共查询到20条相似文献,搜索用时 15 毫秒
1.
T Arakawa 《Analytical biochemistry》1985,144(1):267-268
Larger elution volumes of proteins in gel filtration in the presence of polyethylene glycol than in its absence were explained in terms of the unfavorable interactions between polyethylene glycol and both the proteins and the gel matrix. This should favor the protein molecules being in the cavity of the gel matrix, leading to their slower elution, since, in this situation, the unfavorable interactions are decreased. 相似文献
2.
以聚乙二醇为层析伴侣同时制备SOD、过氧化氢酶和血红蛋白 总被引:2,自引:0,他引:2
发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。 相似文献
3.
A high molecular weight fraction of gelatin precipitated from a solution of Difco gelatin with polyethylene glycol has several favourable properties in gel exclusion chromatography. To manufacture the gel exclusion agent, the gelatin was dissolved in hot water, allowed to set in the cold and rendered insoluble by reaction with formaldehyde. When finely disrupted and used as bed material in columns this gel is capable of separating protein components from mixtures of proteins of widely different molecular weights. Favourable properties include elasticity at relatively low concentration, its availability and its inexpensive nature. 相似文献
4.
A. Poison Margaret Donnelly Margaret Hodgkiss 《Preparative biochemistry & biotechnology》2013,43(6):421-430
A high molecular weight fraction of gelatin precipitated from a solution of Difco gelatin with polyethylene glycol has several favourable properties in gel exclusion chromatography. To manufacture the gel exclusion agent, the gelatin was dissolved in hot water, allowed to set in the cold and rendered insoluble by reaction with formaldehyde. When finely disrupted and used as bed material in columns this gel is capable of separating protein components from mixtures of proteins of widely different molecular weights. Favourable properties include elasticity at relatively low concentration, its availability and its inexpensive nature. 相似文献
5.
A multi-enzyme complex of mammalian aminoacyl-tRNA synthetases was isolated from rabbit reticulocytes, and purified by polyethylene glycol fractionation and gel filtration on Biogel A15m and affinity chromatography on tRNA-Sepharose. The synthetase complex contains nine synthetase activities, and the corresponding proteins as analyzed by SDS polyacrylamide gel electrophoresis. Three of the proteins showed the identical subunit molecular weights to those of the reticulocyte's elongation factor eEF1H. The eEF1 alpha protein could not be removed by second tRNA-Sepharose column chromatography, or gel filtration on Biogel A5m or Biogel A15m. Antibodies against eEF1 alpha react with the purified synthetase complex on the basis of dot blot analysis. This finding should provide new clues for elucidating the structural organization of the mammalian protein biosynthetic machinery. 相似文献
6.
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. 相似文献
7.
Binding of immunoreactive radioiodinated human beta-endorphin (125I-beta-EP) to rat serum was demonstrated by gel filtration of 125I-beta-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, alpha 2- and beta 2-macroglobulins, and the second peak at the fraction of albumin. Binding of 125I-beta-EP to albumin was directly proved by gel filtration of 125I-beta-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of 125I-beta-EP with serum proteins, because of the intense nonspecific adsorption to the semipermeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of 125I-beta-EP in sera from rats and humans, we utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of 125I-beta-EP in rat serum. Binding of 125I-beta-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of 125I-beta-EP was concentration independent over the concentration range studied (1-1000 nM). 相似文献
8.
Alpha 2-macroglobulin from horse plasma. Purification, properties and interaction with certain serine proteinases 总被引:1,自引:0,他引:1
alpha 2-macroglobulin was isolated by polyethylene glycol precipitation, gel filtration on Sephacryl S-300 and DE-52 cellulose chromatography, with 20% yield. The preparation obtained was homogenous as tested by biochemical and immunological criteria. Its molecular mass was estimated at 800,000, comprising of four identical subunits. The isoelectric point of our preparation was 4.8 and two molecules of serine proteinases per 1 molecule of inhibitor were bound. 相似文献
9.
Homogeneous cytosolic pyruvate kinase from endosperm of germinating castor oil (Ricinus communis L. cv Hale) seeds was potently activated by polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the pyruvate kinase reaction mixture caused a 2.6-fold increase in maximal velocity and 12.5- and 2-fold reductions in Km values for phosphoenolpyruvate and ADP, respectively. Glycerol, ethylene glycol, and bovine serum albumin also enhanced pyruvate kinase activity, albeit to a lesser extent than polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the elution buffer during high-performance gel filtration chromatography of purified cytosolic pyruvate kinase helped to stabilize the active heterotetrameric native structure of the enzyme. A higher degree of inhibition by MgATP, but lower sensitivity to the inhibitors 3-phosphoglycerate and fructose- 1,6-bisphosphate, was also observed in the presence of 5% (w/v) polyethylene glycol. It is concluded that (a) plant cytosolic pyruvate kinase activity and regulation, like that of other regulatory pyruvate kinases, is modified by extreme dilution in the assay medium, probably as a result of deaggregation of the native tetrameric enzyme, and (b) ATP is probably the major metabolic effector of germinating castor endosperm cytosolic pyruvate kinase in vivo. 相似文献
10.
Small and preparative gel filtration columns were studied for high pressure liquid chromatography of myelin proteins in sodium dodecyl sulfate. The preparative column proved useful for isolating and purifying proteolipid protein almost free (0.3–0.5%) of myelin basic protein as demonstrated by SDS-PAGE, MBP RIA, and immunoblotting. The small columns were not as useful as SDS-PAGE for analytical purposes. 相似文献
11.
Y Piquet D Fizet J Bousquet D Platel F Cabantous J P Dubecq J Moulinier 《Comptes rendus des séances de la Société de biologie et de ses filiales》1983,177(2):175-182
A non specific immunosupressive factor able to block an in vitro cytotoxicity reaction is demonstrated in the serum of pregnant women. A solution containing this blocking factor is obtained by gel filtration and precipitation of plasma by polyethylene glycol 4 000. Then after immunisation of rabbits the immune serum can be used for affinity chromatography. An alpha 2 glycoprotein has been separated which inhibits the in vitro cytotoxicity reaction and whose molecular weight determined by gel filtration and polyacrylamide gel is about 200 000 daltons. 相似文献
12.
A purified potato virus S was prepared using precipitation by the solution of 35% polyethylene glycol 4000 in the presence of an electrolyte. The mixture for precipitation of the potato virus S had to contain 11% polyethylene glycol and 0·25m NaCl. The S virus was extracted by 0·01m phosphate buffer, pH 7·5, from the precipitation separated by centrifugation. One part of the extract was further purified by means of differential centrifugation and the other by means of gel filtration on Sephadex G-100. The ultraviolet absorption measurements of both preparations showed that the differential centrifugation gave a purer preparation than the gel filtration. 相似文献
13.
Anders Aksnes Tore Skotland Torgeir Flatmark Torbjørn Ljones 《Neurochemical research》1979,4(3):385-398
Dihydropteridine reductase has been purified to homogeneity from bovine liver and bovine adrenal medulla by precipitation with polyethylene glycol, ion exchange chromatography, gel filtration, and affinity chromatography on 5-AMP-Sepharose 4B. The enzymes from the two tissues seem identical by the criteria of gel filtration chromatography, affinity chromatography, polyacrylamide gel electrophoresis in the presence and absence of dodecyl sulfate, isoelectric focusing, amino acid analysis, and binding of NADH. Fluorescence studies show two independent binding sites for NADH and a dissociation constant of 10 nM at pH 6.8. Isoelectric focusing of the enzyme as purified in the presence of NADH revealed three different bands, which by removal of this coenzyme were converted into a single band, corresponding to pI 5.7. The enzyme contains no carbohydrate or zinc. 相似文献
14.
Dynamic light scattering (DLS) is a technique capable of determining the hydrodynamic radius of proteins. From this parameter, a molecular weight can be assessed provided that an appropriate calibration curve is available. To this goal, a globin-based calibration curve was used to determine the polymerization state of a recombinant hemoglobin-based oxygen carrier and to assess the equivalent molecular weight of hemoglobins conjugated with polyethylene glycol molecules. The good agreement between DLS values and those obtained from gel filtration chromatography is a consequence of the high similarity in structure, shape, and density within the globin superfamily. Moreover, globins and heme proteins in general share similar spectroscopic properties, thereby reducing possible systematic errors associated with the absorption of the probe radiation by the chromophore. 相似文献
15.
N. S. Melik-Nubarov I. D. Grozdova G. Yu. Lomakina M. V. Pokrovskaya V. S. Pokrovski S. S. Aleksandrova O. Yu. Abakumova O. V. Podobed D. V. Grishin N. N. Sokolov 《Applied Biochemistry and Microbiology》2017,53(2):165-172
N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures. 相似文献
16.
The structure-function relationship of mitochondrial malate dehydrogenase was investigated at low enzyme concentration, as well as in the presence of polyethylene glycol (PEG 6000) and structure making ions. Previous reports claimed the dimeric enzyme to undergo dissociation in dilute solution, and PEG-induced pairing of dimers in the crystalline state. Sedimentation analysis and gel filtration in 0.1 M sodium phosphate pH 7.6 plus 1 mM EDTA and 1 mM dithioerythritol prove the enzyme to be a stable dimer at c greater than or equal to 0.2 microgram/ml (5 nM). In the presence of 8-20% (w/v) PEG 6000, association of the dimer to tetramers and higher aggregates is observed. At 20% (w/v) polyethylene glycol, ultracentrifugal analysis yields up to 50% tetramers; chemical cross-linking by glutaraldehyde confirms the association in a qualitative way. The enzymatic properties of mMDH (specific activity, Km for oxaloacetate and NADH) in the absence and in the presence of PEG 6000 are indistinguishable. At high polyethylene glycol concentrations (greater than or equal to 20%), the thermal stability of the enzyme is found to be increased. The fluorescence emission, as well as the far-UV and near-UV circular dichroism remain unaffected. Accumulated evidence from equilibrium experiments at low enzyme concentration and reconstitution kinetics (after dissociation at acid pH) proves the active species of mMDH to be the dimer. 相似文献
17.
Two distinct components having potent heparin cofactor activity have been separated from human blood plasma and partially purified by means of precipitation with polyethylene glycol, adsorption on Al(OH)3 gel and ion exchange chromatography on DEAE-cellulose and hydroxyapatite. The first component, cofactor A, is distinguished from the second, cofactor B, by its very low activity in the progressive antithrombin assay. Ion exchange rechromatography experiments indicate that cofactors A and B are not interconvertible, and that neither is an artifact of the polyethylene glycol precipitation step. Molecular weights, estimated by gel filtration were found to be 105,000 and 87,000 daltons for cofactors A and B respectively. Except for the molecular weight, the bulk of evidence suggests that cofactor B and the antithrombin III purified by Abildgaard (3) are identical. 相似文献
18.
Glycogen phosphorylase from Neurospora crassa: purification of a high-specific-activity, non-phosphorylated form.
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A highly active glycogen phosphorylase was purified from Neurospora crassa by polyethylene glycol fractionation at pH 6.16 combined with standard techniques (chromatography and salt fractionation). The final preparation had a specific activity of 65 +/- 5 U/mg of protein (synthetic direction, pH 6.1, 30 degrees C) and was homogeneous by the criteria of gel electrophoresis, amino-terminal analysis, gel filtration, and double immunodiffusion in two dimensions. The enzyme had a native molecular weight of 180,000 +/- 10,000 (by calibrated gel filtration and gel electrophoresis) and a subunit molecular weight of 90,000 +/- 5,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Each subunit contained one molecule of pyridoxal phosphate. No phosphoserine or phosphothreonine was detected by amino acid analysis optimized for phosphoamino acid detection. The enzyme isolated from cells grown on high-specific-activity 32Pi (as sole source of phosphorus) contained one atom of 32P per subunit. All the radioactivity was removed by procedures that removed pyridoxal phosphate. Thus, the enzyme could not be classified as an a type (phosphorylated, active in the absence of a cofactor) or as a b type (non-phosphorylated, inactive in the absence of a cofactor). The level of phosphorylase was markedly increased in mycelium taken from older cultures in which the carbon source (glucose or sucrose) had been depleted. The polyethylene glycol fractionation scheme applied at pH 7.5 to mycelial extracts of younger cultures (taken before depletion of the sugar) resulted in co-purification of glycogen phosphorylase and glycogen synthetase. 相似文献
19.
Biological and physicochemical properties of keyhole limpet hemocyanin (KLH)-induced guinea pig lymphotoxin (LT) were compared to those of LT induced by another antigen, ovalbumin, or by the mitogen, phytohemagglutinin (PHA). Mitogen- and antigen-induced LT had similar colony inhibitory activities toward guinea pig and rat tumorigenic cells but none toward nontumorigenic cells. Conversely, human nontumorigenic but not tumorigenic cells were inhibited by the three LT preparations. The molecular weight of each LT was 45,000 daltons on gel filtration chromatography but sucrose density gradient ultracentrifugation indicated greater heterogeneity. The KLH-, ovalbumin-, and PHA-induced LTs were resolved into multiple peaks upon preparative column isoelectric focusing (IEF) with IpH's in the range of 4.5 to 5.3. Analytic IEF of the KLH-induced LT revealed the presence of three distinct biologically active peaks with IpH at 4.77, 5.02, and 5.20. LT activity was stabilized during preparative IEF by inclusion of 0.1% polyethylene glycol (4000 molecular weight) in the ampholine gradient. This resulted in 90% recovery of LT activity with a concomitant removal of 96% of the KLH used as the antigenic stimulus for LT production. Fractionation of LT activity during diafiltration and IEF when exogenous proteins were included as protective agents altered the number and the isoelectric characteristics of the peaks obtained suggesting that LT was being bound by the added protein. Colony inhibitory (cytostatic) and 3H-release (cytolytic) LT activities copurified during fractionation. Thus, this study demonstrated that high yields (approximately 50% of the starting material) of LT, free of antigen or exogenous serum proteins, can be obtained by a combination of diafiltration and IEF performed in the presence of polyethylene glycol. 相似文献
20.
Urea amidolyase was purified to homogeneity from extracts of Candida utilis. The purification involves protamine sulfate precipitation, ammonium sulfate precipitation, polyethylene glycol precipitation, Sepharose 6B gel filtration, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography. The final preparation is pure as judged by disc-gel electrophoresis. The molecular weight of urea amidolyase, as determined by gel filtration and disc-gel electrophoresis, is between 500,000 and 520,000. Treatment with sodium dodecyl sulfate results in two peptides with molecular weights of 70,000 and 170,000. The urea carboxylase and allophanate hydrolase activities of urea amidolyase may be distinguished from one another on the basis of (a) the effect of the stabilizers, urea and glycerol, (b) the effect of storage pH on activity, and (c) selective inhibition by sulfhydryl reagents. 相似文献