The origin and consequences of concentration dependence in gel chromatography

Concentration dependence of elution volume was determined for Blue Dextran 2000, Dextran 500, Dextran sulphate 500 and bovine serum albumin on columns of Sephadex G-100 equilibrated with sodium phosphate buffer, I 0.1, pH6.8. From the results for Dextran 500, it was shown that a linear relation exists between elution volume and the corresponding osmotic pressure calculated for the same concentration and incorporating the term containing the second virial coefficient. This relationship was used to predict the concentration dependence of elution volume for bovine serum albumin and myoglobin, proteins that partially penetrate Sephadex G-100. Possible consequences of osmotic effects are considered in relation to various types of column experiments, including differential chromatography.     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.     

Purification to apparent homogeneity of inactive kallikrein from rat urine

Inactive kallikrein was purified from rat urine by a procedure including ammonium sulfate fractionation, DEAE cellulose chromatography, phenyl-Sepharose CL-4B chromatography, and gel filtration on Sephadex G-100 and Sephadex G-75 columns. The resulting preparation was essentially homogeneous, as assessed by polyacrylamide gel electrophoresis. This preparation migrated as a single protein band on a SDS-polyacrylamide gel and the molecular weight was 41000. The purified material underwent marked activation by trypsin, but not by deoxycholate, Triton X-100, SDS or acidification. These results indicate that the purified inactive kallikrein is the precursor rather than a complex with a substance binding to the active form of kallikrein.     

The osmotic behaviour of Sephadex and its effects on chromatography

1. Sephadex in bead form shows reversible changes of inner volume when immersed in solutions of a non-penetrating solute. These changes are in accordance with the theory of Flory (1953) for the swelling of gels. This makes possible the use of single beads for measuring the osmotic pressures of polymer solutions, up to or beyond 1kg./cm.(2). 2. Measurements of the inner volume of Sephadex G-200 by equilibrium dilution, at various concentrations of dextran 500, gave values in agreement with those obtained from the dimensions of single beads. 3. Measurements by chromatography of the inner volume of Sephadex G-200 gave values that differed slightly but significantly from those obtained by the other methods. Similar small disagreements were found between the dilution and chromatographic values for bovine serum albumin measured in the presence of various concentrations of dextran 500. The major factor that affects the dependence of the chromatographic elution volume (of dextran 500) on concentration is shown to be the change of the inner volume of the Sephadex, with dynamic factors playing a minor role. 4. Contrary to the findings of Ackers (1964), the equilibrium dilution and chromatographic methods gave closely agreeing values for the distribution coefficient, K(av.), of bovine serum albumin on Sephadex G-200; this cast doubt on his explanation of the nature of chromatographic separations on Sephadex.     

Purification and physical-chemical properties of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver

Acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver was purified by protamine sulfate precipitation, ion exchange chromatography on DEAE-A-25 Sephadex, gel filtration on Sephadex G-75, amethopterin-AH-Sepharose 4B affinity chromatography, and finally, gel filtration on Sephadex G-100. The enzyme preparation was homogeneous as judged by ultracentrifugation studies, SDS-polyacrylamide gel electrophoresis and gel filtration. The N-terminal amino acid was detected to be histidine and the complete amino acid composition is reported. The enzyme contains one disulfide bridge and two cysteine residues/mol monomer. The isoelectric point was estimated to be 4.8. The molecular weight was determined to be 32900 by high-speed sedimentation equilibrium analysis, 33000 by Sephadex G-100 gel filtration and 31600 by SDS-disc gel electrophoresis. The sedimentation coefficient from conventional sedimentation velocity runs was 3.1 S observed by ultraviolet optics. 'Active enzyme centrifugation' showed a sedimentation constant of 5.0 and 4.8 S for the purified enzyme and crude extract from pigeon liver, respectively, indicating that the enzyme forms a dimer under conditions of catalysis. It could be demonstrated that the inhibitor amethopterin was noncompetitive with respect to the acetyl donor and the acetyl acceptor. Acetyl-CoA:arylamine N-acetyltransferase was examined in different organs of pigeon. The enzyme was not inducible by 1,3-phenylenediamine and hexobarbital in vivo.     

Isolation and characteristics of a nerve growth factor from the venom of the Central Asiatic viper Echis multisquamatus

The nerve growth factor (NGF) was isolated from the Echis multisquamatus venom by ultrafiltration on PM-10 filter, chromatography on TSK-55 gel, ion-exchange chromatography on CM-cellulose and gel filtration on Sephadex G-75. The protein exhibited a marked nerve growth activity within the concentration range of 10-15 ng/ml in cultures of chicken embryo spinal ganglia. The molecular mass of NGF is equal to 33,000-37,000 Da according to Sephadex G-75 gel filtration data; however, according to SDS electrophoresis data its Mr is 13,000 Da. Isoelectrofocusing data suggest that the pI of the isolated factor lies in the region of 7.0-7.2; sugar content is 1-2%.     

Starch phosphorylase from tapioca leaves: Absence of pyridoxal phosphate

Starch phosphorylase from tapioca leaves has been purified to homogeneity, using the technique of ammonium sulfate fractionation, heat treatment, DEAE-cellulose chromatography, filtration through Sephadex G-100 and Sephadex G-200, and DEAE-Sephadex chromatography. The enzyme has a molecular weight of 450,000, as determined by gel filtration through Sephadex G-200 and contains 22 sulfhydryl groups per mole of the enzyme protein. Several types of evidence indicate the absence of pyridoxal 5′-phosphate as a prosthetic group of the enzyme. The kinetic data show a sequential type of the reaction mechanism. The enzyme activity is inhibited by tyrosine (K_i = 2.15 mm).     

Purification and Properties of a Dehyrodicaffeic Acid Dilactone-forming Enzyme from a Mushroom,Inonotus sp. K-1410

A dehydrodicaffeic acid dilactone-forming enzyme was purified from the mycelia of a mushroom, Inonotus sp. K-1410 by calcium acetate treatment, ammonium sulfate precipitation and column chromatography on Sephadex G-100, DEAE-Sephadex A-50 and caffeic acid-bound AH-Sepharose 4B. The enzyme was purified about 1200-fold from a crude extract and shown to be almost completely homogeneous by polyacrylamide gel electrophoresis. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G-100 to be approximately 39,000. The optimal pH for the enzymic conversion of caffeic acid to dehydrodicaffeic acid dilactone is around 6.0. The enzyme is stable up to 60°C and preincubation of the enzyme at 40°C for 10 min gives 1.5-fold activation compared with preincubation at 0°C. The optimal temperature for the enzyme reaction is 40°C.     

Purification and characterization of a collagenase from the mackerel,Scomber japonicus

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.     

Purification of the nitrogenase proteins from Clostridium pasteurianum

A method is described for the purification of the nitrogenase proteins from Clostridium pasteurianum by two polyethylene glycol precipitations and chromatography on columns of DEAE-cellulose, Sephadex G-100 and Sephadex G-200. The Mo-Fe protein and the Fe protein have been purified 70–80-fold from the crude extract, and they appear essentially pure when tested by anaerobic polyacrylamide gel electrophoresis.     

A thermophilic extracellular -amylase from Bacillus licheniformis

A strain of Bacillus licheniformis isolated from soil produced an extracellular α-amylase(s) with unusual characteristics. The enzyme was purified 126-fold by starch adsorption, DEAE-cellulose treatment, and CM-cellulose column chromatography. Four active protein bands were detected by disc electrophoresis in poly-acrylamide gel although the enzyme behaved as a single peak during both ultracen-trifugation and chromatography using CM-cellulose and Sephadex G-100. The enzyme showed a very broad pH-activity curve and had substantial activity in the alkaline range. The optimal temperature was 76 °C at pH 9.O. The enzyme was stable between pH 6 and 11 at 25 °C, and below 60 °C at pH 8.0. Using Sephadex G-100 gel filtration, a molecular weight of 22,500 was estimated for the enzyme. The action pattern on amylose and amylopectin is unique in that the predominant product during all stages of hydrolysis is maltopentaose.     

Purification and characterization of thiamine triphosphatase from bovine brain.

Soluble thiamine triphosphatase (EC 3.6.1.28) of bovine brain has been purified 68,000-fold to an electrophoretically homogeneous state with an overall recovery of 5.5% by hydrophobic chromatography on Toyopearl HW-60, Sephadex G-75 gel filtration, DEAE-Toyopearl 650M chromatography and Blue Sepharose CL-4B chromatography. The enzyme has an absolute specificity among thiamine and nucleoside phosphate esters for thiamine triphosphate and shows no nonspecific phosphatase activities. Thiamine triphosphatase is composed of a single polypeptide chain with molecular mass of 33,900 kDa as estimated by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 8.7 and is dependent on divalent metal ions. Mg2+ has been found to be the most effective among cations tested. A study of the reaction kinetics over a wide range of thiamine triphosphate concentrations has revealed a biphasic saturation curve being described by higher-degree rational polynomials.     

Production and characteristics of inhibitory factor of raw starch digestion from Aspergillus niger

Summary The glucoamylase preparation of Aspergillus niger 19 inhibited the raw starch digestion by it at high enzyme concentration. The inhibitory factor (IF) was isolated from the glucoamylase preparation by heat treatment and purified by DEAE-Sephadex A-25 column chromatography, an initial Sephadex G-50 gel filtration followed by SP-Sephadex C-25 column chromatography (twice) and then second Sephadex G-50 gel filtration. The IF thus purified was homogenous in polyacrylamide gel electrophories. The inhibitory activity of IF increased with the increasing IF concentration but decreased with an increasing quantity of raw starch or enzyme concentration. The IF had no effect on the hydrolysis of boiled soluble starch. It was completely adsorbed onto raw starch. The IF had a molecular weight of about 10,500. It was abundant in hydroxy amino acids such as threonine and serine. Xylose, mannose, glucose, galactose, and galacturonic acid were present in it.     

Cadmium-binding component in Escherichia coli during accommodation to low levels of this ion.

An inducible cadmium-binding protein was isolated from Escherichia coli cells accommodated to 3 X 10(-6) M Cd2+ but not from normal or unaccommodated cells. Sephadex G-100, metal chelate affinity chromatography, and disc gel electrophoresis were used in the purification procedure. The molecular weight of the Cd2+-binding protein was estimated to be about 39,000 by Sephadex G-100 chromatography, making it different from the conventional, much smaller metallothionein.     

Study of the translational diffusion of macromolecules in beads of gel chromatography by the FRAP method

We measured the translational diffusion of fractions of dextrans labelled with fluorescein isothiocyanate, in Sephadex gel beads permeated by aqueous solutions of these molecules. The molecular weights of these fractions were between 5400 and 200,000 and measurements of their diffusion coefficients inside a gel bead (D) and in the free solution (D0), were performed using the fluorescence recovery after photobleaching method (FRAP). We also determined the coefficient of partitioning (Kav) of these fractions between the gel and the free solvent, with a new microfluorimetric method. We found that, for Sephadex G-50, G-75, G-100, G-150 and G-200 gels, Kav varied with the Stokes radius (rs) of the dextran molecules, in agreement with the formula of Laurent and Killander (J. Chromatogr. 14 (1964) 317). For Sephadex G-100, G-150 and G-200 gels, D/D0 varied with rs, according to the theory of Ogston et al. (Proc. R. Soc. Lond. 333 (1973) 297). In addition, these theories predict a relation linking D/D0 to Kav which was well verified. Our work is the first systematic study of the translational diffusion of macromolecules in a chromatography gel. These measurements should allow a better evaluation of the factors which influence the resolution in exclusion chromatography. In addition, the diffusion of macromolecules in gels may provide models for the diffusion of these molecules in the cytoplasm of living cells and in connective biological tissues.     

Invertase inhibitor from potatoes: purification, characterization, and reactivity with plant invertases

Invertase inhibitor was extracted from potato tubers and purified nearly 1000-fold. The purification procedure involved precipitation at pH 4.0, fractionation with ammonium sulfate, adsorption on alumina Cγ gel, and gel filtration on Sephadex G-100 and DEAE-Sephadex A-50. The product obtained was homogeneous to electrophoresis on polyacrylamide gel. Exclusion chromatography on Sephadex G-100 indicated a molecular weight of about 17,000. The inhibitor did not inhibit yeast, Neurospora, and several plant invertases. It completely inhibited potato tuber invertase and a number of other plant invertases. Some plant invertases were partially inhibited.     

Purification and some physicochemical properties of staphylococcal enterotoxin D.

A method was developed for the isolation of staphylococcal enterotoxin D in highly purified form from cultures of Staphylococcus aureus strain 1151m. The method involves removal of the toxin from the culture supernatant fluid with the ion-exchange resin CG-50 followed by chromatography on carboxymethylcellulose (twice) and by gel filtration on Sephadex G-75 (twice). The purified toxin is homogeneous by polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and double gel diffusion tests. It is a simple, colorless, antigenic protein with an isoelectric point of 7.4 as determined by isoelectric focusing. Its molecular weight was determined to be 27 300 +/- 700 by molecular sieve chromatography on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its serological activity is stable over a wide range of pH values (1.2--10.7). The enterotoxin consists of 236 amino acid residues and contains no free sulfhydryl groups. End-group analysis showed serine to be the NH2-terminal amino acid and lysine to be the COOH-terminal amino acid.     

Interactions between Peroxidases, Polyphenoloxidases and Auxin-Oxidases

Root extracts, prepared from young Lens culinaris seedlings, are analysed after a gel chromatography on Sephadex G-25 and G-100. IAA-oxidase, peroxidase and polyphenoloxidase activities were found in the same fractions.     

De novo biosynthesis of FSH like peptide by the human prostate

Human benign prostatic hyperplasia tissue is able to synthesize immunoreactive FSH (IR FSH) in vitro. The prostatic FSH is similar to pituitary FSH as evident by co-elution on Sephadex G-100. High performance gel filtration chromatography and Western blot analysis. Immunocytochemical localization studies indicate positive staining in the cytoplasm of prostatic epithelial cells. There is a two fold increase in the concentration of immunoreactive FSH in benign hyperplastic tissue as compared to normal prostate.     

Polypeptide antibiotic 26a from Bacillus subtilis. II. Isolation and purification procedures.

Both the analytical and preparative methods by which the preparations of 26a bydrochloride salt with a high antibacterial activity and 20--30% recovery have been obtained from the fermentation fluids of Bacillus subtilis are presented. On an industrial scale the antibiotic can be yielded by absorption of bioactivity on Amberlite CG-50I column and precipitation with picric acid of crude substance from active elutes as adduct which was divided on equilibrated CM--cellulose and finally purified by gel filtration on Sephadex G-25 column. The purified preparation gave a single band by SDS-polyacrylamide gel electrophoresis and one ninhydrin-positive spot by thin-layer chromatography on silica gel G plates corresponding to single zones of bioactivity on bioautograms, and moved as a single peak of almost constant antibacterial activity on Sephadex G-75, G-100 and G-200 columns. The potency of the purest preparations, lot Sephadex G-25, was 6,500--7,000 arbitrary units/mg, and were characterized as follows: purification factor, 57; purity of 98--100% by densitometer scans of SDS-polyacrylamide gels; MIC for Sarcina lutea by twofold agar dilution assay, 0.306 microgram/ml.