SU1752187A3 - Method for delipidation of lyophilized lipoproteins of high density from human blood serum - Google Patents

Abstract

Use: for preparing preparative amounts of apolipoproteins that are part of human serum lipoproteins. SUMMARY OF THE INVENTION: To 2 g of lyophilized high-density lipoprotein is added 200 ml of a mixture of chloroform-methyl alcohol (3: 1) which is cooled to a temperature of and mixed for 30 minutes. The suspension is mixed with 800 ml of a diethyl mixture cooled to 0 ° C. ether, stirred for 30 minutes at 0 ° C, filtered and the filter cake was dried under reduced pressure. 1 ex.

Description

The invention relates to biological chemistry, namely to methods for the preparation of preparative amounts of apolipoproteins that are part of human serum lipoproteins.

There is a known method for delipidation of lyophilized lipoproteins, which consists in the following. that 20-50 mg of lyophilized lipoproteins are extracted at -20 ° C for 30 min 3 x 35 ml ethyl alcohol-diethyl ether mixture (1: 1 by volume), after each extraction, the precipitate of lipoprotein proteins from insufficient liquid is centrifuged at 2000 rpm for 30 min at 4 ° C. Then 35 ml of diethyl ether are added to the precipitate, incubated for 3 minutes at -20 ° C, centrifuged at 2000 rpm for 30 minutes at 4 ° C, after which the supernatant is drained and the precipitate is dried in a stream of nitrogen.

However, this method of delipidation of lyophilized lipoproteins is poorly productive, multistage, and very labor intensive.

The closest to the present invention is a method of delipidation of lyophilized high density lipoproteins from human serum, which includes extraction steps in a 50 ml centrifuge tube of lyophilized high density lipoproteins with chloroform-methyl alcohol (1: 2 by volume), based on 50 ml of mixture per 200 mg of protein for 30 minutes at -10 ° C and stirring once every 10 minutes by inverting the tube; centrifuging the resulting mixture at 2000 rpm for 10 min at 6 ° C and decanting the supernatant These operations are repeated six times, then the precipitate is extracted in the same tube with diethyl ether at the rate of 50 ml of the mixture per 200 mg of protein for 30 min at -10 ° C and stirring once every 10 min by transferring

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with

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Ca

After centrifuging the tubes, centrifuge the mixture at 2000 rpm for 10 minutes at 6 ° C, decant the supernatant, repeat these operations again, and dry the precipitate in air.

The disadvantages of this method are the time consuming and manual labor in obtaining preparative quantities of high-density, delipidated lipoproteins, long-term use of a centrifuge with an integrated refrigeration unit. For centrifugation at a low temperature, the need for constant human participation during delipidation for a long time.

The aim of the invention is to simplify and speed up the process of delipidation of high density lyophilized lipoproteins from human serum.

The goal is achieved by the fact that the proposed method for the delipidation of high-density lyophilized lipoproteins from human blood serum at the stage of multiple extractions with chloroform-methyl alcohol and the subsequent washing of the precipitate of apolipoproteins with diethyl ether is combined into a single stage, without the accumulated sample from the volcanic acid, with the ethyl ether being combined with diethyl ether in a single step without the separation of the precipitates from the alphanoproteins by diethyl ether in a single step without the separation of the precipitates from the volcanic acid by diethyl ether. also using the method of mechanical filtration under reduced pressure to determine the final product (fine apolipoproteins of precipitate) from the mixture of organic solvents,

New in the proposed method of delipidation of high-density lyophilized lipoproteins is the separation of finely dispersed sediment of apolipoproteins from a mixture of organic solvents by mechanical filtration under reduced pressure and the combining of extraction and washing steps of the precipitate of apolipoproteins without first separating the precipitate of apolipoproteins from chloroform-methylomethylomethylomethylamine-apolipoprotein precipitate without prior separation of the apolipoprotein precipitate from the chloroform-methanol precipitate from the chloroform-methylene precipitate.

Example. High-density lipoproteins (1.063-1.210 g / cm3) were isolated from human serum by sequential ultracentrifugation. The resulting fraction of high-density lipoproteins in the form of a clear, light yellow liquid is transferred to a dialysis bag, dialyzed against 9 l of 10 mM ammonium bicarbonate solution for 15 hours at 4 ° C, after which the solution of high-density lipoproteins is transferred to a round bottom flask, freeze while rotating the flask in the cooling mixture (dry ice - acetone or liquid nitrogen) and lyophilize for 15 h.

The resulting lyophilized high density lipoproteins (2 g) in the form of

a dry, light yellow, fluffy mass is placed in a round-bottom flask, 200 ml of chloroform-methyl alcohol mixture (3: 1 by volume) is added and cooled to 0 ° C and stirred vigorously with a magnetic stirrer at 0 ° C (ice on a bath) for 30 min, after which the resulting mixture in the form of a thin suspension of light yellow color is mixed with 800 ml of diethyl ether cooled to 0 ° C and the resulting mixture is vigorously stirred with a magnetic stirrer at 0 ° C for 30 minutes. The resulting mixture in the form of a thin suspension of light yellow color is filtered using a glass funnel with a porous

filter number 3 under reduced pressure (vacuum water-jet pump). The residue of the apolipoproteins in the form of white powder is dried under reduced pressure (vacuum of a water-jet pump), tightly closing the glass funnel with a soft rubber plate.

To determine the protein content before delipidating a portion of high-density lyophilized lipoproteins

(10 mg) is dissolved in 1 ml of potassium phosphate buffer (10 mM KH2RS, pH 7.4) containing 0.9% sodium chloride NaCl. The resulting solution is analyzed for protein content by the method of Lowry. The concentration of the total protein in the 4.6 mg / ml solution of the solution, which in terms of the initial 2 g of lyophilized high-density lipoprotein, is 0.92 g of the total protein (46% by weight).

For the analysis of the apolipoproteins obtained after the delipidation by the proposed method, the residue of the apolipoproteins obtained after the delipidation of 2 g of high lyophilized lipoproteins

density, weigh balance weight 0.90 g

A portion of the residue (10 mg) was dissolved in 1 ml of potassium phosphate buffer (10 mM KH2P04, pH 7.4) containing 0.9% NaCl, and the resulting solution was analyzed for content

protein as indicated. The concentration of the total protein in the sample is 9.9 mg / ml of solution, which, in terms of the baseline, 9 g of the residue of api-dipoteins is 0.891 g of the total protein (99% by weight). Extraction rate

apoliproteins with delipidation by the proposed method of 97%. The resulting value is a ratio of 0.891 g (total protein content in the dry residue of apoliproteins after delipidation) to 0.92 g (total content

protein-lyophilized high-density lipoproteins before delipidation).

The amount of non-protein impurities in the dry residue of apoliproteins does not exceed 1%.

The proposed method for the delipidation of high-density lyophilized lipoproteins from human serum has been repeatedly and successfully used to isolate high purity apolipoproteins A-I and A-P from high-density lipoproteins. The isolated preparations of proteins A- and A-I were used successfully in various diagnostic and immunological studies.

In the proposed method of delipidation of lipoproteins from human serum compared to previously known ones, the amount of manual labor is minimized, centrifugation at a low temperature is completely excluded, the amount of organic matter used is reduced

solvents and total time spent for delipidation (approximately 8-20 times)

Claims (1)

Invention Formula Method for delipidation of lyophilized high-density lumpoproteins from human serum, including primary extraction of lipids with chloroform-methyl alcohol mixture at low temperature and reextraction of the mixture obtained with diethyl ether, characterized in that, in order to simplify and speed up the process, for the primary extraction, a mixture of chloroform-methyl alcohol with a content of methyl alcohol of 25-60% by volume in an amount of 100-300 ml per 1-3 g of lyophilized lipoproteins, for re-extraction - two to six times the volume of diethyl ether, extractions are carried out with stirring for 15-45 minutes at (-10) -40 ° C. the precipitate is then filtered off and the target product is dried in vacuo.