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Journal ArticleDOI

Glutamate labelling pattern obtained from acetate-1-C 14 and sodium bicarbonate-C 14 by incubation with rat liver slices

01 Jan 1958-Naturwissenschaften (Springer-Verlag)-Vol. 45, Iss: 21, pp 521-522
About: This article is published in Naturwissenschaften.The article was published on 1958-01-01. The article focuses on the topic(s): Sodium bicarbonate & Labelling.
Topics: Sodium bicarbonate (60%), Labelling (52%), Incubation (51%)
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Journal ArticleDOI
TL;DR: In the present investigations of the chromatographic separation of amino acids, it has been observed that, when the color development is carried out in tubes exposed to the air, these difficulties appear to result primarily from the influence of dissolved oxygen.
Abstract: For the investigations on the chromatographic separation of amino acids outlined in the preceding communication (1)) it was necessary to have available a suitable quantitative method for the determination of the concentration of amino acids in the effluent from the column. For this purpose, the method should be sufficiently general to include the determination of most of the amino acids and peptides likely to be encountered in protein hydrolysates or other material of biological origin. The method should have as high a sensitivity as possible to permit the determination of low concentrations of amino acids in the effluent from the chromatogram. Also the laboratory procedure should be fairly simple to permit the method to be applied conveniently to large numbers of effluent samples. It appeared probable that a photometric method would best fulfil these requirements. The two calorimetric methods of this type which had received the most study were the procedures based on the use of /3-naphthoquinonesulfonic acid and ninhydrin (triketohydrindene hydrate) as reagents. For reasons which will be described, the ninhydrin reaction was selected for further investigation. The color reaction between CY-NH2 acids and ninhydrin has been studied extensively in the past. It has been established that colored compounds are formed not only with amino acids, but also with peptides, proteins, and other classes of substances possessing free amino groups. The reaction is known to be extremely sensitive for qualitative work. In earlier attempts to render the color reaction quantitative (2-S), however, it has been found that the color yield per microgram of amino acid decreased markedly as the concentration of amino acid was reduced. In addition, the results have not been reproducible. In the present investigations, it has been observed that, when the color development is carried out in tubes exposed to the air, these difficulties appear to result primarily from the influence of dissolved oxygen. Improved results can be obtained when the reaction is performed in tubes evacuated to 20 mm. Under these conditions, the relationship between color yield and amino acid concentration is more nearly linear, although the deviations are still marked. By the addition of a strong reducing agent directly to the reaction medium, however, the oxidative side reaction has been eliminated. In preliminary experiments

2,803 citations


Journal ArticleDOI
Abstract: In the present method the CO2 evolved by decarboxylating amino acids with ninhydrin is transferred to standard barium hydroxide and titrated. The conditions for making the ninhydrin reaction quantitative are those previously worked out for applicat,ion in the manometric method (6, 7). The transfer of the CO, to the barium hydroxide is done by a distillation in vacua which is completed in 2 to 3 minutes. The titration, with added barium chloride to insure complete precipitation of the barium carbonat,c formed, follows a principle used by Krogh and Rehberg (2). After the present method was completed Christensen, West, and Dimick (1) published a procedure in which Van Slyke and Dillon’s (6) conditions for the quantitative ninhydrin reaction were applied, and, as in the present method, the CO2 was distilled into standard barium hydroxide and tit.rated. We nevertheless publish the present method because it is carried out somewhat more rapidly, and with cheaper and simpler apparatus. A pair of small Erlenmeyer flasks attached to a U-tube provides all the special apparatus required for the reaction, the distillation, and the titration. The titration yields results of the same order of constancy as the manometric method in the macroand microanalyses. In the submicroanalysis, however, (carboxyl carbon under 0.1 mg.) the titration shows less constancy than the manometric procedure. When the manometric apparatus is available, the analysis based on it will usually be preferred, particularly for small amounts. The manometric procedure requires fewer precautions against

168 citations




Journal ArticleDOI
TL;DR: Experiments of a similar nature involving glutamic acid as the target molecule will be described in this paper, and it is predicted that carboxyl-labeled 4-carbon dicarboxylic acids (oxalacetic), 3-carbon glycolyt,ic acids (pyruvic), or their biological precursors (aspartic acid, alanine) would also give rise to glutamic acids labeled only in carbon 1.
Abstract: The isotope pattern found in rat liver glycogen following administration of a variety of carbon-labeled compounds has been used by Wood and his coworkers (l-6) in studies concerning carbon metabolism. Experiments of a similar nature involving glutamic acid as the target molecule will be described in this paper. Its relative abundance, ease of isolation, and interconversion in ti~o with cY-ketoglutarate, a key intermediate in the Krebs tricarboxylic acid cycle, make glutamate a useful compound for studying metabolic pathways. Such studies with microorganisms (7-11) and rats (12-14) have been described. However, no data are available concerning the complete isotope pattern found in glutamate derived from mammalian tissue following the administration in wivo of carbon-labeled substrates. With the exception of purine synthesis, those in viva mammalian CO* fixation reactions which result in synthesis of carbon to carbon bonds involve compounds of the Krebs tricarboxylic acid cycle. The principal reaction is the formation of oxalacetic (15) or malic acid (16). Fixation of C1402 in this manner gives rise to a-ketoglutarate, and hence glutamate, labeled only in carbon 1 (17-Zl).’ It would be predicted, therefore, that carboxyl-labeled 4-carbon dicarboxylic acids (oxalacetic), 3-carbon glycolyt,ic acids (pyruvic), or their biological precursors (aspartic acid, alanine) would also give rise to glutamic acid labeled only in carbon 1. However, administration2 of nn-glyceric acid-1-C14, bicarbonate-Cl4 (12), and C1402 to rats (13), and nn-aspartic acid-4-C14 to rabbits: resulted in tissue glutamic acid having as low as 70 to 83 per cent of its total radioactivity located in carbon 1. Since in none of these investigations was a complete degradation of glutamat,e accomplished, the location of activity other than in carbon 1 is not known.

45 citations