Trichloroacetic acid, extraction

The walls of Gram-positive bacteria, unlike the membranes, sometimes do not contain teichoic acids. However, there are numerous examples of walls which do contain these compounds in substantial proportions, although in relatively few cases has detailed structural work been carried out. The purification of wall teichoic acids is often much more readily achieved than for the membrane polymers. Addition of ethanol to trichloroacetic acid extracts of walls gives a precipitate which is usually pure teichoic acid. Extraction is effected at low temperatures, and for a reasonably short time, in order to avoid extensive degradation of the polymers under the acidic conditions. Consequently, although walls may contain 20-50% by weight of teichoic acid, practiced yields are generally rather low. 

Trichloroacetic Acid Extraction. The remaining 10 insects injected with Na2S3504 were homogenized in 5% (w./v.) trichloroacetic acid and the homogenate was kept at 100°C. in a water bath for 30 minutes. The homogenate was centrifuged, and the residue was washed once with 5 ml. of 5% trichloroacetic acid and then with distilled water. These fractions were added to the original supernatant solution and the combined mixture was dried in vacuo. The proteinaceous residue was then washed three times with a total of about 25 ml. of acidified ethanol (2... 

Preparation of Extracts for Chromatography. The dried ethanolic extract was dissolved in 2 ml. of 10% (v./v.) 2-propanol, shaken with an equal volume of chloroform, and centrifuged. The chloroform layer was discarded. The trichloroacetic acid extract was taken up in 2 ml. of water and extracted four times with an equal volume of ether to remove trichloroacetic acid and lipides. The ether extract was discarded. 

Fig. 6.—Quantitative, Precipitin Curves of Rat-liver Glycogen with Con A. (O, Potassium hydroxide-extracted glycogen A, trichloroacetic acid-extracted glycogen , water-extracted glycogen. Con A, 48 /xg of nitrogen. After Ref. 369.)...

In addition to the usual adenine nucleotides (such as adenosine 5-phosphate, adenosine 5-pyrophosphate, and adenosine 5-triphosphoric acid) present in bacterial extracts, several adenine nucleotides of uncertain structure have been reported. These substances, termed adenylyl peptides have been isolated from trichloroacetic acid extracts of Staphylococcus aureus ... 

It was found that most of the 2-deoxyribosylic compounds in a trichloroacetic acid extract of the bacteria are present chemically in such a combination that snake-venom treatment is required in order to make them microbiologically active. Furthermore, when the extract was tested without venom pretreatment but in the presence of thymidine, the growth-promoting effect of the 2-deoxyribosylic compounds was 10 times that displayed without thymidine and reached 60% of the activity obtained after venom treatment. The nature of the substances responsible for this effect has not yet been described. The acidic nature of the 2-deoxyri-bosylic compounds was shown by the fact that 98% of the mixture was adsorbed to a Dowex anion exchanger. In the eluted fractions, thymidine 5-phosphate, thymidine 5-pyrophosphate, and thsmiidine 5-triphosphoric acid were identified. From the major 2-deoxyribosylic fraction, thymidine rhamnosyl pyrophosphate (XIX) has been isolated. This substance was also identified in the thymidine-requiring mutant of Escherichia coli 15 T-. 

Two major metabolites were isolated by paper chromatography (from trichloroacetic acid extracts of the cells) and were identified as 8-azaguano-sine 5-phosphate and 8-azaguanosine, respectively. The two compounds were present in the ratio of 4 1. The structure of the 8-azaguanosine 5-phosphate was indicated by chromatographic and electrophoretic comparison of it with (a) the 5-nucleotide prepared by the action of snake venom on the bacterial ribonucleic acid containing 8-azaguanine, and (b) the 3-nucleotide prepared by alkaline hydrolysis. 

Leggett GE and Westeemann DT (1973) Determination of mineral elements in plant tissue using trichloroacetic acid extraction. J Agric Food Chem 21 65-69. 

A compound having an aspartic acid moiety attached to the 5-position of a uracil ring has been isolated from the trichloroacetic acid extract of pea seedlings [439]. The exact structure is not known. 

Fio. 1-5. Nucleotides of the human erythrocyte. Trichloroacetic acid extracts of fresh erythrocytes were chromatographed on a column of Dowex-1 resin (formate form) eluted with a gradient of ammonium formate. [From G. Bartlett, Patterns of phosphate compounds in red blood cells of man and animals, in Adwinces in. Experimental Medicine and Biology (G. J. Brewer, ed.), Vol. 6, p. 245. Plenum Press, New York, 1970. Reproduced with permission.)... 

Folic acid can be determined in methods designed for multivitamin analysis either from multivitamin preparations (90-93) or from fortified samples such as infant milk (94). In these methods trichloroacetic acid extraction of liquid and powdered infant milk followed by ion pair chromatography with reversed-phase Cig column was applied. Satisfactory separation was achieved with octanesulfonic acid (5 mM) with triethylamine (0.5%) in methanol-water (15 85 v v) at pH 3.6 (Fig. 6). UV detection and wavelength switching were used for six vitamins giving sensitivity of 1 ng folic acid per injection (282 nm for folic acid). Total run time of this isogratic separation was 55 min. This approach has not been reported for nonfortified samples. 

Subsequently, Schneider (94) suggested the addition of the hot trichloroacetic acid extraction step to the acid-soluble fraction remaining at the end of the ST procedure, thus permitting more direct isolation of the DNA fraction. This procedure (the Schmidt-Thannhauser-Schneider, or STS, procedure), which permits the use of either phosphorus or sugar assays as the measure of nucleic acid, has become a standard rapid tissue assay. Certain refinements necessary for conversion of a tissue assay method to an isotope assay method, involving more precise segregation of cellular constituents, can be regarded as- extensions or adaptations of the basic procedure. For these reasons, the procedure is described in detail. 

Logan et al. (66), in a careful study, found that the ST and S methods yielded high phosphorus values in nerve tissue presumably due to the presence of the lipoprotein described by Folch et al. (46). In addition, trichloroacetic acid hydrolyzates (93) of brain and nerve tissue contain chro-mogenic material that interferes with the Dische and Mejbaum tests. Comparative ultraviolet spectrophotometry (see Sections II,1,E and III,4) of trichloroacetic acid solutions, with proper blanks, was found to yield reliable results perchloric acid extracts gave high results. Di Carlo and co-workers (37-39) were also successful in measuring spectrophotometri-cally the total nucleic acid derivatives found in hot trichloroacetic acid extracts. 

Fio. 9. Effect of azaeerine on the pyridine nucleotide level of mouse liver. Determinations were made on a 6% (w/w) trichloroacetic acid extract of mouse liver. Each value represents the pooled samples of two mice. Curve 1, saline control curve 2, asaserine-injected animals. From Narrod et al. (ttO). 

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