SU1131506A1 - Method of obtaining mitolectin (phytohemagglutinin) from bean seeds - Google Patents

Abstract

METHOD FOR PRODUCING MITOLEKTINA (FITOGEMAGGLNLININA) FROM SEEDS BS -J2rj 3i: FASSLI bean flour by extraction with hydrochloric acid, followed by a two-step ammonium sulfate salting out, dialysis and purification, characterized in that, in order to reduce the time of preparation and povppeni title product activity, extraction carried out in the presence of 2 mmol of sodium metabisulfine and for 1 h, salting out. ammonium sulphate was carried out with a flaton during the first salting out of 40Z, and subsequent purification was carried out by filtering through a cut of Sephadex G-100 in a 10% solution of sodium chloride at pH 7.0.

Description

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ate

About ot The invention / relates to biology, in particular, to the production of a biologically active glycoprotein from bean seeds, and can be used in cyto- and immunogenetic analysis, in membranology, and immunology. A known method for producing mitolithin by extracting the bean flour with dilute hydrochloric acid, two-stage salting out with ammonium sulfate followed by dialysis and purification with acetone f 11 However, the known method does not provide sufficient activity of the imje product and is time consuming and laborious. The purpose of the invention is to increase the activity of the target product and reduce the time it is received. This goal is achieved in that according to the method of obtaining mitolelectin (phytohemaglutinin) from bean seeds by extracting flour from salt with a solution of hydrochloric acid followed by a two-step salting out with ammonium sulfate, dialysis purification, extraction is carried out in the presence of 2 mmol sodium metabisulfite for 1 hour , salting out is carried out with ammonium sulphate with saturation with salting out 40%, and subsequent purification is carried out by filtration through Sephadex G-100 in 10% sodium chloride solution at pH 7, Example Bean Seeds Red n In 2005, 15–19 of the 1982 harvest was ground and sifted through a sieve so that the average particle size of the flour was 250300 microns. 2.5 l of cold distilled water was added to 0.5 kg of flour (all operations were carried out at 4 ° C with stirring and the pH of the mixture was adjusted with concentrated hydrochloric acid 1 (pH was controlled by reddening the paper of the universal pH indicator) Extraction was carried out in the presence of 2 mmol of antioxidant ( sodium bis-sulfite) for 1 hour with constant stirring. The mixture was centrifuged in a Kg-70 centrifuge (GDR) at 4000 rpm for 10 minutes. Pressed (to remove large lumps) crystalline ammonium sulfate X was added to the supernatant or OFC in the amount of 304 t in 1 l of the mixture with stirring. After 1 h, the mixture was centrifuged for 2 h at 4000 rpm, the precipitate was discarded, and the supernatant was sprinkled with protein a second time by adding ground ammonium sulfate so that the final salt concentration ( taking into account the first addition of salt) was 380 g in 1 liter of the mixture (taking into account the second addition of salt). After 1 h of stirring, the mixture was centrifuged for 2 h at 4000 rpm (as after the first protein salting out), the supernatant was discarded, and the sediment was subjected to . dial ive distilled water (1:20 by volume, changing the water 3) 48 hours. The mixture was centrifuged at 4000 rev / min for 20 minutes. the precipitate was discarded and the supernatant was adjusted to pH 5.4. The deposited precipitate was removed by centrifugation for 20 minutes at 4000 rpm, and bidiscated water (1:10 by volume) was added to the supernatant. The mixture was immediately centrifuged for 1 hour at 4000 rpm, the resulting precipitate was dissolved in 10% sodium chloride solution with pH 7 (in a volume equal to 1/20 of the volume of the column with Sephadex G-100) and gelfiltrated through a column with Sephadex G -100 cm in size and 20 cm in height. The first mitolectin peak emerging from the column was collected and lyophilized. The yield of mitolectin (phytohemagglutinin) was 1.45 g from 1 kg of bean seed flour. The proposed method is illustrated in Table. 1 and 2. Thus, the proposed method allows to increase the mitogenetic activity of the target product and shorten the time of its production.

The corresponding mitogenetic

saturation of the extrinsic activity of celeract sulfate product,

ammonium, %% RBTL

3570.1

/ 4080.2

Time h

2424

eleven

Table 1

table 2

1st salting out | 2nd salting out

Claims (1)

METHOD FOR PRODUCING MITOPECTIN (PHYTOGEMAGLUGININ) FROM SEEDS BEANS by extraction of bean flour with a solution of hydrochloric acid followed by two-stage salting out with ammonium sulfate, dialysis and purification, characterized in that, in order to reduce the production time and increase the activity of the target product, the extraction is carried out in the presence of 2 mmol sodium metabisulfite for 1 h, salting out carried out with ammonium sulfate with a saturation at the first salting out of 40%, and subsequent purification is carried out by filtration through Sephadex G-100 in a 10% solution of sodium chloride at pH 7.0.