Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing.

A method for preparative isoelectric focusing of 0.1-10 g amounts of proteins is described. For anticonvective stabilization of the pH gradient, layers of granulated gels (E.G. Sephadex or Bio-Gel) of variable length, width and thickness were used either on glass plates or in troughs. Load capacity, defined as the amount of protein per ml gel suspension, was determined to be 5-10 mg per ml for total protein, irrespective of the pH range of the carrier ampholytes. For single proteins load capacities of 0.25-1 mg per ml were found for pH 3-10 carrier ampholytes, and 2-4 mg per ml for narrow pH range ampholytes. Experiments on a quartz plate followed by densitometric evaluation in situ at 280 nm have demonstrated that it is possible to proceed from analytical thin-layer isoelectric focusing to preparative separations without loss of resolution, just by changing the dimension of the gel layer and increasing the protein load. Improved resolution which facilitates isolation of isoelectrically homegenious components could be achieved on a 40 cm long separation distance. The geometry of a layer is favourable to heat dissipation and this permits the use of high voltage gradients. Recovery of the focused proteins is high an elution simple. The efficiency of the method is illustrated by examples showing separations of single proteins and protein mixtures.     

Isolation of aldehyde oxidase (EC 1.2.3.1) from potato extracts by preparative page

A method is proposed for the isolation (and purification) of enzymes, with retention of their activity, from solutions or gels of preparative PAGE runs. It is based on the inclusion of Sephadex G-25 as a supporting medium for a collector buffer in otherwise normal disc-PAGE gels. The collector buffer has a lower pH and higher concentration than the stacking gel buffer. This makes the proteins concentrate in a very narrow, slowly moving band in the Sephadex on electrophoresis, and makes their recovery easy. The method is illustrated by the isolation of aldehyde oxidase from potato extracts (which was unsuccessful by classical methods), and of one isoenzyme from commercial lipoxygenase after preparative PAGE. Recovery of chicken egg albumin after PAGE was over 90%.     

Immunochemical study of the antigenic structure of bacteria of genus Bordetella. IV. Fractionation of B. pertussis extracts and study of the immunochemical and biological properties of the isolated fractions]

Fractionation of an extract of pertussis microbes was carried out with the aid of gel-filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose, and preparative electrophoresis. Fractions differing in serological, immunogenic activity and the content of antigenic components were isolated. In using the method of gel-filtration of sefadex G-100 the greatest serological, immunogenic and histamine-sensitizing activity was possessed by the high-molecular fraction containing 8 of 11 antigenic components detected in the initial extract. The antigenic components were distributed into 5 fractions by the ion exchange chromatography on DEAE-cellulose. The greatest serological activity was possessed by fractions exiting from the column at the 0.01--0.04M interval of the phosphate buffer concentration. A method of preparative electrophoresis from the pertussis microbes extract was applied and two fractions were isolated from the anode and the cathode zones, each containing 4 antigenic components only, but possessing serological and immunogenic activity and having no histamine-sensitizing properties.     

Isolation of all proteins from the 50S subparticles of ribosomes of Escherichia coli

The paper proposes a method of preparative isolation of all proteins from the 50S subparticle of E. coli ribosomes. The method is based on (1) preliminary fractionation into protein groups and ribonucleoprotein particles by a consecutive treatment of the 50S particles with increasing LiCl concentrations, and (2) chromatographic separation of protein groups on DE- and CM-cellulose and gel-filtration of separate fractions. The method allows to obtain any protein required for studies in preparative amounts avoiding many chromatographic stages. A detailed scheme of isolation of all proteins is given together with quantitative data of yields of individual proteins calculated per 6 g of the 50S subparticles.     

Characterization of the apolipoproteins of rat plasma lipoproteins.

Purified fractions of three major rat high-density lipoproteins (HDL) and one rat very low-density lipoprotein (VLDL) were isolated by Sephadex gel chromatography or preparative sodium dodecyl sulfate gel electrophoresis. These proteins were characterized by amino acid analysis, end-group analysis, molecular-weight determination, polyacrylamide gel electrophoresis, and circular dichroism. One of these rat proteins, of molecular weight 27 000, appears to be homologous with the human A-I protein. However, rat HDL possesses two additional major components not reported in human HDL - an arginine-rich protein of molecular weight 35 000 and a protein of molecular weight 46 000. The arginine-rich protein of the rat is similar in size and amino acid analysis to the arginine-rich protein reported in human VLDL. A major component of rat VLDL of 35 000 molecular weight appears similar or identical to the arginine-rich protein in rat HDL by every criterion employed for their characterization.     

Gradiflow as a prefractionation tool for two-dimensional electrophoresis

The Gradiflow trade mark, a preparative electrophoresis instrument capable of separating proteins on the basis of their size or charge, was used to separate whole cell lysates, prepared from bakers yeast (Saccharomyces cerevisiae) and Chinese snow pea seeds (Pisum sativum macrocarpon), into protein fractions of different pH regions. Both broad and narrow range (with a difference of approximately 1 pH unit) pH fractions were obtained. Analysis of the protein fractions by isoelectric focusing gels and two-dimensional (2-D) polyacrylamide gel electrophoresis indicated minimal overlap between the pH fractions. Further, when the prefractionated acidic samples were analyzed on pH 4-7 immobilized pH gradient 2-D gels, improved resolution of the proteins within the chosen pH region was achieved compared to the unfractionated samples. This study demonstrates that the Gradiflow could be used as a preparative electrophoresis tool for the isolation of proteins into distinct pH fractions.     

beta-Glucosidase of Penicillium funiculosum. I. Purification

A strain of Penicillium funiculosum, isolated in this laboratory, produced in high yield both endo- and exo-glucanases and beta-glucosidases, which were suitable for the saccharification of cellulosic materials. The isolation of the beta-glucosidase of this organism, which differs from other beta-glucosidases of fungi in its substrate specificity, by preparative electrophoresis, is described in this article. The organism was grown on a lactose-casein medium and the culture filtrate concentrated by ammonium sulfate precipitation and dialysis. Electrophoresis was carried out on large slabs of polyacrylamide gel in an anodicrun in the presence of borate at pH 7. After elution of active fractions, a cathodic run was made at pH 6.0. Two precipitations with ammonium sulfate resulted in a homogeneous enzyme (specific activity 174 IU/mg). A second isozyme was also produced by P. funiculosum on cellulose-wheat bran medium. This isozyme was purified by electrophoresis at pH 7.0 in the absence of borate and was obtained free from other isozymes of beta-glucosidase and cellulases.     

Purification and characterization of ethylene inducing proteins from cellulysin

Ethylene inducing proteins were partially purified and characterized from the cell wall digesting enzyme mixture, Cellulysin. Purification included binding to Sephacryl S-200, isoelectric focusing, molecular sieving on Sephadex G-75, agarose electrophoresis, and sizing using a Superose 12 column. At least three active proteins were obtained from the Sephadex G-75 fraction that move towards the cathode during nondenaturing agarose electrophoresis. These three protein fractions separated by preparative agarose electrophoresis contain polypeptide patterns that are very similar on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions contain three main Coomassie blue stained bands of about 10, 14, and 18 kilodaltons. Gel filtration of the major fraction on a Superose 12 column yields an active peak with an apparent molecular weight of 27,000. Proteolytic enzymes, in the presence of urea, destroy the ethylene inducing activity. We conclude that the ethylene inducing factor (EIF) that we have isolated from Cellulysin is protein. Similar ethylene inducing factors are present in Cellulase RS. Ethylene inducing components from pectinase, Pectolyase, and Rhozyme do not bind to Sephacryl like EIF from Cellulysin. Thus, the components responsible for the ethylene inducing activity in these latter enzyme preparations differ from that of EIF.     

The application of acetylated Sephadex for the separation of proteolipids.

The finding that acetylated Sephadex G 200 swells in chloroform/methanol mixture and still acts as a molecular sieve has permitted for the first time the separation of several proteolipid fractions from brain white matter, two of which appear almost free of phospholipids. This new procedure will be useful not only for a rapid purification of proteolipids and separation of lipids but also for general purposes of protein chemistry in organic media. However, it is predictable that one of the main uses of the method will be in the isolation of membrane carrier and of cholinergic receptor proteins, since these components appear to be of proteolipid(-like) nature.     

Preparation and some properties of strongly acidic proteins from calf-thymus nucleohistone.

The isolation of acidic proteins from calf-thymus nucleohistone (starting from purified nuclei) is reported. The method involved dissociation in 1 M KCl solution. Denaturating agents were not used at all. After electrophoresis on polyacrylamide gel, fractions containing a small number of components were obtained. The fractions display high ratios of acidic to basic amino acids, the ratios ranging from 4.0 to 1.5. In all fractions, the major components were of molecular weights in the ranges 12000-15000 and 24000-28000 as determined by gel-disc electrophoresis in dodecylsulphate and by equilibrium ultracentrifugation. Minor components of high molecular weights were also present. Amino-acid analyses are also reported. The tryptophan content was determined by a fluorometric method. Circular dichroism spectra depict a very low content of alpha-helicity that did not increase at higher ionic strength. A marked RNA-polymerase activity was found in one fraction.     

Analysis of glycated hemoglobin A1c by capillary electrophoresis and capillary isoelectric focusing

Two capillary electrophoretic methods were developed and evaluated for measurement of glycated hemoglobin A1c (HbA1c). First, a capillary electrophoresis analysis is performed with a sodium tetraborate buffer (pH 9.3) as background electrolyte in a neutrally coated capillary. HbA1c is separated from HbA0 due to specific interactions of borate anions with the cis–diol pattern in the saccharide moiety of glycohemoglobin. Second, a capillary isoelectric focusing method, which exploits a difference in pI values of HbA0 and HbA1c, is performed with Servalyt pH 6–8 or alternatively with Biolyte pH 6–8 carrier ampholytes spiked with a narrow pH cut of pH 7.2 prepared by preparative fractionation of Servalyt pH 4–9 carrier ampholytes. Both methods reflect recent developments in the methodology of capillary electrophoresis. They allow quantifying HbA1c in generic capillary electrophoresis analyzer with specificity that is consistent with previously reported electrophoretic assays in slab gels and capillaries.     

Purification of a High Molecular Weight Kininogen from Rat Plasma

A high molecular weight kininogen has been isolated from rat plasma and purified. At each preparative step the kininogen concentration and purity were monitored by assay on the perfused isolated rat uterus in terms of bradykinin equivalents formed per mg protein following incubation of the plasma fractions with rodent acid protease for 24 hours at 37 and pH 4.0. Kinin formation by crystalline trypsin and human pancreatic kallikrein also was compared. Citrated rat plasma first was precipitated with 43% ammonium sulfate. The kininogen fractions then were subjected to a series of gel filtration ion exchange chromatographic columns that included G-200 Sephadex, G-200: G-100 Sephadex interconnected columns, DEAE-A50 Sephadex, and hydroxylapatite. The kininogen fractions finally were subjected to preparative polyacrylamide gel electrophoresis, resulting in a final purification of 92.9-fold compared to the initial rat plasma. A single major kininogen protein band and a minor band of protein impurity were obtained on disc gel electrophoresis. Only the pancreatic kallikrein did not form kinin from this purified kininogen. The apparent molecular weight was estimated by SDS polyacrylamide gel technique to be 110,000.     

Simple method for isolation of 4-deoxynivalenol from rice inoculated with Fusarium graminearum.

A new method for preparative isolation of 4-deoxynivalenol (DON) is presented. This method avoids the loss of material during purification on silica gel by column chromatography. DON and 3-acetyldeoxynivalenol in crude extracts of rice inoculated with Fusarium graminearum were converted to triacetyldeoxynivalenol; the acetylated product was easier to purify by silica gel chromatography than DON is. After hydrolysis and further purification on a charcoal-alumina column, the 71% pure DON was recovered in yields as high as 450 mg of DON per kg of rice. Subsequent separation on a Sephadex LH20 column yielded DON that was greater than 90% pure.     

Chemical characterization and study of the autodigestion of pure collagenase from Achromobacter iophagus.

Only one collagenase (EC 3.4.24.3) is produced by the non-pathogenic Achromobacter iophagus strain. The chromatography of the crude enzyme on DE-32 cellulose followed by gel filtration on Sephadex G-100 in the presence of 1 M sodium chloride led to the isolation of a homogeneous enzyme. Its specific activity (1.642 mukat/mg) represents the highest value ever obtained for a bacterial collagenase. The amino acid composition of A. iophagus collagenase differs from that of Clostridium histolyticum mainly in the sulfur-containing amino acids. 1 mol of zinc was found for 1 mol of enzyme of molecular weight 104 000. The autodegradation of the A. iophagus collagenase results in the formation of at least three active fractions which can be separated by preparative polyacrylamide gel electrophoresis as well as rechromatography on DE-32 cellulose. They are active towards the synthetic substrate as well as towards the native collagen. The results of ORD have shown that the digestion of the last one occurs in the helical parts of the substrate.     

Isolation and Characterization of Neutral Peptides in Soy Sauce

A soy sauce sample was fractionated by gel filtration on a Sephadex G–15 column, then the fractions were subfractionated on the basis of acidity by ion exchange chromatography on a QAE-Sephadex A–25 column. After preliminary fractionation, the components in the neutral subfractions were transformed to copper salts, and the salts were chromatographed on a DEAE-Sephadex A–25 column with a borate buffer and aqueous acetic acid to separate neutral peptide substances from a large amount of free amino acids. The peptide fractions were further fractionated on a preparative amino acid analyzer and by paper chromatography to separate the peptide substances.

Three glycodipeptides and eight dipeptides were isolated and characterized as the major neutral peptide components in soy sauce. However, it was difficult to anticipate any direct contribution of these peptides to the flavor construction in soy sauce on the basis of their taste intensities and contents.     

Identification of protective pneumococcal t(h)17 antigens from the soluble fraction of a killed whole cell vaccine

Mucosal or parenteral immunization with a killed unencapsulated pneumococcal whole cell antigen (WCA) with an adjuvant protects mice from colonization by a T(H)17 CD4+ cell-mediated mechanism. Using preparative SDS gels, we separated the soluble proteins that compose the WCA in order to identify fractions that were immunogenic and protective. We screened these fractions for their ability to stimulate IL-17A secretion from splenocytes obtained from mice immunized with WCA and adjuvant. We identified 12 proteins within the stimulatory fractions by mass spectrometry; these proteins were then cloned, recombinantly expressed and purified using an Escherichia coli expression system. The ability of these proteins to induce IL-17A secretion was then evaluated by stimulation of mouse splenocytes. Of the four most stimulatory proteins, three were protective in a mouse pneumococcal serotype 6B colonization model. This work thus describes a method for identifying immunogenic proteins from the soluble fraction of pneumococcus and shows that several of the proteins identified protect mice from colonization when used as mucosal vaccines. We propose that, by providing protection against pneumococcal colonization, one or more of these proteins may serve as components of a multivalent pneumococcal vaccine.     

The preparative isolation of endosome fractions: a review

The endosome is an intracellular, acidic, membrane-bound, subcellular compartment to which endocytosed ligands, receptors and plasma membrane proteins are conveyed before sorting and delivery to destinations elsewhere in the cell. The preparative isolation of elements of this compartment has been achieved successfully using various appropriate combinations of density gradient ultracentrifugation, electrophoretic, gel filtration and immunoaffinity techniques. These methods for isolating endosome fractions are reviewed together with the difficulties of establishing markers for such fractions. The isolation of an endosome fraction from the pathway of polymeric IgA transcytosis in rat liver is discussed to exemplify successful isolation procedures and appropriate subcellular markers.     

Simple radioassay for uridine phosphorylase and 5'-nucleotidase

A method is described whereby a large number of chromatographic fractions containing protein or enzyme components may be run on a single-starch gel. A multiple punch for making the sample holes, gel slab support, and a gel slicer used to cut several slices from the one gel for staining for the various enzyme or protien constituents are described. An example is given of a run using fractions obtained from a Sephadex G-200 column chromatography run of a herring muscle extract. Densitometry may be used to assay the stained gels. In another example a commercially obtained muscle lactate dehydrogenase preparation was chromatographed on a Sephadex G-200 column. The electrophoretic run of the fractions revealed the presence of both heart-type A and muscle-type B lactate dehydrogenase subunits and showed that the column run had effected a partial separation of the various tetramers.     

Preparative separation of the saponin lancemaside a from Codonopsis lanceolata by centrifugal partition chromatography

Introduction. Lancemaside A is a saponin that inhibits decreases in blood testosterone level and thus prevents or ameliorates symptoms associated with male climacteric disorder. Our initial attempt to preparative isolation of lancemaside A from the saponin fraction of Codonopsis lanceolata roots by a preparative HPLC did not give a clear result. Objective. To develop a simple and efficient method for the preparative isolation of lancemaside A from the hot water extract of C. lanceolata roots using centrifugal partition chromatography (CPC). Methodology. The saponin fraction obtained from the hot water extract of C. lanceolata roots was used as the sample for preparative‐scale separation of lancemasides by CPC using n‐hexane:n‐butanol:methanol:0.1% aqueous formic acid (3:4:1:6, v/v) as the two‐phase solvent system. The upper phase (organic phase) of the two‐phase solvent system was used as the mobile phase, and 0.5 g of saponin fraction was applied for separation by CPC. Each fraction that was separated by CPC was analysed by HPLC, and the fractions containing each of the separated compounds were pooled together, and then were purified by simple preparative HPLC. Results. The demonstrated separation sequence, hot water extraction, DIAION HP‐20 column chromatography, CPC and preparative HPLC, yielded lancemaside A, foetidissimoside A and astersaponin Hb in their pure forms. Conclusion. The simple and efficient method for the preparative isolation of lancemaside A along with two other saponins, foetidissimoside A and astersaponin Hb, from the saponin fraction of C. lanceolata was established using CPC.     

Studies on phytohemagglutinins: XXII. Isolation and characterization of a lymphocyte receptor for concanavalin A

A receptor glycopeptide for concanavalin A was isolated from calf thymocytes by a method originally devised for the isolation of a lectin receptor from human erythrocytes (Kubánek, J., Entlicher, G.; and Kocourek, J. [1973] Biochim, Biophys. Acta 304, 93–102). The method consisted of pronase digestion of the lipid-depleted thymocyte membrane material followed by ethanol fractionation, separation on Sephadex and preparative paper electrophoresis. The isolated glycopeptide contains 10.4% of neutral sugar. The molar ratios of the sugar components mannose, galactose, glucosamine, glucose, fucose and sialic acid are 3 : 2 : 2 : 1 : 1 : 1. The minimum molecular weight calculated from the sugar composition is about 12 000.Concanavalin A receptor activity of the glycopeptide was demonstrated in three different ways: (i) Reduction of the ¹²⁵I-labeled concanavalin A binding to thymocytes. (ii) Prevention of concanavalin A induced agglutination of calf thymocytes. (iii) Inhibition of concanavalin A stimulated DNA synthesis in calf and rabbit thymocytes and rabbit lymph node lymphocytes cultivated in vitro.The isolated glycopeptide seems to be involved in the interaction of lymphocytes with concanavalin A and the subsequent stimulation.