Thermoresponsive properties of sugar sensitive copolymer of N-isopropylacrylamide and 3-(acrylamido)phenylboronic acid

The copolymer of N-isopropylacrylamide and 3-(acrylamido)phenylboronic acid (82:18, (M) over bar (n)=47000 g. mol(-1)) was prepared by free radical polymerization. The copolymer showed typical thermal precipitation behavior in aqueous solutions, its precipitation temperature (T-P) being increased from 23 to 32degreesC by increasing the pH from 6.5 to 9.7, because of ionization of the phenylboronate units. The pK(a) was evaluated as 8.9+/-0.1 from the effect of pH on T-P. At pH>9, i.e., in the anionic form of the copolymer, T-P was affected by a very low concentration of glucose (5.6 muM, DeltaT(P)=1-1.5degreesC), because of complex formation with a high binding constant. At a higher concentration of polyols (560 muM, pH>8) the increase of T-P was maximal for the copolymer complexes with fructose (7-10degreesC) and decreased in the order: fructose>glucoseapproximate tomannitol>pentaerythritol>galactose>Tris>glycerol. Di- and oligosaccharides (lactose, sucrose, and dextran) caused a slight increase of T-P at pH 7.5-8.7 while no effect was observed at pH>9. Isothermal dissolution of the copolymer suspension in water (27degreesC, pH 8.5) was possible in the. presence of fructose or mannitol but required higher concentrations (1.4-3.6 x 10(3) muM) as compared to those which enabled the shift of T-P in the soluble copolymer. The dissolution rate increased with fructose concentrations

Effects of polyols, saccharides, and glycoproteins on thermoprecipitation of phenylboronate-containing copolymers

The copolymer of 3-(acrylamido)phenyl boronic acid and N-isopropylacrylamide (82:18. M-n = 47000 g/mol) was prepared by free radical polymerization. The copolymer showed typical thermoprecipitation behavior in aqueous solutions; its phase transition temperature (T-p) 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 T-p (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 T-p, (ca. 2 degrees C at 0.56 rnM concentration and pH 9.2), while the effects of other sugars are well expressed at the higher concentrations (16 and 80 rnM) and decreased in the order xylose approximate to galactose >= N-acetyllactosamine >= mannose approximate to fucose >> N-acetylglucosarnine. 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

Purification of His(6)-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography

Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His(6)-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)-polyacrylamide cryogel (PAA) and Me2+-N,N,N'-tris (carboxymethyl) ethylendiamine (TED)-PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA-PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA-PAA provided the highest level of selectivity for His(6)-OPH. Comparative analysis of purification using Co2+-IDA-PAA and Ni-nitrilotriacetic acid-agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration

Purification and substrate specificity of peroxidase from sweet potato tubers

Previously the screening of tropical plants demonstrated a high peroxidase activity in sweet potato (Ipomoea batatas) tubers. The major peroxidase pool is localized in peel. Using peel of sweet potato as a source, the sweet potato peroxidase (SPP) has been isolated and purified to homogeneity. The enzyme purification included homogenization, extraction of colored compounds and consecutive chromatographies on Phenyl-Sepharose and DEAE-Toyopearl. The purified SPP had specific activity of 4900 U mg(-1) protein, RZ (ratio of absorbances at 403 and 280 nm, respectively) 3.4, molecular mass of 37 kDa and isoelectric point of 3.5. The spectrum of peroxidase from sweet potato is typical for plant peroxidases with a Soret maximum at 401 nm and the maxima in the visible region at 497 and 638 nm, respectively. The substrate specificity of SPP is distinct from the specificity of other plant peroxidases, ferulic acid being the best substrate for SPP

Metal-chelate affinity precipitation of proteins using responsive polymers

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