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PubMed1982

Determination of the molecular weight of alpha-amylase, as the enzyme-inhibitor complex, using thin layer gel filtration on Sephadex.

Thin layer gel filtration on Sephadex was performed as a simple method for recognition of the small molecular weight differences of human alpha-amylases from different sources. Activity was located with dry chromogenic substrate, using a replica technique. Undesirable interaction between the gel matrix and substrate binding sites on the enzyme, which causes an anomalous decrease in the migration rate of the enzyme protein, was suppressed by preincubation of the enzyme with appropriate inhibitor. Gradual masking of substrate binding sites of the enzyme by increasing concentrations of amylase inhibitors resulted in two distinct migration rates for the enzyme in thin layer gel filtration. This suggests the existence of two substrate binding sites in the enzyme molecule. Together with thin layer gel affinity chromatography on a mixture of Sephadex and ConA-Sepharose, the method yielded useful data on the molecular weight of amylase and its glycosylated forms and served as a valuable tool for the differential diagnosis of macroamylasaemia.

Amylases↗
PubMedDate not supplied

Partitioning of flunitrazepam into model membranes studied by temperature controlled gel filtration chromatography.

The use of gel filtration chromatography with Sephadex as a separation medium was used in order to study flunitrazepam (FNTZ) partitioning into artificial model membranes consisting of dipalmitoyl-phosphatidylcholine (dpPC) vesicles, under controlled temperature conditions. In this system two phenomena are taking place simultaneously: the ligand-liposome interaction and the lipid self-aggregation to form the liposome. The liposome-FNTZ interaction was evidenced by the non-enantiography of the first derivative of FNTZ elution peak in frontal chromatography through Sephadex G-75. On the other hand, the presence of FNTZ reduced liposomes mean size and increased their size dispersion as evidenced by molecular filtration through Sephadex G-200. The dpPC-buffer FNTZ partition coefficient determined in zonal chromatography through Sephadex G-10 increased about 33% when the temperature rose above the temperature of dpPC transition from the liquid crystalline to the fluid phase. Gel filtration chromatography seems a suitable technique to study lipid liposome-FNTZ interactions at a qualitative level. In addition, this technique has the advantage over other methods of giving the possibility of observing the mutual effects exerted between the drug and the self-aggregating structure.

Chromatography, Gel↗
PubMed1986

[Effect of roasting on the chromatographic profile obtained by gel filtration of Arabian coffee extract].

Gel filtration chromatography under a high pressure system was used to investigate green and roasted coffee aqueous extracts. Chromatographic columns used were of the type TSK-PW 4000, in series. Chromatography of the extracts was monitored either using a refractive index (RI) detector in series with an ultraviolet (UV) detector at 420 nm or with two UV detectors at 280 and 325 nm, respectively. The utilization of RI detection showed significant differences in the molecular weight profile between green and roasted coffees. UV detection at 280 and 325 nm revealed similar chromatographic patterns, suggesting that phenolic compounds were probably bound to proteins. Pigment formation after roasting was monitored by detection at 420 nm, showing that colored material was distributed throughout the molecular weight range studied. The method provided a more rapid analysis compared to traditional gel filtration chromatography and proved to be useful for monitoring changes in molecular weight profile which occur during coffee roasting.

Chromatography, Gel↗
PubMed2001

[Removal of PEG and salt from recombinant human tumor necrosis factor-alpha(rhTNF-alpha) with serial operation of gel filtration chromatography].

A serial gel filtration operation with the combination of G25 and G100 in one column was developed to remove salt and PEG from upper phase rich in recombinant human tumor necrosis factor alpha after simultaneous cell disruption and two-phase aqueous extraction. Buffer exchanging and primary purification were also achieved at same time. The purification factor of 4.4 was obtained with an activity recovery of 97% by one single step of serial operation.

Chromatography, Gel↗
PubMed1995

Deconvolution of gel filtration chromatographs of human plasma lipoproteins.

Gel filtration chromatographs of lipoproteins represent a superposition, or convolution, of the intrinsic polydispersity of the solute and the dispersion due to transport phenomena. We describe a deconvolution technique for improving the resolution of gel filtration chromatographs applicable to lipoproteins and other polydisperse solutes. A matrix of spreading functions, characterizing the dispersive properties of the column, was determined by fitting chromatographic data from a series of monodisperse standards with the solution to the transport equations and interpolating between the fit parameters. A successive approximation scheme was used in which a test distribution was incrementally corrected by an amount proportional to the error between the measured chromatograph and that derived from the test distribution. A nonlinear relaxing function was used to constrain the correction term such that the solution remained physically realizable (i.e., nonnegative absorbance) as it evolved. Deconvolved chromatographs of lipoproteins provided resolution of peaks that were obscured by spreading in the original data. The distribution of particle sizes within each fraction was calculated and verified experimentally by further separating the contents of fractions by gradient gel electrophoresis. Our technique, however, provided comparable resolution of the peaks without the additional experimental procedure.

Algorithms↗
PubMed1978

Gel filtration of sialoglycoproteins.

The role of sialic acid in the gel-filtration behaviour of sialoglycoproteins was investigated by using the separated isoenzymes of purified human liver alpha-L-fucosidase and several other well-known sialic acid-containing glycoproteins (fetuin, alpha1-acid glycoprotein, thyroglobulin and bovine submaxillary mucin). For each glycoprotein studied, gel filtration of its desialylated derivative gave an apparent molecular weights much less than that expected just from removal of sialic acid. For the lower-molecular-weight glycoproteins (fetuin and alpha1-acid glyocprotein), gel filtration of the sialylated molecules led to apparent molecular weights much larger than the known values. The data indicate that gel filtration cannot be used for accurately determining the molecular weights of at least some sialoglycoproteins.

Chromatography, Gel↗
PubMed1980

Macroamylasemia: variable response of a 7S and a 11S macroamylase to phosphate-benzoic acid buffer during gel filtration.

During thin layer or macrocolumn gel filtration with Sephadex G 200, a 7S macroamylase dissociated completely to normal-sized amylase when the eluant was phosphate-benzoic acid buffer. In contrast a 11S macroamylase was not affected by the buffer and amylase activity was associated with 11S proteins. Since phosphate-benzoic acid buffer is used in a thin layer screening method described by Peeters et al. (9), some macroamylases may go undetected. After treatment of the 7S macroamylase with phosphate-benzoic acid buffer, the amylase-free 7S proteins obtained formed macroamylase with added pancreatic amylase. This indicates that the buffer did not inhibit the activity of the macroamylase, but caused a dissociation of the macroamylase during gel filtration. The difference in the effect of phosphate-benzoic acid buffer on the two types of macroamylases may be attributed to the difference in affinity of binding proteins for the amylase molecule.

Amylases↗
PubMed2002

Microplate gel-filtration method for radioligand-binding assays.

A thin-layer gel-filtration chromatographic method has been developed in a 96-well format to separate free and protein-bound ligand in radioligand-binding assays. The mobile phase in the gel-filtration plate is removed via quick centrifugation before samples are applied. Protein-bound ligand is recovered via centrifugation into another 96-well plate for radioactivity measurements. The method exhibits excellent recovery of protein-ligand complexes and less opportunity for dissociation of the complexes since it eliminates major dilution effects from the mobile phase of a column and from elution steps in conventional gel-filtration chromatography. It offers other advantages: simple, rapid, inexpensive, quantitative, and able to handle a large number of samples as required in drug discovery and clinical settings. This microplate gel-filtration method was optimized in studies of receptor-ligand interactions using estrogen receptors as examples and can be used in other radioligand-binding assays.

Binding, Competitive↗
PubMed2005

A gel filtration assay to determine glycogen synthase activity.

We developed a gel filtration assay for the determination of glycogen synthase activity in cultured cells or tissue homogenates. Compared to the commonly used filter paper assay, the gel filtration assay resulted in a more than 5-fold reduction of background levels leading to an--at least--twofold increase in precision. These benefits allow the gel filtration method to detect differences of +/-5% in enzyme activity out of 300 microg total cell protein. In addition to high precision and sensitivity, the method's additional salient advantages include lesser expenditure of time and labour and reduced exposure time of the personnel to radioactivity.

Animals↗
PubMed1976

Self-association of cytochrome b5 in aqueous solution. Gel filtration and ultracentirfugational studies.

The aggregation state of detergent-extracted cytochrome b5 was examined by gel filtration and sedimentation equilibrium ultracentrifugation. Both techniques indicated the presence of a mixture of monomer and octomer. The proportion of monomer was decreased as the buffer salt concentration was raised and was approximately 1 muM in 10 mM Tris acetate/0.1 mM EDTA. The monomeric cytochrome b5 was isolated by gel filtration and showed a decreased tendency to aggregate in dilute buffer, although its other physical properties were identical to those of the original cytochrome b5. The monomer was found to have a Stokes radius of approximately 26 A, as determined by classical gel filtration and by an equilibrium saturation method where the sample was monitored in the gel by measuring the absorbance of the gel column directly in a dual wavelength spectrophotometer. The gel filtration and ultracentrifugation data together with the studied on the monomeric cytochrome b5 suggest the octomer is not a rapid equilibrium with monomer and this observation should be taken into account in lipid binding experiments with cytochrome b5.

Animals↗
PubMed1965

The gel-filtration behaviour of proteins related to their molecular weights over a wide range.

1. Correlation between elution volume, V(e), and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 (alpha-crystallin) on Sephadex G-200 columns at pH7.5. 2. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free globular proteins fitted a smooth V(e)-log(mol.wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. 3. V(e)-log(mol.wt.) curves based on results with the three gels are taken to represent the behaviour of ;typical' globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. 4. Some glycoproteins, including gamma-globulins and fibrinogen, do not conform to the standard relationship. The effect of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. 5. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. 6. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually >10(6). 7. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250000. The free sub-units readily lose enzymic activity. 8. Recognition of the atypical gel-filtration behaviour of gamma-globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140000; milk alkaline phosphatase, 180000.

Chemical Phenomena↗
PubMed1983

Gel filtration in sodium dodecyl sulphate of hydrophobic muscle proteins on Sephacryl S-400 Superfine.

Gel filtration has been applied to muscle proteins in the presence of the detergent sodium dodecyl sulphate (SDS). This detergent not only solubilizes a variety of proteins but also increases their hydrodynamic volumes, which is a disadvantage for the process of gel filtration. Nevertheless, appropriate choice of gel type, in this case Sephacryl S-400 Superfine, and optimal elution conditions make SDS gel filtration a useful tool in the separation of heterogeneous protein mixtures in the molecular weight range of 20,000-200,000.

Acrylic Resins↗
PubMed1989

Soluble complex formation of bovine immunoglobulin G2 with staphylococcal protein A studied by gel filtration chromatography.

The binding property of bovine IgG2 to staphylococcal Protein A was investigated by the methods of gel filtration chromatography and affinity chromatography. High performance gel filtration chromatography was carried out using TSK gel G3000SW and G2000SW columns, and immobilized Protein A column was used for affinity chromatography. Although bovine IgG2 did not form any precipitin lines with Protein A by double diffusion method on agar gel, IgG2 could bind to immobilized Protein A column. Moreover, by gel filtration chromatography, peaks of the complex between bovine IgG2 and Protein A were observed in addition to the IgG2 monomer peak. Thus, it is concluded that bovine IgG2 interacts with staphylococcal Protein A and forms "soluble complexes". Carbethoxylated IgG2 lost its affinity to Protein A indicating that histidyl residues in IgG2 is essential for the binding to Protein A.

Animals↗
PubMed1991

Comparison of the separation of bovine heparin by strong anion exchange and by gel filtration chromatography.

Heparin has been fractionated by strong anion exchange chromatography followed by elution of the pools on a gel filtration column. This resulted in the expected inverse relationship in the elution order for the pools run by the two methods. Also chromatography of heparin was performed in the reverse order: gel filtration first, followed by anion exchange of the pools. For this order of separation four of the five gel filtration pools of different molecular weights eluted at a similar LiCl concentration. The specific activities of different pools of heparin were evaluated using a colorimetric microwell kinetics assay using antithrombin and thrombin. For the pools separated by ion exchange first, there was an exponential increase of specific activity with increasing molecular weight for all pools. For the pools isolated by gel filtration first, the specific activities became level after an initial increase in relation to molecular weight. Thus, unique pools of heparin species are being isolated by different modes of chromatography.

Animals↗
PubMedDate not supplied

Factors in biological fluids affect gel filtration behavior of epidermal growth factor.

There have been conflicting reports on the presence of multiple forms of epidermal growth factor (EGF) in human saliva. The present study was initiated to study the gel filtration behavior of EGF in saliva and other biological fluids. In addition, studies were performed to determine if there were factors in saliva or other biological fluids that would influence the gel filtration behavior of EGF. Gel filtration studies with saliva and urine demonstrated EGF migration consistent with a molecular weight of 35 kDa with no heterogeneity observed. The addition of radiolabeled EGF to either saliva or urine resulted in an altered migration on gel filtration. These results provide evidence for a protein-peptide interaction in saliva and other biological fluids that alters the apparent physical properties of mature EGF.

Chromatography, Gel↗