RU2129429C1 - Method of isolating complex destabilase-prostaglandin from medicinal leech - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves purification of treated raw by affinity chromatography in reversed columns containing antibodies raised to 6-ketoprostaglandin-F_1α and immobilized on water-insoluble carrier. Invention can be used in hematology, biochemistry and medicinal industry. EFFECT: increased yield of the end product volume, improved purity. 1 tbl

Description

 The invention relates to medicine and can be used in biochemistry, hematology, medicine and the medical industry.

 A known method of producing destabilase from a leech, we took as a prototype (1), including the preparation of aqueous extracts of medical leeches, extraction with ethyl acetate. As raw materials, the secretion of the salivary glands of leeches, the powder of lyophilized dried leeches and the extract from the culture of the symbiotic bacterium of medical leeches are used.

 However, this method has the following disadvantages: the final product is not a homogeneous preparation, because it contains hirudin and a kallikrein inhibitor.

 The aim of the invention is to increase the yield of the final product and increase its purity.

This goal is achieved by the fact that affinity chromatography is performed on antibodies to 6-keto-prostraglandin F _1α immobilized on a water-insoluble carrier.

 We give examples of the method.

Example 1. Lyophilized antiserum to 6-keto-PG was immobilized on bromine-activated agarose. 5 ml of an aqueous extract of lyophilized dried leeches was applied to the column (recyclin system for 2 hours). The column was washed with distilled water. The desired product was eluted with a buffer containing 0.2 M glycine, 0.15 M HCl pH 3.0. The collected fractions were made basic with 0.1 N NaOH to pH 8.0. The volume of the eluate is 8.5 ml (protein 0.03 mg / ml). The eluate was dialyzed against distilled water for 48 hours at 4 ^° C. through a 2000 Da pore membrane, and then lyophilized. The resulting preparation corresponds to the target product, has fibrinolytic activity, contains prostanglandins and does not have the activity of hirudin and a plasma kallikrein inhibitor. When electrophoresis is manifested in the form of a dominant band corresponding to a protein with an MM of 12.3 kDa (Table 1).

 Example 2. All products, as in paragraph 1, only as raw materials used the secret of the salivary glands of leeches in an amount of 5 ml, and the elution buffer was a solution of acetic acid with a pH of 4.0. The resulting preparation corresponds to the target product (table. 2).

 Example 3. Everything, as in example 1, only as a raw material was used an aqueous extract of a culture of the symbiotic bacterium of medical leeches, and the eluting solution was a solution of acetic acid with a pH of 3.5, containing 02 M glycine. The resulting preparation corresponds to the target product.

 Example 4. Everything, as in example 1, only as an elution buffer used a solution of acetic acid with a pH of 5.0. Activity reduced by 80%. The resulting preparation does not match the target product, because complete elution did not occur.

 Determination methods.

 1. Spectrophotometric method for determining protein (2).

 2. Hirudin activity was determined by the extension of thrombin time and expressed in international ATNIH units. The activity of a kallikrein inhibitor in blood plasma was analyzed by lengthening the time of plasma recalcification and expressed in conventional antiprocoagulant units (3).

3. The content of prostaglandins was analyzed according to the recipe firm Amersham (USA) using the radiommunological method for the determination of 6-keto-P ₆ F _1α ,
4. Electrophoresis was carried out in the Lemley system in SDS page under decotouring conditions.

 5. Fibrinolytic activity was determined by the size of lycine zones on stabilized fibrin plates (3).

 The proposed method allows to obtain the final product with a higher frequency and in a larger volume.

Sources of information
1. DAN USSR, 1987, v. 292, N 6.

 2. Kochetov G. A. A Practical Guide to Eximology. - M .: Higher school, 1971.

 3. Kudryashov B.A. A large workshop on the physiology of humans and animals. - M.: Higher School, 1984.

Claims (1)

A method of isolating a destabilization-prostaglandin complex from a medical leech, comprising preparing an aqueous extract followed by purification of the processed raw material, characterized in that the purification is carried out by affinity chromatography on antibodies to 6-keto-prostaglandin F _lα immobilized on a water-insoluble carrier.

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