phosphatidylethanolamine; (iii) (carbohydrate-spacer)n-biotin-polyacrylamide, biotinylated probes; (iv) (carbohydrate-spacer)n-polyacrylamide-(macroporous glass), affinity sorbents based on macroporous glass, covalently coated with polyacrylamide. An almost quantitative yield in the conjunction reaction makes it possible to insert in the conjugate a predetermined quantity of the ligand(s). Pseudopolysaccharides proved to be a suitable form of antigen for activation of polystyrene and poly(vinyl chloride) plates (ELISA) and nitrocellulose membranes (dot blot), being advantageous over traditional neoglycoproteins. Polyvalent glycolipids insert well in biological membranes: their physical properties, particularly solubility, can be changed in a desired direction. Biotinylated derivatives were used as probes for detection and analysis of lectins.

Blood group B antigenic trisaccharide-beta-aminopropyl glycoside (B-TSAP) covalently attached to poly-N-(2-hydroxyethyl)acrylamide-coated porous glass interacts with anti-B monoclonal antibodies faster than the ligand coupled to CNBr-activated Sepharose 4B and Affi-Gel 10. Rates of hydrophobic adsorption of antibodies on the butyl derivatives of the same supports were measured to evaluate the diffusion input to overall kinetics. The lowest average affinity adsorption time [t1(aff) = 250 s] observed for polymer-coated glass probably arises because of the flexibility of the extended segments of chemisorbed N-substituted polyacrylamide acting as effective spacer arms.

Wide porous glass (WPG) chemically coated with a poly-N-(2-hydroxyethyl)acrylamide layer is proposed as a carrier of biospecific ligands in affinity chromatography. The method of WPG chemical modification includes synthesis of the gamma-aminopropyl derivative followed by chemical adsorption of poly(p-nitrophenyl acrylate). Ester groups of the polyacrylate-coated WPG can be used for coupling the ligands bearing primary amino groups. Condensation of esters with ethanolamine yields a poly-N-(2-hydroxyethyl)acrylamide-coated support with non-specific adsorption properties resembling those of Sepharose 4B. Human IgG immobilized on the polyacrylate support was used for isolation of the first complement component from human serum and for its separation into subcomponents C1r, C1s and C1q by a one-step method. An unbound part of serum may be used as the R1 reagent for determining haemolytic C1 activity. The stepwise elution of C1r, C1s and C1q from the column reflects the course of C1 breakdown after its activation on immune complex formation.