Gel filtration technique

Finally, radiochemical methods of analysis may be used to follow rates of detritiation. This method is particularly useful for very slow reactions (where it is impractical to collect data for any appreciable extent of reaction) as an initial rate approach may then be employed. Separation difficulties, at least for aqueous solutions, may be overcome by using the freeze-drying method or the more recent countercurrent dialysis and Sephadex gel filtration techniques. ... 

G. Ackers, Methods Enzymol. 27, 441-455 (1973), Academic Press (New York). Studies of protein ligand binding by gel filtration techniques. 

J. Robyt and B. White, Biochemical Techniques Theory and Practice (1987), Brooks/Cole (Monterey, CA), pp. 88-95. An introduction to gel filtration techniques. 

The binding of various ligands to rabbit liver FDPase has been studied in detail in Pontremoli s laboratory, using the gel filtration technique of Hummel and Dreyer (55). 

The availability of gel filtration techniques during the 1960s allowed fractionation to be achieved on the basis of size differences. The most noteworthy work using these techniques is attributed to Cameron et al. (1972) (see Swift, 1985). They, using gel filtration and discrete pore size membranes, fractionated a HA extract into 11 different size fractions and determined the molecular weight of the fractions using ultracentrifugation techniques (see Section 1.4.7). 

Figure S. Enzymatic analysis of inhibitor glycopeptides. Glycopeptide and oligosaccharide products were isolated by ion-exchange and gel filtration techniques. Monosaccharide constituents were determined by GLC of their glycitol acetate derivatives following Dowex-SO [H J catalyzed acid hydrolysis ("26, 27). Liberated mannose was determined by direct GLC of its glycitol acetate A. niger a-l,2-mannosidase and fl-mannosidase were prepared by the procedures of Swami-nathan et al. (31) and Elbein et al. (32), respectively S. griseus endoglucosamini-dase teas prepared as described by Tarentino and Maley (33).

Andreatta and Rink U9) synthesized model-peptides on a linear polystyrene of molecular weight 20,000, employing the gel filtration technique for the separation of the polystyrene-bound peptides from excess reagents. These investigators observed that the maximum leading to maintain satisfactory solubility properties of the polymer-peptide was 0.5 mmol peptide per gram of polymer. 

Adsorption chromatography and gel filtration techniques have also been utilized for quantitative measurements of the partitioning of solubilizates between the micellar phase and the bulk solvent (Kaufman, 1962 Herries et al., 1964 Dunlap and Cordes, 1968 Romsted and Cordes, 1968), but like distribution techniques these methods are completely ineffectual for elucidating the location of the solubilizate in the micellar phase. 

Other groups also used gel filtration exchange to measure middle molecules and perform in vitro toxicity tests (C7, D21). However, many of the middle molecular weight substances isolated by these techniques proved to be much smaller than anticipated. This discrepancy was due to the intrinsic inadequacies of the standard gel filtration techniques for the isolation of middle molecules, as pointed out by Furst et al. (F13) and later by Schoots et al. (S13). These investigators used analytical techniques to demonstrate that middle molecular fractions obtained by gel filtration comprised many low-molecular-weight solutes, such as carbohydrates, amino acids, polyols, aromatic substances, and other UV-absorbing solutes, and also sodium chloride, acetate, phosphate, and sulfate (S10). Thus these fractions do not exclusively represent middle molecules. 

Early workers showed that BSP and rose bengal were protein bound (R18a, R19). Subsequently, salting out, electrophoresis, and gel filtration techniques have indicated that this association is primarily with the albumin fraction (B3, B17). More recently it has been reported that a considerable proportion (30-60%) of BSP is bound to ai-lipoprotein (B2). This protein fraction has an even higher affinity for indocyanine green, 90% of which is bound. However these studies on the binding of BSP to protein are not definitive because nonphysiological and nonequilibrium conditions were used. 

The apoproteins made from human and bovine erythrocuprein have been examined in several laboratories (69, 70, 72, 74, 76, 78). The purest apoerythrocuprein was obtained using the gel-filtration technique (76). This bovine apoerythrocuprein was metal-free and homogeneous as seen from several parameters. The numerical value of the molar absorption coefficient at 259 nm was only 37% of the corresponding value of the native protein (72, 74) and the fine structure of the apoprotein was well resolved. Six maxima were detectable at 250, 258, 262, 264, 268, and 275 nm. Apoproteins prepared by the dialysis procedure displayed a much higher absorption coefficient for example, apoerythrocupreins prepared from human and bovine erythrocuprein showed 70% (69) and 54% (72), respectively, of the absorption of the native protein (Fig. 19). 

An alternative to dialysis methods is the gel filtration technique of Hummel and Dreyer (57), as modified by Price (58). A column of Sephadex G-25 is equilibrated with a buffer containing a desired concentration of calcium and 45CaCl2, and is used for gel filtration of the binding protein. As the process of gel filtration proceeds, the protein migrates in the excluded volume of the column, removing calcium ions from the column buffer until equilibrium is reached. The protein peak, in the void volume of the column, contains above-base line amounts of calcium. The amount of calcium bound to the protein can be found by dividing the molar concentration of calcium above the base line value by the molar protein concentration. A separate gel filtration run is used to determine each point on the binding plot. This is at once time-consum-... 

Gel Filtration Technique. Cellulases were among the first proteins to be separated by the gel filtration technique. Cellulolytic enzymes from Polyporus versicolor were thus separated by Pettersson et ah (39) on Sephadex G-75 (Pharmacia Fine Chemicals, Uppsala, Sweden) and simultaneously Whitaker et ah (56) applied the same technique in studies on the cellulolytic enzymes from Myrothecium verrucaria. The gel filtration method has since been so frequently used in separation and... 

The eflFect of metals on hydrogen exchange in a number of other proteins determined by the gel-filtration technique is summarized in Table IV. The difference after one hour in the number of hydrogens exchanged in each protein, in the presence and absence of the appropriate metal, is expressed both as total hydrogens per mole and as percent of peptide... 

We believe the most feasible explanation of the bimodal behavior in linewidth to be two distinct molecular weight fractions. The inability to separate the two may be due to an equilibrium between them, or unknown inadequacies in the gel filtration techniques used. The B-polysaccharide sample which we passed through an analytical column (l cm x 90 cm packed with sepharose liB) was diluted to the extent that the precise data required for a histogram analysis was difficult to obtain. 

Prior to about 1982 it was often assumed that the enzymes of the chloroplast fatty acid synthase would be organized into some sort of multienzyme complex even though the absolute requirement of all known plant fatty acid synthase (FAS) preparations for added acyl carrier protein (ACP) precluded the type of complex observed in yeast and mammalian cells. Then in 1982 three laboratories [1-3] independently showed that the FAS consisted of individual proteins that were readily separable by conventional gel filtration techniques i.e. the plant synthase was of the prokaryotic type, similar to that in E. coli. After that, the concept of a multienzyme complex synthesizing fatty acids in chloroplasts was effectively in limbo. 

With solubilisates having significant water solubility, it is of interest to know both the distribution ratio of solubilisate between micelles and water under saturation and unsaturation conditions. To measure the distribution ratio under unsaturation conditions, a dialysis technique can be employed, using membranes that are permeable to solubilisate but not to micelles. Ultrafiltration and gel filtration techniques can be applied to obtain the above information. The data are treated using the phase-separation model of micellisation (micelles are considered to be a separate phase in equilibrium with monomers). 

Gel filtration techniques were first applied to solubilized systems by Herries et al. [57] and Borgstrom [58]. A development of this technique [59-64] involves tail analysis of the elution curves to obtain information not only about the elution behaviour of the solubilizates but also of the micelles themselves. Fig. 5.4 shows a typical elution curve of methylparaben solubilized in solutions of dodecyl-hexaoxyethylene glycol monoether, Ci2E6 The heights of the plateaux (S)t and (Z>)t correspond to the concentrations of C12E6 methylparaben, respectively, in the original sample. (D)m and (D)f are the concentrations of methylparaben solubilized in the micellar phase and of free methyl paraben in the aqueous phase, respectively. The low plateaux of the elution curve of corresponds to the... 

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