O. Mikeš

Chromatography of biopolymers and their fragments on ion-exchange derivatives of the hydrophilic macroporous synthetic gel spheron.

New ion-exchange materials have been developed from the synthetic glycol methacrylate gel Spheron: carboxymethyl-Spheron (weakly acidic); phosphonyl-Spheron (medium acidic); sulphonyl-Spheron (strongly acidic); diethylaminoethyl-Spheron (Type 1, weakly basic; Type 2, medium to strongly basic). Information is presented on the chromatographic characteristics of these new, macroporous, hydrophilic and rigid ion-exchangers as determined by experiments on the separation of mixtures of proteins, peptides, amino acids, nucleic acids, oligonucleotides, and nucleotides. The possibility is discussed of the use of ion-exchangers of this type in high-performance liquid chromatography of biopolymers.

Ion-exchange derivatives of spheron : I. characterization of polymeric supports

Abstract In order to characterize matrices suitable for the preparationof ion exchangers, three commercially available types of glycol methacrylate macroreticular gels, SpheronTM P-100, P-300 and P-1000 (particle size, 20–40 μm), have been characterized by their bulk rate, degree of swelling, working volume and inner surface area. Prior to modification, the gels were extracted with dilute acid, dilute alkali. 8 M urea, pyridine and hot organic solvents. Extracted and dried Spherons have been characterized by elemental analysis, and by the determination of the inner surface area, exclusion limit, specific pore volume, the most frequent pore diameter, specific unpenetrable volume, number of unpolymerized double bonds and capacity for small ions. The particle-size distribution of Spheron P-300 has also been determined. Porosimetric data obtained by nitrogen sorption and desorption measurements are compared with those obtined by mercury porosimetry. The inner structure of Spheron is discussed in relation to the experimentl results (electron microphotography and the course of thermal vacuum depolymerization, ion-exchange capacity after the highest attainable ionogenic substitution, unpenetrable volume and particle porosity). Extracted Spheron P-300 exhibits an advantageous, approximately level dependence of the height equivalent to a theoretical plate on the flow-rate within the range 25–280 ml/h. The suitability of a Spheron matrix for the preparation of ion exchangers is demonstrated.

Ion-exchange derivatives of spheron

Abstract The preparation of diethylaminoethyl derivatives of Spheron (DEAE-Spheron 300) having different capacities for small ions (nominal capacities 0.11, 0.26, 0.60, 1,23, 1.63, 2.05 and 2.20 mequiv./g) is described. Routes leading to a high degree of ionogenic substitution are discussed. The ion exchangers were characterized analytically and by titration curves, which indicate that the ion exchangers are homoionic. The chromatographic properties of the DEAE-Spherons were tested in the chromatography of serum albumin, of an artificial mixture of proteins (lysozyme, chymotrypsinogen and serum albumin), blood plasma, a mixture of peptides and of glutamic, aspartic and cysteic acids using an amino acid analyzer and also in the chromatography of adenosine phosphates. In all cases the best separations were obtained with anion exchangers having the highest nominal capacities. The effect of degree of ionogenic substitution on the decrease of the partial hydrophobicity of the Spheron matrix is discussed. It is shown that at a nominal capacity exceeding 1.2 mequiv./g the effect of hydrophilic iogenic groups balances the partial hydrophobicity of the Spheron matrix, so that no hydrophobic sorption of proteins takes place.

Rapid chromatographic separation of technical enzymes on spheron ion exchanges

Abstract New types of ion exchangers with a hydroxyalkyl methacrylate matrix (Spheron) that are suitable for the the sorption and high-performance liquid chromatography of technical enzymes are described. Their use is illustrated by examples of the sorption and desorption of a microbial protease and by examples of the rapid, semi-preparative chromatography of technical enzymes (protease, glucose oxidase, pectinase).

High-performance liquid chromatography of pectic enzymes☆

Publisher Summary Various methods have been used for the separation of pectic enzymes, such as ion-exchange chromatography on DEAE-cellulose, CM-cellulose, cellulose phosphate, gel chromatography, and affinity chromatography on pectic acid cross-linked by epichlorhydrin and its amino derivative, or on poly(hydroxyalkyl methacrylate) containing glycosidically bound oligo-D-galactosiduronic acids. Because of the variability in the composition of pectic enzyme complexes and the new trends in some industrial processes to apply preparations containing certain components of the pectolytic complex or mixtures of definite composition, rapid and efficient separation procedures on an analytical and preparative scale are required in research and industry. High-performance liquid chromatography (HPLC) of biopolymers, especially of enzymes has permitted the application of this rapid method to the separation of pectic enzymes of microbial and plant origin. Microbial pectic enzymes from three commercial preparations of pectinases were separated in 35–70 min on various types of ion-exchange derivatives of hydrophilic polymer Spheron using a combination of isocratic and linear gradient elution.

High-performance anion-exchange chromatography of organic acids

Abstract The advantages of a partially quaternized diethylaminoethyl derivative of Spheron™/Separon HEMA™ in comparison with classical microporous polystyrene anion exchangers for the rapid anion-exchange chromatography of organic acids are demonstrated. An investigation of the effects of the composition and pH of the mobile phase, of the column temperature and of the flow-rate resulted in two optimized procedures. The first is suitable for the separation of some organic acids frequently occurring in agricultural and food samples of plant origin. These organic acids are eluted in about 20 min at 60°C using 0.6 M sodium sulphate as the eluent. The second method employing a more dilute mobile phase (0.1 M sodium sulphate) is proposed for the analysis of oligo(galacturonic acids) in pectin hydrolysates.

Chromatography of sugars on deae-spheron☆

Abstract Chromatography of borate complexes of sugars was carried out on diethylaminoethyl derivatives of glycol methacrylate macroporous gel, SpheronTM in borate form. The dependence of the separation of sugars on the exchanging capacity of the ion exchanger was tested. Sugar mixtures were best separated on an ion exchanger of maximum capacity 2.2 mequiv./g. The effect of the molarity of boric acid as well as the effects of temperature, flow-rate thourght the column, and boric acid gradient on the quality of the separation were investigated, and expressed by resolution and efficiency values (number of theoretical plates) and also by elution volume. For chromatography of the investigated mixture of sugars the following conditions were selected: buffer concentration 0.1 M, pH 8.5, temperature 50°, eluent flow, through a 500 x 6 mm I.D. column, 50 ml/h. The separation of a mixture of sugars on DEAE-Spheron under these conditions was compared with the separation on Aminex A-15. The separation efficiency for individual sugars was also compared on the basis of the number of theoretical plates achieved, and the values of un-reduced heights of the theoretical plates.

Serine-containing active center of alkaline proteinase of Aspergillus flavus☆

Abstract 1. 1. From the soluble portion of the tryptic digest of oxidized diisopropyl [ 32 P]-phosphoryl-derivative of alkaline proteinase of Aspergillus flavus a radioactive fragment was isolated and its amino acid composition determined to be Asp 7 , Met 1 , Thr 5 , Ser 8 , Glu 4 , Pro 3 , Gly 7 , Ala 9 , Val 6 , Ile 5 , Leu 6 , Tyr 2 and Phe 2 . 2. 2. Four radioactive peptides were isolated from a partial acid hydrolysate of the slightly soluble portion of the tryptic digest. These peptides provides evidence of the amino acid sequence Gly-Thr-Ser-Met-Ala around the reactive serine residue of alkaline proteinase. This amino acid sequence represents a part of the large tryptic fragment. 3. 3. Similarities in the amino acid sequences of serine-containing active centers of microbial proteinases are discussed.

Ion-exchange derivatives of spheron : IV. Phosphate derivatives

Abstract Medium acidic phosphate derivatives of the hydroxyethyl methacrylate macroporous gels Spheron 300 and 1000 were prepared by several procedures. The resulting cation exchangers had nominal capacities of 0.20—4.08 mequiv./g and were characterized by their capacity for small ions, static and dynamic capacities for proteins, elemental analysis, operating volume and inner surface area. Some samples were titrated potentiometrically to the first and second dissociation degrees and the results discussed in terms of the phosphorus content. The relationships between the nominal capacity and phosphorus content, between nominal capacity and static capacity for proteins and between the static and the dynamic capacity for proteins were investigated and discussed. Chromatographic experiments on a mixture of serum albumin, chymotrypsinogen and lysozyme showed a dependence of retention volumes on the nominal capacity. Applications to the separations of egg-white proteins and of cellulolytic enzymes from a cultivation liquid of Trichoderma viride-reesei are described. Experiments with the cation exchanger Spheron 300 phosphate, used as an “immobilized acid” for catalysis (esterification of alcohol and inversion of saccharose), are also reported.

Medium-pressure liquid chromatography of Leozym, a pectic enzyme preparation, on ion-exchange derivatives of spheron

Abstract Leozym, a commercial pectic enzyme preparation. was subjected to medium-pressure liquid chromatography on weakly, medium, and strongly acidic cation exchangers and on weakly and strongly basic anion exchangers, and to hydrophobic chromatography on the ionogenically unsubstituted glycomethacrylate macroreticular gel of the Spheron type. The course of the gradient chromatography was examined by contiouus measurement of the absorbance of the effluent at 285 and nm, by pH and conductivity measurements on the fractions collected and by specific measurements of selected enzyme activities. Fractions containing endo- D -galacturo-nanse, pectin-lyase and pectin-esterase activity were successfully resolved. The presence of multiple forms of endo- D -galacturonanase and pectin-lyase in the preparation examination was shown. The technological importance of these findings is briefly discussed.