Sephadex, DEAE

Abras precatorius. Purified by successive chromatography on DEAE-Sephadex A-50, carboxymethylcellulose, and DEAE-cellulose. [Wei et al. J Biol Chem 249 3061 1974.]... 

Adenosine 5"-[P-thio]diphosphate tri-lithium salt [73536-95-5] M 461.1. Purified by ion-exchange chromatography on DEAE-Sephadex A-25 using gradient elution with 0.1-0.5M triethylammonium bicarbonate. [Biochem Biophys Acta 276 155 7972.]... 

Coiicin E (from E.coli) [11032-88-5], Purified by salt extraction of extracellular-bound colicin followed by salt fractionation and ion-exchange chromatography on a DEAE-Sephadex column, and then by CM-Sephadex column chromatography [Schwartz and Helinski J Biol Chem 246 6318 1971],... 

Dibydropteridine reductase (from human liver) [9074-11-7] Mr52,000 [EC 1.6.99.7]. Purified to homogeneity on a naphthoquinone affinity adsorbent, followed by DEAE-Sephadex and CM-Sephadex... 

Reverse transcriptase (from avian or murine RNA tumour viruses) [9068-38-6] [EC 2.7.7.49]. Purified by solubilising the virus with non-ionic detergent. Lysed virions were adsorbed on DEAE-cellulose or DEAE-Sephadex columns and the enzyme eluted with a salt gradient, then chromatographed on a phosphocellulose column and enzyme activity eluted in a salt gradient. Purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 7977 Smith Methods Enzymol 65 560 1980 see commercial catalogues for other transcriptases.]... 

A) A solution of (SMI (320 mg) in trifluoroacetic acid (7 ml) was kept under nitrogen at room temperature for 15 minutes. Ether (100 ml) was added and the precipitate filtered, washed thoroughly with ether and dried. This material (280 mg) was added to concentrated sulfuric acid (20 ml), cooled at -20°C. The solution was kept in the dry ice-acetone bath at -20°C for 75 minutes. The sulfuric acid solution was poured into ice water (80 ml). The precipitate was centrifuged, resuspended in ice water (30 ml) and 4N sodium hydroxide was added until a clear solution was obtained. After reacidification to pH 4 with dilute sulfuric acid, the precipitate formed was centrifuged, washed twice with ice water and dried. Yield 155 mg. Chromatograph of DEAE Sephadex (with ammonium carbonate buffer) yielded the desired octa-peptide sulfate ester 30 mg. 

Several L-amino acids are produced on a large scale by enzymatic resolution of N-acetyl-D,L-amino adds (Figure A8.4). Acylase immobilised on DEAE-Sephadex is for example employed in a continuous process while Degussa uses the free acylase retained in a membrane reactor. In the latter process the advantage of reuse of the enzyme and homogeneous catalysis are combined. 

To develop a continuous process, the immobilisation of aminoacylase of Aspergillus oryzae by a variety of methods was studied, for example ionic binding to DEAE-Sephadex, covalent binding to iodo-acetyl cellulose and entrapment in polyacrylamide gel. Ionic binding to DEAE-Sephadex was chosen because the method of preparation was easy, activity was high and stable, and regeneration was possible. 

Purification of Pholas luciferase (Michelson, 1978). Acetone powder of the light organs is extracted with 10 mM Tris-HCl buffer, pH 7.5, and the luciferase extracted is chromatographed on a column of DEAE Sephadex A-50 (elution by NaCl gradient from 0.1 M to 0.6 M). Two peaks of proteins are eluted, first luciferase, followed by a stable complex of luciferase and inactivated pholasin. The fractions of each peak are combined, and centrifuged in 40% cesium chloride... 

Step 4. Anion-exchange chromatography on a column of DEAE-Sephadex A-50. The column had been equilibrated with the basic buffer containing 0.12 M NaCl. The NaCl in the photoprotein solution was removed by gel filtration, and then the solution was added onto the column. The photoprotein adsorbed on the column was eluted with the equilibration buffer. 

Isolation from cultures of Eupenicillium hrefeldianum NRRL 5734. a Amberlite IRA-411/pH 10. b Chromatography on DEAE-Sephadex A-25. 

Att eZcven y-cJuiin voA nts, discovered thus far, exhibit a change In electrophoretic mobility, and starch gel electrophoresis Is the recommended method for their detection. Quantitation of the variant can best be done by chromatography on columns of either DEAE-Sephadex or CM-Cellulose. The quantities of some variants In heterozygotes differ greatly. For Instance, the relative amount (expressed In %F /Fxotal) varies from 20-25% (F-Malta-I) to 10-15% (most Y C >aln variants) to 5-6%... 

The, chain voAiantS are characterized by the presence of two abnormal components, an abnormal Hb-F (02 /2) and an abnormal Hb-A (tt2 32) Of these two, the 02 2 component dominates and the 02 32 component Is often difficult to detect. The methods of choice are starch gel electrophoresis and anion-exchange chromatography using DEAE-Sephadex or DE-52 Cellulose. Chain analyses of these Isolated hemoglobin components will lead to a definitive Identification. 

Macrochromatographlc Methods. These procedures use cation exchangers, such asAmberllte-IRC-50, carboxymethyl-Cellulose (CMC) and carboxymethyl-Sephadex (CMS), and the anion exchangers diethylamlnoethyl-(DEAE)-Cellulose and DEAE-Sephadex. 

Chromatography of hemoglobins on columns of DEAE-Cellulose (DE-52 mlcrogranular, preswollen Whatman) often results In excellent separations of many variants, because the hemoglobins are eluted as sharp, narrow zones generally widely separated from each other The hemoglobin zones are eluted from the DE-52 columns at a distinctly higher pH value than from a similar DEAE-Sephadex column ... 

Figure 12, Heat denaturation curves of hemoglobin from three members of a family with Hb-Leslie, a newly discovered unstable variant with a deletion of residue 131 of the p-chain, G.P.Sr. has Hb-Leslie p-thalassemia the %Hb-Leslie is 85% (DEAE-Sephadex chromatography) Gr.P. has Hb-LesUe- Hb-C %Hb--Leslie is 28% M.B. fm Hb LesUe trait %Hb Leslie is 28%,...

This electrophoretlcally fast-moving variant was readily Isolated by DEAE-Sephadex chromatography. Hybridization analyses with canine Hb confirmed the suggestion that the abnormality was located In the a-chaln. The a-chaln was separated from the 3-chaln on 1.7 X 15 cm columns of CM-Cellulose using the method of Clegg tt aJL ( ). The CM-Cellulose used was CM-52 (Reeve Angel, Clifton, N.J.) and the developers were 8 M urea-3-mercaptoethanol phosphate buffers, pH 6.7 - 7.1. 

Heinz body preparations are positive as Is the heat stability test Electrophoretic examination of hemolysate showed the presence of a fast-moving variant which was readily separated from Hb-A by chromatography on DEAE-Sephadex The abnormal 3- chain was Isolated by CM-Cellulose chromatography as described before, and converted into the S-2-amlnoethyl (AE) derivative... 

As an example, consider the separation of the creatine kinase isoenzymes, MM, MB, and BB. Mercer has used classical ion-exchange chromatography (DEAE - Sephadex - A50) for the resolution of these three isoenzymes (44) To speed up the separation and ultimately to allow an automated analysis,... 

Light scattering (nephelometry) was used as a detection system for gly-cosaminoglycans from urine, eluted from a DEAE Sephadex (Pharmacia Biotechnology Uppsala, Sweden) A-25 column.68 This technique has been more recently applied to protein characterization.69 Interferometry was used for analysis of dextran eluted from a size exclusion column.70 One of the problems of electrochemical detection is that it is relatively insensitive to polymers. Because many of the materials discussed below (DNA, proteins, and polysaccharides) are polymeric, a brief mention of some alternative... 

Almost simultaneously, a pectinesterase was isolated from tomatoes of the Immuna variety.97 After extraction with 2% sodium chloride at pH 7.8, and fractional salting-out with ammonium sulfate, chromatography on DEAE-Sephadex A-50 removed a substantial proportion of colored contaminants and accompanying acid compo-... 

Fig. 4. — Monitoring of the Multiple Molecular Forms of Tomato Pectinesterase by Starch-gel Electrophoresis.98 [ENZ, detection of pectinesterase activity by paper print with pectin and Bromothymol Blue PROT, protein staining with nigrosin O, origin. Key A, 1 crude tomato extract after ammonium sulfate salting-out, and dialysis 2 pectinesterase fraction from column of DEAE-Sephadex A-50 3 and 4 pectinesterase fractions from column of Sephadex G-75. B, Two parts of the same gel after horizontal slicing 1, 500 fig of the isolated form of pectinesterase from a column of CM-Seph-adex C-50 with 175 mM phosphate-sodium chloride buffer 2, active fraction at 150 mM buffer 4 and 5, 250 fig and 1 mg of the isolated form of pectinesterase, respectively.]...

Four forms of pectinesterase were found in different tomato varieties (Marion, Homestead, and Pixie)83 by separation on columns of DEAE-Sephadex A-50, using elution with 150 mM sodium chloride at pH 6.0. By gel filtration on columns of Sephadex G-100, the molecular weights of the individual forms were found to be 22,000-27,000, and 35,000. 

By use of starch-gel electrophoresis, the total extract of bananas, and the fractions obtained after separation on DEAE-Sephadex A-50, were found to contain six multiple forms of pectinesterase having electrophoretic patterns different from those of tomato pectinesterase.103... 

Miller and Macmillan49 purified pectinesterase produced by Fusarium oxysporum f. sp. vasinfectum by chromatography on DEAE-Sephadex A-25, Sephadex G-75, CM-Sephadex C-50, and CM-cellulose. The homogeneity of the pectinesterase obtained was confirmed by disc electrophoresis an apparent molecular weight of 35,000 was estimated by its behavior on a column of Sephadex G-75 (Superfine). 

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