Ether protein precipitation

Very little information exists on the phenolic protein-conplexing resins, except for that on creosote bush and sane arctic plants. In contrast to condensed tannins and hydrolyzable tannins, these are typically ether-soluble compounds this may allow their entry into the body across cell membranes, and thios give them the potential for action outside the gut lumen. Reports for animals consuming seaweed indicate that astringent protein-precipitating substances (presumably phlorotannins), are potentially important in marine plant-herbivore interactions. 

The fish tissue sample containing AMO residues was homogenized with phosphate buffer (pH 4.5), followed by protein precipitation with TCA and SPE on a C18 cartridge. Trace amounts of nonpolar interfering substances present in the SPE eluate were removed by LLE using ether. The final extract was reacted with formaldehyde and TCA at 100°C for 30 min. A fluorescent derivative was extracted with ether three times, and the extracts were combined, evaporated, and reconstituted in the mobile phase. No interfering peaks from the control fish extract were observed. A proposed chemical structure of the fluorescent derivative was reported and confirmed by both MS and NMR experiments (73). 

It is not possible, currently, to offer an explanation for these observations, but perhaps it is permissible to speculate. The low efficiency with methylene chloride might be explained by the fact that proteins precipitate when plasma is extracted with this solvent. Since A9-THC is bound to protein, it may co-precipitate and not be extracted from precipitate. However, co-precipitation with proteins cannot be invoked to explain the results with diethyl ether since this solvent does not cause protein precipitation. Furthermore, it is also necessary to explain the observation on extraction with toluene and the differences between rabbit and human plasma. The coprecipitation argument is simply not applicable to these phenomena. 

Figure 5.34 Infusion chromatograms covering the LC/MS assay time (2.5 minutes), obtained using the post-column infusion method shown in Figure 5.33, comparing the ability of different sample preparation methods to remove endogenous sample matrix components that interfere with the ionization of phenacetin. Panels (a) - (f) show the variation with time of the MS signed specific for the infused standard (phenacitin) following on-column injection of 10 p,L of a blank plasma sample prepared by one of the tested sample preparation methods, (a) Protein precipitation, (b) Oasis SPE. (c) Methyl-tertbutyl ether (MTBE) hquid-liquid extraction, (d) Empore C2 disk SPE. (e) Empore C8 disk SPE. (f) Empore Cl 8 disk SPE. Reproduced from Bonfigho, Rapid Commun. Mass Spectrom. 13,1175 (1999), with permission of John Wiley Sons, Ltd.

Dioctyl sebacate (DOS) with relative permittivity e of 3.9 and 2-nitrophenyl octyl ether (NPOE) with e = 23.9 are the traditionally used sensor membrane plasticizers. The choice of a plasticizer always depends on a sensor application. Thus, NPOE appears to be more beneficial for divalent ions due to its higher polarity, but for some cases its lipophilicity is insufficient. Furthermore, measurements with NPOE-plasticized sensors in undiluted blood are complicated by precipitation of charged species (mainly proteins) on the sensor surface, which leads to significant potential drifts. Although calcium selectivity against sodium and potassium for NPOE-based membranes is better by two orders of magnitude compared to DOS membranes, the latter are recommended for blood measurements as their lower polarity prevents protein deposition [92],... 

Tyrothricin was obtained by acidification of the fermentation broth of Bacillus brevis to precipitate the antibiotic activity along with various proteins and then dissolving the antibiotic complex in alcohol. The alcohol was removed under vacuum, the residue was washed with ether, then redissolved in alcohol and finally reprecipitated with 1% sodium chloride5 6. 

The reactions were carried out in each case with a 0-1 per cent protein solution in phosphate buffer (pH 6 8), to which the radioactive phosphorofluoridate was added as a concentrated solution in dry ethanol. At the end of the reaction time, the product was dialysed for 20 hr. against running water, and precipitated at 0° by addition of two volumes of acetone. The precipitate was spun off and washed at —5° with ethanol and ether, and dried in air or over sulphuric acid. Samples of 25-50 mg. of dry powder were used for radioactivity determinations, and compared with a standard prepared by hydrolysing a weighed amount (ca. 1 mg.) of the phosphorofluoridate in n sodium hydroxide, neutralizing and drying. 

Cohn, and their associates in Harvard during World War II in their efforts to isolate serum proteins for clinical use. Traces of the organic solvents were removed during the freeze-drying of the different preparations. In the U.K. plasma fibrinogen was obtained by Kekwick and his colleagues by low-temperature precipitation with ether. Serum proteins acquired by these chemical methods were mixtures. 

Organic solvents. Addition of organic solvents decreases the solubility of proteins by reducing the dielectric constant of the medium. For the precipitation of enzymes, methanol, ethanol or propanol are mostly used, but acetone and diethyl ether can also be employed. The principal disadvantage of organic solvents is their tendency to cause stmctural damage of enzyme molecule. 

A similar procedure was developed for NFZ, FZD, and FTD residues in porcine muscle tissue and eggs. The tissue samples were extracted twice with dichloromethane ethyl acetate, and the combined organic layers were evaporated to dryness. The residue was dissolved with dichloromethane and petroleum ether. Egg proteins were precipitated with MeCN, and the supernatant was extracted with dichloromethane and petroleum ether. The organic layers were applied directly on a silica SPE cartridge. The optimal recoveries (84-111 %) were achieved after elution with dichloromethane ethyl acetate (146). 

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