SU566873A1 - Chemical agent for qualitative assessment of proline - Google Patents

Description

This invention relates to the microbiological industry and concerns the use of thiosemia, carbazone-3 1-methylisatin and its derivative 1,6-bis - (1 - methyl isatin thiosemicarbazone - 3 - yl-4) hexane of the general formula, -Mi - with -NHR 0S where R is hydrogen or - (CH2) b-, as organic reagents for the qualitative determination of proline, in particular for the specific manifestation of the proline spot on chromatograms, and can be used in biochemical and clinical laboratories. Unsubstituted thiosemicarbazone-3 1-methylisatin, known as Metisazop, is currently used only as a drug 1. A lot of reagents are used to identify amino acids, of which ninhydrin, alloxan, unsubstituted isatin, quinisatin, indandion are most known. -1.3 2. These compounds are not specific for individual amino acids and are used most often for the polychromic manifestation of these acids against thin-layer chromatograms. The aim of the invention is to identify a range of isatin derivatives of compounds that could be used as specific amino acid spots in chromatograms. This is achieved by using a known drug, metizazone and its derivatives, as specific reagents only on prolias, which make it possible to identify its presence even in mixture with other L-amino acids. When Proline interacts with thiosemicarbazone-3 Methylisatin, a compound is formed, for which the maximum of Ymax max of 586 is determined (in methanol). Example 1. Filtrak No. 14 chromatographic paper was applied with a micropipette of 0.01 ml, 0.02 ml, ... to 0.07 ml of an aqueous solution containing 0.02 mg of proline in 1 ml. After short drying with 50 ° C, paper is pulled through a 0.4% solution of methisazone in acetone acidified with acetic acid (a mixture of 96 ml of acetone and 4 ml of glacial acetic acid). The paper is then placed for 3-5 minutes in a thermostat heated to 115 ° C, while spots containing 1 mg or more of proline are colored green. Similarly, the proline spot exhibits in the case of 1,6 - bis - (1 - methylisatin thiosemicarbazone - 3 - yl - 4) -hexane garbage, more

strongly acidified (until a clear solution is obtained) preparation solutions. Feel ,, n-r-NlcH ,, - ai-c-3JiT-N. A.-H

SNS

Example 2. One drop of an aqueous solution of amino acids, containing 1 mg of DL-a-alanine, L-arginine hydrochloric acid, aminoacetic acid, L-asparagine, DL-p-alanine, Bb-p-phenyl-a-ala.nin each , DL-valEna, L-histidine hydrochloride, L - glutamic acid, DL - leucine, DL - lysine hydrochloric acid, DL-methionine, DL-norvaline, DL-norleucine, DL-ornithine hydrochloride, L - cysteine hydrochloride, DL-serine, DL-tryptophan, L-hydroxyproline and proline are applied on Filtrak No. 14 chromatographic paper. The paper is dried and chromatographed in a solvent | Telly n-bu system. yl alcohol - glacial acetic acid - water (4: 1: 5 upper layer). After 18 h, the chromatogram is dried in a thermostat at 70 ° C and sprayed with solutions (as in Example 1) of thiosemicarbazone-3 1-methylisatin and its hexamethylene bis-derivative. After heating the chromatogram for 3-5 minutes at 115 ° C, only the proline spot appears.

The proposed reagents do not produce colored spots on chromatograms with L-amino acids (except for proline), even in an amount of 20 mg. At the same time, the proline gives a noticeable green spot already at 1.0 mg, at 20 mg the spot is dark blue-green, the background of the chromatogram is light orange.

The accuracy of proline detection is almost the same as the previous method.

The proposed reagents allow faster and better quality than the existing methods to analyze and determine the presence of proline in the amino acid mixture.

Claims (2)

1. US Patent No. 3253991, cl. 424-274, 1966. 2. Analytical methods of protein chemistry. M., publishing house of foreign literature, 1963, p. 491-560.