Gel Filtration on Sephadex Columns

In order to determine the approximate sequence and range of molecular sizes of the macromolecular materials contained in native gastric juice, as well as to dissociate some naturally forming large molecular 

Kakei and Kubo (G13, G14) in our laboratory further demonstrated that vitamin Bi2 binders from the gastric juice were eluted into the excluded volume of Sephadex G-lOO and G-200 eolumns. Bound radioactive Bi2 was excluded from the gels and formed one large symmetrical peak, appearing between the first and second peaks of proteins (Fig. 39). 

Electrophoretic analysis of these fractions revealed that radioactivity corresponded to the area of the electropherogram in which the primary and secondary intrinsic factor-related B12 binders were localized (see Section 1.9.3). Administration of material containing bound radioactive vitamin B12 to patients with pernicious anemia in remission demonstrated its high intrinsic factor activity on urinary excretion test (Table 4). This indicates that intrinsic factor in gastric juice and intrinsic factor-related vitamin Bi2 binders either have a molecular weight above 200,000 or, 

Gel filtration on Sephadex G-200 of Co T.vitamin Bj2 bound to normal gastric juice. From Glass et al., in preparation. 

Sephadex columns were also used by Chosy and Schilling (C4) and Grasbeck et al. (G28) for purification of intrinsic factor from human gastric juice. The gel filtration raised the Bi2-binding activity of the purified product several times over the activity of the starting material (see Section 3.3). 

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G45. Glass, G. B. J., Kakei, M., Kubo, K., and Stephanson-Liounis, L., Fractionation of mucosubstances and proteins in human gastric juice by gel filtration on Sephadex columns combined with paper electrophoresis, 7 Congr. Intern. Assoc. Soc. Natl. Europ. et Mediterran. Gastro-enterol., Bruxelles, 1964, pp. 158-174, Imprim. des Sciences, Bruxelles, 1964. 

Dried shrimp was ground, defatted with benzene, and then extracted with cold water. The luciferase extracted was purified first by a batch adsorption onto DEAE cellulose (elution with 0.4 M NaCl), followed by gel filtration on a column of Sephadex G-150, anion-exchange chromatography on a column of DEAE-cellulose (gradient elution 0.05-0.5 M NaCl), and gel filtration on a column of Ultrogel AcA 34. The specific activity of the purified luciferase was 1.7 x 1015 photons s 1 mg-1, and the yield in terms of luciferase activity was about 28%. 

Fig. 8.2 Gel filtration on a column of Sephadex G-100 at pH 8 (both panels) of the crude extract of Gonyaulax polyedra cells prepared at pH 8 (upper panel) and prepared at pH 6 (lower panel). The activities of the 35 kDa and 130 kDa luciferases are measured by the addition of an excess of luciferin at pH 6.3 ( ) or at pH 8(A). The activity of the luciferin-bound LBP (luciferin-binding protein) in the upper panel is measured after the addition of an excess of 35 kDa luciferase at pH 6.3 ( ). In the lower panel, the LBP activity can be obtained by the addition of an excess of luciferin at pH 8, followed by the removal of unbound luciferin with a small column of Sephadex G-25 before the luminescence assay of bound luciferin at pH 6.3 (see the Section 8.2.8). The Overlap in the upper panel is the light emission resulting from the mixing of an aliquot of the fractions with pH 6.3 buffer. From Fogel and Hastings, 1971, with permission from Elsevier.

Purify the reduced antibody using gel filtration on a column of Sephadex G-25. Concentrate the protein to lOmg/ml using centrifugal concentrators. 

Various chromatographic techniques may be utilized to purify urokinase further. Commonly employed methods include anion-exchange (DEAE-based) chromatography, gel filtration on Sephadex G-100 and chromatography on hydroxyapatite columns. Urokinase is a relatively stable molecule. It remains active subsequent to incubation at 60 °C for several hours, or brief incubation at pHs as low as 1.0 or as high as 10.0. 

Delincee and Radola100 used a commercial preparation, as well as fresh tomatoes, for the preparation, purification, and characterization of tomato pectinesterase. The tomatoes were pressed and then homogenized directly with ammonium sulfate at 70% saturation. The precipitate obtained was extracted with 0.3 M phosphate and repeatedly salted out with ammonium sulfate, and the product was separated on a column of Sephadex G-75. The pattern of separation was similar to that in preceding work.50,97 A detailed study of the size properties of pectinesterase was conducted by gel-filtration and sedimentation analysis.100 By column and thin-layer gel-filtration on Sephadex G-75, the approximate molecular weight of a number of preparations of tomato pectinesterase was determined, values of 24,000 and 27,000 being obtained. A possible interaction of the... 

The purification of glutamine cyclotransferase from papaya latex has been carried out by Messer and Ottesen (116). A batch procedure was used for the removal of impurities by passage of a papaya latex extract through a thin layer of carboxymethyl-Sephadex the active protein was separated by selective elution. Additional purification was achieved by chromatography on a column of carboxymethyl-Sephadex and by gel filtration on Sephadex G-100. The purified enzyme was homogeneous by the criteria of paper electrophoresis, ultracentrifugation, and gel filtration on Sephadex G-100 columns and chromatography on carboxy-methyl- or DEAE-Sephadex. The electrophoretic behavior of the enzyme indicates that it is a basic protein with an isoelectric point near... 

Remove unreacted SMPT and reaction by-products by gel filtration on Sephadex G-25. Pool fractions containing SMPT-activated antibody (the first peak eluting from the column) and concentrate to 10 mg/ml using centrifugal concentrators with a molecular weight cut-off of 10,000. 

To purify the SATA-modified avidin or streptavidin use gel filtration on a column of Sephadex G-25 or dialyze against 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, containing 10 mM EDTA. At this point, the derivative is stable and may be stored under conditions that favor long-term avidin activity. 

The peptide resin prepared above was treated with a 20-fold excess of anhydrous hydrazine in DMF (20 ml) at laboratory temperature for 24 hours, and the mixture was filtered and evaporated to dryness. This procedure also removed the BrZ protecting group from the Tyr moeity. The residue was purified by gel filtration on a column (LH 20 Sephadex) using a 20 1 v/v mixture of water and acetic acid as eluant. There was thus obtained Pyr-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg(H+)-Pro-NH-NH2. The structure of which was confirmed by amino acid analysis and mass spectroscopy. 

Interaction with Adenosylcobalamin. It has been considered generally that adenosylcobalamin or its analogs binds to the apoprotein of diol dehydrase or other adenosylcobalamin-dependent enzymes almost irreversibly (4). However, we found that the holo-enzyme of diol dehydrase was resolved completely into intact apoen-zyme and adenosylcobalamin when subjected to gel filtration on a Sephadex G-25 column in the absence of K+ (9, 10). Among the inactive complexes of diol dehydrase with irreversible cobalamin inhibitors, those with cyanocobalamin and methylcobalamin also were resolved upon gel filtration on Sephadex G-25 in the absence of both K+ and substrate, yielding the apoenzyme, which was reconstitutable into the active holoenzyme (II). The enzyme-hydroxocobalamin complex, however, was not resolvable under the same conditions. The enzyme-cobalamin complexes were not resolved at all by gel filtration in the presence of both K+ and substrate. When gel filtration of the holoenzyme was carried out in the presence of K+ only, the holoen-... 

If a further purification is desired, then the supernatant (above) can be subjected to ammonium sulfate fractionation, gel filtration on Sephadex G-200, and finally on a-aminopropane-agarose affinity column. On polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, the final product migrated as a single band with an estimated molecular weight of 113,000. Upon sedimentation equilibrium velocity ultracentrifugation, an estimated molecular weight value of near 117,000 was obtained. 

The purification of many enzymes has been achieved by use of gel-filtration chromatography, ion-exchange chromatography, or other chromatographic procedures. Eluates from such columns may be analyzed for carbohydrate and protein content, as well as for enzymic activity. Such analyses have revealed that, for glycoenzymes, the enzymic activity, the carbohydrate content, and the protein content are distributed identically in the eluates from the columns. Results of this type of experiment for ribonuclease B are shown in Fig. 1. In these experiments,8 ribonuclease B and a hydrolyzate of the enzyme were subjected to gel filtration on Sephadex, and the eluates were analyzed for protein and for carbohydrate by appropriate colorimetric methods. A control experiment containing ribonuclease A and D-man-nose was treated similarly. The data in Fig. 1 show that the carbohydrate component in the unhydrolyzed sample of ribonuclease B... 

As Streptomyces contain high proportions of proteases, a protease inhibitor, diphenylcarbamoyl chloride, was added to the buffer during purification of the dTDP-dihydrostreptose synthase. A partially purified, enzyme preparation from S. griseus could be obtained by removal of nucleic acids with streptomycin and fractionation with ammonium sulfate.40 However, when this enzyme preparation was subjected to gel filtration on a column of Sephadex G-100, enzyme activity was completely lost. By combining certain fractions of the column eluate, enzyme activity could be partially restored. 

Figure 9.151 Determination of strictosidine synthetase activity by HPLC. Codeine (a), tryptamine (b), and strictosidine (c) were separated on a 4.0 (i.d.) X 250 mm LiChrosorb RP-8 Select B column at a flow rate of 1.0 mL/min. Incubation was for 30 minutes at 30°C with enzyme from Catharanthus roseus after ammonium sulfate precipitation (35-50% saturation) and gel filtration on Sephadex G-25, in the presence of 100 mM fi-D-gluconolactone. Injection volume was 8 pL and the UV detector was set at 0.02 AUFS. (From Pennings et al., 1989.)...

The efficacy of purification of the branched chimera containing oligotuftsin depended on the pattern of the HPLC chromatogram. A large difference between the retention time values of the epitope dimer and chimeric peptide favoured HPLC separation. However, when the peaks are close, reduction of the dimer with DTT could be helpful. The reduced epitope peptide can be separated by HPLC or by gel filtration on Sephadex GIO. Sometimes a change of HPLC column from Cjg to C4 provided a good solution. 

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