SU805146A1 - Method of quantitative analysis of protein in bio-liquid - Google Patents

Description

(54) METHOD FOR QUANTITATIVE DETERMINATION OF PROTEIN IN THE BIOLOGICAL TEKIDOSTY, the reduction and reduction potential is in the range of pH 6,, 7. In the process of recovery of the heteropoly acid, gradual neutralization of the spiky components of the mixture hydrolys by the acid components of the Farnna-Cheokolte reagent occurs. At the same time, the composition of the hydrolizuccine mixture includes ingredients with various pK-from pK S (sodium) and (pK input of soda carbonate reagent) to 10 (pK sodium carbonate) and, therefore, during the reaction, depending on the quantities of alkaline hydrolyzing mixture and acidic reagent Fo 0 on Cheokolte, the pH of the medium can be stabilized, taken from within wide limits 13-7-10,2. This, in turn, can ensure the inadequacy of the process of phospho-molybdate framing hetero-polypotting with the predominant involvement in the process, the restoration of phospho-molybdeic or phopholoframe polyacids with a corresponding change in the spectral characteristics obtained by heteropoly lines and, ultimately, distorting the amount of quantities. Therefore, in order to ensure the restoration of only one of the polyacids that make up the Foil-Cheocolte reagent, and the stability of the spectral characteristics, the peaKJuaio must be brought in such a way that by the end of the analysis its alkaline state will stabilize. This limit will ensure the constant involvement and reaction of one of the polyacid mixtures and the stability of the resulting heteropolysini. This is achieved by equimolar neutralization of the alkaline and acidic components of the hydrolyzing reagent (a mixture of sodium carbonate and sodium hydroxide (1), and the Folin-Chekoko solution; dissociation of these conditions of the HCO ion with 10 pK2 pH 10.2, 0.5 ml of Folin-Cheokopte reagent diluted to an acidity 2.24 times the alkalinity of the reagent 1), popstory neutralize sodium hydroxide with pK iO, which is part of 2 ml of reagent 1), and sodium carbonate with, Under these conditions x the pH of the mixture is stabilized within 1010, 5 due to the dissociation of the HCOj ion, which ensures the selectivity of the restoration of the phosphomolybdate-tungsten geopolyacid; the stability of the spectral characteristics and the optical density of the heteropolisini formed; the possibility of carrying out the reaction at eiicoKofi temperature; the acceleration of the recovery process. Under these conditions, the selectivity of the heteropolyacid reduction by cyclic amino acids is achieved: cystine cysteine, in concentrations of 100 mg per 10O ml of protein solution, ascorbic acid and sucrose, in concentrations of 5OOm per 100 ml of protein are replaced by the reduction process. In these cases, the need for protein binding to the biurethane complex is also eliminated, which eliminates this step from the analysis and, therefore, simplifies the reaction. Example 1.; The Fopin-Cheokolte reagent: 100 g of sodium tungstate and 25 g of sodium molybdate are dissolved in 700 ml of distilled water, 50 ml of 8S% phosphoric acid and ml of concentrated hydrochloric acid are added. The mixture is boiled in a round-bottomed flask equipped with a reflux condenser for 10 hours. The resulting reagent is titrated with phenolphthalein or with reagent 1 controlled by a pH meter. The working solution is prepared by diluting reagent 2 with distilled water to a pH of 2.24 times that of the reagent. 1. And a curable solution of protein (serum, plasma extracts of organs and tissues, solutions of protein preparations - diluted to a content of no more than 10 mg of protein, 1 ml of solution). Example 2. About 2 mp of the protein solution to be tested is added to 2 ml of reagent 1, the samples are incubated for 10 minutes in a boiling water bath, cooled and 0.5 MP of the working solution 2 are added. The refill is maintained in boiling water 40 s, cool and photomet at 640-74 O n in cuvettes of 3 mm. The obtained optical density of the samples is compared with a standard graph of absorbance versus protein concentration. When conducting an analysis using this method, the colored complex is maintained for 48 hours, the resolution of the method is 2 micrograms in 0.2 ml of solution.

58051464

Disturbance of the effects of various additives on protein concentration by the compared methods

Table

Cysteine

Cystine

Tryptophan

Ascorbic acid

Sucrose

Water (protein benchmarks) are statistically significant differences from the optical density of samples when determining the protein concentration by the ttatfir method is higher than that determined by the Lowry method, which allows reducing the width of the colorimetric layer from 1O to 3 mm. The Lambert-Bera law is preserved both in the proposed method and in the Lowry method to a protein concentration of 1%. Solutions with greater concentration must be diluted before determination. Whether or not human albumin solutions have been prepared with a content of 2O, 4O, 6O, 8O, 10O, 2OO, 600, and 8OO micrograms of protein in the sample. At each of the dilutions in 6 parallels, the protein content is determined by this method and the Lowry method. 20 4.5 0.252 0.101 5.6 6.7 40 6.25 0.188 0.075 3.0 8.5

200+ 3.8

252 i 4,1 24Oi-3,9 2О2 ± 2,7

286 ± 4,3

222 ± 2.4 12О ± 6., О

201t 4.2 2 lot 4.4 20O ± 3.6

2OOt5,620О ± 2, В benchmarks. In order to standardize yciiOBiitft. Photometric measurements in both methods used a cuvette of 3 mm, and for: ease of calculation, only 1 absorption was expressed in whole numbers. The results of determining the optical density of protein solutions in each of the dilutions were subjected to statistical processing, calculating the arithmetic mean (M), the standard deviation of a single determination (G), the mean error {t}, and the variation coefficient (V%). The data obtained are given in Table 2. Table 2 0.32 0.1228 4.8, 0.544 0.217 6.4

805146 8

Continued table. 2

Claims (1)

Claim The method of quantitative determination of protein in biological fluid by treating a sample of the analyzed fluid with 40 aqueous solution of a mixture of sodium carbonate and sodium hydroxide, followed by processing the resulting solution with Folin – Cheokolte reagent and photometry of the obtained colored compound, about 45 and 45. Part one with the fact that, with a chain of increasing the accuracy of the method and its simplification, the Folina-Cheokolte reagent is treated at a pH of 9.8-10.2. 50 Sources of information taken into account in the examination 1. Pushkin N. I. ’Biochemical research methods, Medgiz, 1963, p. 10 (prototype). / Circulation 918 Subscription Branch of PPP Patent * ', Uzhhorod, st. Project, 4