Gel Bed Research Articles

Localization and partial characterization of soybean lectin-binding polysaccharide of Rhizobium japonicum.

Immunoelectron microscopy was combined with partial characterization of isolated exopolysaccharide to study binding of soybean lectin by Rhizobium japonicum strain USDA 138. Lectin-binding activity resided in two forms of exopolysaccharide produced during growth: an apparently very high-molecular-weight capsular form and a lower-molecular-weight diffusible form. At low-speed centrifugation, the capsular form cosedimented with cells to form a viscous, white, cell-gel complex which was not diffusible in 1% agar, and the diffusible form remained in the cell-free supernatant. Electron microscopic observation of the cell-gel complex after labeling with soybean lectin-ferritin conjugate revealed that capsular polysaccharides, frequently attached to one end of the cells, were receptors for lectin. The outer membrane of the cell bound no lectin. Various preparations of exopolysaccharide isolated from the culture supernatant were tested for lectin binding, interaction with homologous somatic antigen, and the presence of 2-keto-3-deoxyoctonate and were chromatographed in Sepharose 4B and 6B gel beds. Lectin binding was restricted to a polysaccharide component designated as lectin-binding polysaccharide. This polysaccharide, as present in the cell-free culture supernatant, was a diffusible acidic polysaccharide devoid of 2-keto-3-deoxyoctonate, with a molecular weight of 2 X 10(6) to 5 X 10(6). It was concluded that the soybean lectin-binding component of R. japonicum is an extracellular polysaccharide and not a lipopolysaccharide and that the diffusible lectin-binding polysaccharide probably differs from the very high-molecular-weight lectin-binding polysaccharide of the loose capsule (slime) only in the degree of polymerization.

Altered antigenic expressions of human C3 and C5, their subunits and fragments following exposure to denaturing conditions

The present study deals with the antigenic nature of the subunit polypeptide chains of C3 and C5. The purified proteins were reduced and dissociated in 0.01 M dithiothreitol and 6 M guanidine-HCl (GuHCl). Their subunits were separated by filtration over a Biogel A-15 beaded agarose gel bed (width × efficient length = 2.5 × 450 cm) equilibrated with the same reducing-dissociating agents. A comparative analysis using rabbit antisera raised against native C3 or C5 (anti-native C3-C5 antisera) or against the isolated subunits (anti-subunit antisera) gave the following general reaction pattern: anti-subunit antisera formed specific immune precipitates against isolated C3α, C3β and C5β [solubilized in GuHCl or sodium dodecyl sulfate (SDS)] but failed to recognize the native unreduced parent proteins; anti-native C3-C5 antisera showed a reversed reactivity with specificities restricted to the native parent proteins and with no reactivity against the isolated subunits; upon pretreatment with SDS, sodium desoxycholate (DOC) or glycine buffer of a pH of 2.8, unreduced C3 and C5 underwent profound antigenic alterations leading to the full reactivity with the homologous anti-subunit antisera and the concurrent partial loss of reactivity against anti-native C3-C5 antisera. Like the parent protein, various fragments generated from C3 by the ‘aging’ of whole serum at 37°C or by trypsin digestion proved to be susceptible to a similar antigenic transition in the presence of SDS. The subunit origin of these fragments could therefore be identified immunochemically with the aid of anti-C3α-β antisera. The observed immunochemical modification of C3-C5 was apparently effected by detergent binding. Full antigenic transition occurred when C3 was exposed to SDS to yield a maximal binding of approximately 1000 molecules of SDS per molecule of C3, but also under conditions yielding binding at only 5% of this level. The fact that DOC had the same effect as SDS on C3-C5 suggests that these complement proteins possess hydrophobic binding sites in their native state.

4] Purification of commercial NADH

This chapter describes the study demonstrating the purification of commercial NADH. For the purification of 5 g of commercial nicotinamide adenine dinucleotide (NAD)H (Arzneimittelwerk Dresden, G.D.R.) a 5×100 cm column is used. The separation is carried out at 4° in the dark. DEAE-Sephadex A-25, particle size 40-120/μm (Pharmacia, Uppsala, Sweden), is prepared, according to the manufacturer's instructions. It is transformed into the bicarbonate form, by washing, with a 10-fold volume of l M KHCO3, on a sintered-glass funnel layed out with nylon net. The gel is then rinsed, with twice-distilled water, till neutral. The neutral gel is suspended in 50 mM KHCO3 and fine particles are removed by repeated decantations. The prepared gel is poured into the column, with care, taken to avoid turbulences and inclusion of air bubbles. The column outlet is adjusted so that the hydrostatic pressure amounts to 150 cm of water. The gel bed is allowed to stabilize, by running 4 l of 50 mM KHCO3, through the column. The chromatography takes from 5 to 10 days. This chapter discusses the purification of NADH by thin-layer chromatography. Equipment for the removal of the KHCO3 from eluate is described in the chapter. Characterization of purified NADH is also done.

Preparative gel electrophoresis at high sample load. The effect of some experimental variable on separation performance.

The separation of model protein pairs (hemoglobin/albumin, trypsin/chymotrypsin, hemoglobin A/hemoglobin F) was studied in an apparatus for preparative gel electrophoresis at loads up to 40mg/cm2 of the cross-sectional area of the gel bed. Separation was favored by higher ionic strength and by longer migration path. Under the conditions used and within the load range studied, increasing total protein load had no adverse effect but increased voltage gradient, temperature, or gel strength were all unfavorable.

Isolation of a 70 000 molecular weight antigen of the Novikoff hepatoma.

Previous reports from this laboratory have indicated that a number of cytosol and nuclear proteins of Novikoff hepatoma cells were immunologically related [Yeoman, L. C., Jordan, J. J., Busch, R. K., Taylor, C. W., Savage, H., & Busch, H. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3258; Busch, R. K., & Busch, H. (1977) Tumori 63, 347]. In preparation for analysis of their structure and function, studies were undertaken to purify nuclear antigen 2 from the cytosol of Novikoff hepatoma cells in high yield and purity. It was shown on Ouchterlony gels that cytosol nuclear antigen 2 formed a single immunoprecipitin band of identity with one of the bands extracted from Novikoff nuclear chromatin. In this study, a 70 000 molecular weight antigen was isolated from the cytosol of Novikoff hepatoma cells by ammonium sulfate fractionation, ion-exchange chromatography, and isoelectric focusing in a granulated gel bed. This protein which focused at a pI of 6.3 was labeled with 125I-labeled Bolton-Hunter reagent and purified on an Ultrogel AcA-44 column. As shown by electrophoresis on NaDodSO4-polyacrylamide gels, the antigen in the excluded volume migrated as a single protein with a molecular weight of 70 000. The overall purification over the starting material was 2890-fold.

Preparative and analytical affinity chromatography of neurophysins on methionyl-tyrosyl-phenylalanyl-aminohexyl-agarose

Methionyl-tyrosyl-phenylalanyl-ω-aminohexyl-agarose was synthesized and shown to be suitable for both the affinity chromatographic purification of neurophysins and the measurement of the ligand binding parameters of these proteins by quantitative affinity chromatography. Bovine neurophysin I binds to the tripeptidyl matrix in 0.4 m ammonium acetate, pH 5.7, conditions under which no binding occurs with the parent ω-aminohexyl-agarose. Subsequent elution can be effected with 0.2 m acetic acid. The affinity matrices obtained have capacities for neurophysin of up to 4 mg/ml gel bed volume and therein provide for the convenient purification of the neurophysins by a two-step buffer-acid elution. [ Carbamoyl- 14C]neurophysin I also binds to the ligand-agarose matrix. Using this labeled protein, competitive elution analysis was performed by one-step elution of zones of protein with the binding buffer in the presence of varying amounts of soluble competitive ligand, lysine vasopressin. The characteristic decrease of elution volume of labeled protein with increasing soluble, competing ligand concentration indicates that the affinity matrix interacts biospecifically with neurophysin. This analysis allows the binding affinities for both soluble vasopressin and immobilized tripeptide ligand to be quantitated.

Isolation and partial characterization of magnesium ion-and calcium ion-dependent adenosine triphosphatase activity from bovine brain microsomal fraction

Microsomal fraction was prepared by ultracentrifugation of homogenates of cortical tissue from bovine brains. The preparation displayed ATPase (adenosine triphosphatase) activity in the presence of Mg(2+) (6.4mumol of P(i)/h per mg of protein) and Ca(2+) (3.4mumol of P(i)/h per mg of protein). Kinetic analysis of the activation of the enzyme preparation by Ca(2+) resulted in the demonstration of two apparent K(m) values for Ca(2+) (6.0x10(-8)m and 1.2x10(-6)m). Treatment of the microsomal membranes with Triton X-100 resulted in solubilization of the ATPase, though with some loss of activity. The solubilized microsomal proteins were incorporated into liposomes. By incubation of the liposomes in media containing (45)Ca(2+) an ATP-dependent uptake of Ca(2+) was demonstrated. The solubilized preparation was subjected to preparative isoelectric focusing in granulated gel beds. Two distinct peaks of Mg(2+)- and Ca(2+)-dependent ATPase activity were observed at pH4.8 (peak 4.8) and at pH6.3 (peak 6.3). The material isolated in peaks 4.8 and 6.3 was focused in polyacrylamide gel with pH gradients. The material corresponding to peak 4.8 consisted of a single protein, whereas peak 6.3 contained one major and at least one minor protein. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis confirmed these results and indicated that the major component of peak 4.8 and the protein of peak 6.3 both had a molecular weight of 105000. The material in peaks 4.8 and 6.3 was assayed for ATPase activity in the presence of various concentrations of Ca(2+). Kinetic analysis of the results for peak 4.8 demonstrated an apparent K(m) value for Ca(2+) of 4.1x10(-8)m. The enzyme isolated at pH6.3 had an apparent K(m) value of 3.8x10(-6)m. However, when the material from peak 4.8 was incubated in the presence of 1mm-Mg(2+) the ATPase could not be activated by Ca(2+).

Inclusion Chromatography of Amino Acids on beta‐Cyclodextrin‐Polymer Gel Beds

AbstractThe cyclodextrin‐polymer produced by cross‐linking β‐cyclodextrin in the form of regular beads proved to be well utilizable for fractionating mixtures of amino acids, especially for separating aromatic amino acids from non‐aromatic amino acids and from each other by inclusion chromatography. The relative elution volumes of natural amino acids and HETP (height equivalents of the theoretical plates) values determined on β‐cyclodextrinpolymer gel beds are given.

Vernetzte polymere mit spaltbaren netzbrücken, 4. Untersuchungen zur netzstruktur von copolymeren aus 1,2‐bis(4‐vinylphenyl)‐1,2‐äthandiol und styrol

AbstractCross‐linked copolymers from styrene and 1,2‐bis(4‐vinylphenyl)‐1,2‐ethanediol (1) were synthesized. The concentration of the pendent vinyl groups linked to the polymer was determined by means of IR‐spectroscopy as a function of the initial composition, of the total monomer concentration, and of the yield. The values were compared with previously elaborated theoretical calculations. Differences between experiment and theory are presumably due to an inhomogeneous distribution of the pendent vinyl groups in the polymerization system. Measurements of swelling with crosslinked copolymers from styrene and 1 showed, that the specific gel bed volume can be well correlated with the theoretically calculated concentration of cross‐links, if the total monomer concentration and the fraction of cross‐linking component are kept below certain maximum values and if the yield does not exceed 80% significantly.

Purification of human erythrocyte adenosine deaminase by affinity column chromatography.

Adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) has been purified 468,000-fold from pooled human erythrocytes. The procedure developed was used to isolate the enzyme from up to 23 liters of packed erythrocytes at one time. An easily prepared affinity column bed material employing adenosine as the ligand was used as the final step in the purification. During elution from the affinity column there was approximately a 3:1 partition of adenosine deaminase between gel bed and column buffer. There was no apparent difference in the partitioning of unresolved or partially resolved preparations of the electrophoretically different forms of the enzyme on the affinity column. Gel filtration and electrophoresis on polyacrylamide gels of increasing concentration revealed no differences in the Mr of these electrophoretically different forms. The four bands resolved by electrophoresis of the different forms on polyacrylamide gels under nondenaturing conditions yielded a single band when electrophoresis was carried out in the presence of sodium dodecyl sulfate and 2-mercaptoethanol. Partially resolved preparations of the different electrophoretic forms of adenosine deaminase also gave rise to a single band of the same mobility when electrophoresed on polyacrylamide gels under these conditions. The band had the mobility of a protein of Mr of 36,000. This Mr is approximately the same as estimated for the nondenatured enzyme.

Scaling-up of the procedure for the purification of human urinary kallikrein urinary kallikrein

SCALING-UP OF THE PROCEDURE FOR THE PURIFICATION OF HUMAN URINARY KALLIKREIN URINARY KALLIKREIN AMINTAS F. S. FIGUEIREDO and MARCOS MARES-GUIA Department of Biochemistry, Faculty of Medicine, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil The purification of human urinary kallikrein described by Hial et al. (Biochemistry 13, 4311, 1974) is being scaled up for the preparation of gramquantities of enzyme. In a first approach, 125 liters of male human urine were filtered, diluted to 250 1 with deionized water, adjusted to pH 7.0, and passed through a column of DEAE-Sephadex (13 x 48cm) with 4.84 1 of gel bed. The kallikrein was adsorbed onto the gel in this process. The column was washed with 200 1 ammonium acetate 0.10M, pH 7.0 at a flow rate of 0.48 1/hr, then with 4.5 1 of the same buffer containing 0.20M NaCI. The active fractions had kinin-liberating activity over human kininogen, hydrolized carbobenzoxy-L-tyrosine p-nitrophenyl ester and had practically no hydrolytic activity toward tosyI-L-arginine methyl ester, as previously reported (Diniz et al., Biochem. Biophys. Res. Commun. 21,448, 1965). The pooled fractions were filtered through Sephadex G-25 and lyophilyzed. The active material obtained was then twice chromatographed on a 4.6 x 120 cm column of Sephadex G-150, whereby a large amount of pigment was removed. The final lyophilyzed product was homogeneous upon acrylamide electrophoresis (7.5%) but was microheterogeneous on electrofocusing, as reported by Hial et al. (Ioc. cit.). The initial 125 1 of human urine yielded 1.58g of a lyophilyzed powder containing 0,51 g protein, with a specific activity similar to that previously reported. The process is now being dimensioned for the desalting of 1250 1 of human urine, with the use of Sephadex columns of the KS-370 type. (This work is being supported by BNDE-FUNTEC/199 and by the University Research Council). INHIBITORS OF KININ-FORMING ENZYMES SETSURO FUJI1, .M.D. and PH.D Department of Enzyme Physiology, Institute for Enzyme Research, School of Medicine, Tokushima University Tokushima, Japan. The inhibitory effect of esters of aminocaproic acid, guanidinocaproic acid and trans-4-aminomethylcyclohexane carboxylic acid, and other inhibitors such as A Synops i s o f K i n i n '75 A b s t r a c t s 425 trasylol and leupeptin on trypsin-like proteolyt ic enzymes such as kallikrein, CIr, tissue activator, pancreatin, plasmin and thrombin were studied. The derivatives of phenyl ester of these acids showed potent inhibitory effect on the trypsin-l ike enzymes. The effect of these esters on inflammation induced by croton oil and on experimental pancreatitis in animals will be reported. CI esterase activity and the conversion of CI to CI esterase were competit ively i n h i b i t e d by aromatic esters of guanidinocaproic acid. However, p-aminophenylphenylpropionate, chymotrypsin inhibitor, had no effect on CI esterase activity. Such synthetic inhibitors as described above reversibly inhibited trypsin, while derivatives of p-guanidinobenzoic acid inhibited trypsin irreversibly (E. Shaw; 1969). THE ACTION OF U.V. RAYS ON EXPERIMENTAL CUTANEOUS INFLAMMATION INDUCED BY CANTHARIDIN R. GALLETTI, L. MATASSI, P CHIARINL and F. CORTI Department of Medicine, University of Florence, Florence, Italy In previous research, we observed that histamine, administered intravenously in microdoses (40/~g) in man, activates a self-extinguishing localized cutaneous inflammation. This experimental inflammation in man is represented by a cantharidin blister on the lateral surface of the forearm. Histamine intravenous administration is fol lowed by: 1), an increase in the turgescence of the blister; 2), a sensation of burning pain; 3), an extension of the primary hyperalgesia (erythralgia) and of the secondary hyperalgesia; and 4), an increase of kininogen in the blister fluid. We suggest that histamine acts specifically on the altered microvessels of the local inflammation, thus inducing the passage of the kininogen from the vascular to the interstitial space. Here, there are conditions apt to activate the polypeptide. We carried out another experiment with the same biological meaning. In these subjects, instead of injecting histamine intravenously, we have induced an erythema with U.V. rays (2900-3200 A ~ on a cutaneous area (dorsal section of the thorax) far from the experimental blister. After a mean of 8-10 hours from U.V. irradiation, the above-mentioned signs of reactivation of the cantharidin blister appeared, including the burning pain. Since many active substances of the inflammation are released after U.V. erythema, and among them histamine, we advance the hypothesis that the blister reactivation is mainly due to histamine. This experiment represents a model of a non-specific reactivation of the experimental inflammation in man.

Gel chromatography of glucose oligomers : A study of operational parameters

The effects of solute concentration, column length, flow-rate and temperature upon the elution behavior of glucose oligomers are discussed. A linear relationship between peak height and concentration was established for polymers up to maltoheptaose. Resolution increased considerably by increasing column length. Elution behavior of the homologous series of glucose oligomers was flow and temperature dependent. Gel bed parameters and theoretical data as influenced by operational parameters are presented.

Molecular weight of human factor VIII procoagulant activity

Low molecular weight factor VIII procoagulant activity has been prepared by agarose gel chromatography of highly purified human factor VIII using 0.24 M CaCl 2 buffer. The sedimentation properties of this procoagulant activity in sucrose density gradient centrifugation studies carried out at physiologic ionic strength were consistent with a 6.7S protein. These experiments demonstrate that the elution pattern of factor VIII activity in agarose gel chromatography with high ionic strength buffers is due to separation of low molecular weight material — rather than artifactual retention of large molecules within the gel bed — and that the low molecular weight portion of the large factor VIII complex is sufficient for procoagulant function.

Binding of purine nucleotides to ammonium formate during gel filtration on Sephadex G-10

Gel filtration of a mixture of ATP and ammonium formate on Sephadex G-10 gives rise to two peaks of ATP. The first peak is eluted near the void volume, while the second peak is eluted together with the ammonium formate. More than 50% of the ATP can be present in the second peak, depending on the concentration of ammonium formate and the degree of compression of the gel bed. ATP appears to partition into the salt peak via hydrogen bonding to ammonium formate. Similar results are obtained with GTP but not with ITP.

ZONE SPREADING IN GEL CHROMATOGRAPHY

Axial dispersion in interparticle spaces and intra-gel diffusion are the two unknown factors to control the spreading of a pulse introduced in a dextran gel bed. Macromolecule (blue dextran) which is excluded by gel particle and small molecule (sodium chloride) are used together to separate these two contributions. Axial dispersion is of unusual significance, and intraparticle diffusivities of sodium chloride in Sephadex G-25 and G-50 are 1.55 × 10-6 and 1.5 × 10-5 cm2/sec at room temperature.

The separation of epinephrine from norepinephrine and dopamine from DOPA on Sephadex G-10.

Epinephrine and norepinephrine were separated by acid elution through a Sephadex G-10 column with high recovery (better than 90%), excellent reproducibility, and little overlap (less than 10%). Once packed, the columns could be re-used indefinitely. Total elution time was about six hours and the columns could be left untended since the gravity flow stops automatically once the level of the eluant reaches the gel bed. The resulting dilution was five-fold. 3,4-Dihydroxyphenylalanine (DOPA) was completely separated from 3,4-dihydroxyphenylethylamine (dopamine) by the same procedure.

Purification of methionyl-tRNA synthetase from Escherichia coli K12 by affinity chromatography.

An affinity column for the purification of methionyl‐tRNA synthetase has been prepared. Cyanogen‐bromide‐activated Sepharose 4B was reacted successively with hexamethylene‐diamine and N‐nitrophenylsulphenylmethionine succinimide ester. The nitrophenylsulphenyl protecting group of the polymer was removed under mild conditions with aqueous sodium thiosulfate. The affinity columns were equilibrated in 200 mM potassium phosphate buffer; this ionic strength proved to be sufficiently high to avoid the retention of methionyl‐tRNA synthetase by ion exchange.On columns with high methionine concentration (8–20 μmol/ml gel bed), the enzyme was strongly retained but could be selectively eluted by addition of 20 mM methionine to the buffer. However this resulted in excessive dilution of the enzyme. Highly purified enzyme could be obtained in high yield by lowering the methionine substitution of the polymer to 4.5 μmol/ml gel and by introducing the affinity chromatography after the first DEAE‐Sephadex step of the conventional purification procedure. Under these conditions no retention but sufficient retardation of the enzyme on the column was observed.The possibility of generalizing the method for the purification of other proteins recognizing an amino acid with its free amino group is discussed.

Präparative trennung von proteingemischen durch sephadexgelfiltration bei gleichzeitiger einwirkung von gleichstrom (retardationsfiltration)

Preparative separation of protein mixtures Sephadex gel filtration with simultaneous application of continuous current (electroretardation-filtration) A method is described in which the proteins of human serum are, in one step, separated into thirteen fractions. By this technique, bovine haemoglobin is separated into two different fractions. In this procedure a column especially built for this purpose, which contains coarse Sephadex, is utilised. The principles underlying the function of this column are described in detail. The additional fractions result from coincidental action of continuous current. Because the normal filtration mobility of the protein molecules is greater than the electrophoretic mobility, the electrode of the upper-side must be connected so as to serve as the anode, so that at alkaline pH the forces of separation work in opposite directions. In this way, different degrees of retardation for most protein molecules result from their dependence upon electrical potential. We shall call this method of separation “electroretardation-filtration”. To obtain optimal separation of the protein mixture, it is necessary to work at low V e /h, though it is possible that some proteins may ascend into the anode buffer. In order to examine the direction of mobility we have calculated a quotient (E/W-quotient) from V e /h (ml) and the power (Watts) of the gel bed. Empirical observation indicates that an E/W-quotient of more than 0.15 guarantees the elution of most serum proteins. Using monospecific antiserums, several peaks are identified. Upon immunoelectrophoretic analysis, fractions which contain albumin give an atypical pattern consisting of two or possible three components. IgG is found in two different, remotely separated peaks.

Thermodynamic treatment of partition experiments with special reference to molecular-sieve chromatography

Using thermodynamics an equation (eqn. 8) has been derived for the relationship between distribution coefficients and the parameters characterizing the solute ( e.g. partial molal volume and molecular area) and the gel bed ( e.g. pressure and interfacial tension). When active transport can be neglected the equation might also give a qualitative picture of the factors that determine the distribution of solutes between the internal space of a living cell and its surroundings. Introduction of a series of assumptions, which are all discussed, leads to simplified formulae (eqns. 14 and 15), which, in spite of their approximate nature, seem to be applicable in most experiments. The considerations herein also apply to regular partition expeiments, including A lbertsson's aqueous two-phase systems, recalling that no pressure difference exists between two liquid phases, as between the interior of a gel and the surrounding medium.

Tissue Specific Esterase Expression In Indigenous Chicken Of Bangladesh

The present investigation was done in the esterase gene expression analysis of the Red Jungle fowl Gallus gallus of Bangladesh utilizing the polyacrylamide gel electrophoresis (PAGE) technique. Each allele produces a biochemical message that helps to produce various protein phenotypes those were expressed in the form of bands on the polyacrylamide gel bed. Altogether four different esterase bands were recorded in both Red Jungle (RJ) fowl and Naked Neck (NN) fowl of Bangladesh. These were denoted as Est-1 (2), Est-2(1), Est-3 (21) and Est-4 (13) based on the relative mobility on the gel. Among them Est-2 is very common in almost all tissues. Comparing the esterase in both genetic groups, their expression intensity even the relative mobility provided a unique feature that they were similar. The investigation indicates that esterase gene between the Red Jungle fowl and Naked Neck fowl of Bangladesh has a close relationship. DOI: http://dx.doi.org/10.3329/bjas.v38i1-2.9908 BJAS 2009; 38(1-2): 15-21