RU2262947C2 - Method for obtaining thrombin-like coagulating enzyme - Google Patents

Abstract

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: the present innovation deals with blood testing based upon chromatographic purification of snake's venom, identification and mixing fractions that contain thrombin-like enzyme followed by isolating the target product due to chromatography upon Sepharose leveled with Tris-HCL buffer solution, moreover, snake's venom Agkistrodon halys in 0.05 M sodium chloride solution should undergo chromatography upon a column filled with Sephacryl S-300 HR, after identification and mixing fractions with thrombin-like enzyme they should be concentrated due to ultrafiltration at selecting against molecular weight of 10 Kd, the obtained solution should undergo chromatography upon a column with DEAE Sepharose FF leveled with acetate buffer solution to select and mix fractions with thrombin-like enzyme and repeatedly concentrate due to ultrafiltration, then concentrate should pass chromatography upon a column with SP Sepharose FF gel, fractions that contain the target product should be combined, diluted with distilled water up to activity of 12-15 sec in thrombin time test, then one should add saccharose, sodium azide up to 0.01% to be poured into vials and freeze dried. The method provides broadened quantity of reagents for laboratory diagnostics of disorders in the system of hemostasis. Fraction isolated out of snake's venom Agkistrodon Halys (the Russian Federation) is not inferior to that of snake's venom Bothrops atrox.

EFFECT: higher efficiency.

1 ex

Description

The invention relates to medicine, in particular to laboratory diagnostics, and can be used for blood testing.

It is known that thrombin-like enzyme (TF) is used to diagnose dysfibrinogenemia and hypofibrinogenemia, determine the level of fibrinogen, and to control heparin therapy.

The closest to the achieved positive result is the method of obtaining TF from the snake venom Bothrops atrox (Bothrops family, genus atrox), which consists in isolating a thrombin-like fraction that can cause coagulation of fibrinogen [Holleman WH, Weiss LJ, The thrombin-like enzyme from Bothrops atrox snake venom . Properties of the enzyme purified by affinity chromatography on p-aminobenzamidine-substituted agarose. J Biol. Chem. 1976, Mar. - 25. - 251 (6). - R.1663-9]. Such a reagent called “Reptilase®” was patented by Pentapharm Ltd (Switzerland).

The disadvantages of this method include the low availability and high cost of the poison of the snake Bothrops atrox, not found in the Russian Federation, which lives mainly in South America.

A positive result of the proposed method is the expansion of the arsenal of reagents for laboratory diagnosis of disorders in the hemostatic system.

A positive result of the proposed method is achieved in that the snake venom Agkistrodon halys (family Agkistrodon, genus halys) that lives on the territory of the Russian Federation is used as a source of coagulating thrombin-like fraction.

The method is as follows:

Reagents and equipment:

1. Snake venom Agkistrodon halys (family Agkistrodon; genus halys), 0.01 g, crystalline.

2. Cellulose acetate membranes with a selection (cut-off) of a molecular weight of 10 cd (Sartorius, Germany).

3. Cells for ultrafiltration (Ultrasart Cell 50, Sartorius, Germany).

4. A solution of NaCl (0.05 M).

5. TRIS-HCl buffer (0.02 M, pH = 8.1).

6. Acetate buffer, (0.025 M, pH = 5.5).

7. Gel Sephacryl S-300 HR (Pharmacia, Sweden).

8. Gel DEAE Sepharose FF (Pharmacia, Sweden).

9. Gel SP Sepharose FF (Pharmacia, Sweden).

10. Gel Sephadex G-25 (M) (Pharmacia, Sweden).

11. Reference normal plasma (RNP), freeze-dried firm Technology Standard, Russia.

12. Sodium azide (Sigma, USA).

13. Sucrose, 2% solution (Merk, Germany).

14. Glass tubes with a capacity of 20 ml.

15. Penicillin vials per 10 ml.

16. Chromatographic columns (26 × 800 mm, 26 × 40 mm, 20 × 26 mm and 15 × 200 mm), manufacturer (Pharmacia, Sweden).

17. Analytical balance S-2051 (Sartorius, Germany).

18. Magnetic stirrer MM 3M, Russia.

19. Fraction collector (LKB, Sweden).

20. Spectrophotometer SF-46 (Russia).

21. RN-meter RN-340 (Russia).

22. Coagulometer (KS-4, Germany).

Reagent Preparation

Preparation of Agkistrodon hatys snake venom solution: 5 ml of a 0.05 M sodium chloride (NaCl) solution is added to 100 mg of crystalline Agkistrodon halys snake venom. Agkistrodon halys snake venom solution is stirred for 60 min on a magnetic stirrer at +18 ... + 25 ° С. All subsequent procedures for the preparation of reagents and the selection of TF is carried out at the same temperature.

Agfistrodon hatys snake venom

Step 1. The solution of Agkistrodon halys snake venom is applied to a chromatographic column (26 × 800 mm) filled with Sephacryl S-300 HR gel equilibrated with a 0.05 M sodium chloride solution. Elution is carried out with the same solution at a speed of 60 ml / h. The volume of collected fractions is 10 ml.

Step 2. The TF-containing fractions of the eluate are identified in a thrombin assay. To do this, 0.1 ml of the control normal plasma (incubated for 60 s at + 37 ° C) and 0.1 ml of the studied fraction (in the control the same volume of the elution buffer) are added to the recording cell of the coagulometer and the time of clot formation is recorded. If there is TF in the fraction, the coagulation time is shortened (the degree of shortening depends on the concentration of TF), compared with the control, where 0.1 ml of the studied fraction was replaced with 0.1 ml of elution buffer.

Stage 3. TF-containing fractions of the eluate are mixed and concentrated by ultrafiltration to 1/10 of the initial volume of fractions. Ultrafiltration cells and cellulose acetate membranes with a molecular weight of 10 Cd are used to concentrate the samples.

Step 4. The resulting solution was diluted 10 times with Acetate buffer and applied to a column (26 × 40 mm in size) filled with DEAE Sepharose FF gel, which was equilibrated with the same buffer. The elution is carried out with 0.025 M Acetate buffer containing a linear gradient of sodium chloride (NaCl) from 0.0 to 0.6 M, at a rate of 450 ml / h. The volume of collected fractions is 20 ml.

Step 5. The TF-containing eluate fractions are identified (Step 2), mixed and concentrated by ultrafiltration through cellulose acetate membranes to 1/20 of the original volume.

Step 6. The resulting concentrate of the fractions obtained on the cation exchanger was diluted 10 times with TRIS-HCl buffer and applied to a column (20 × 26 mm in size) filled with SP Sepharose FF gel equilibrated with the same buffer. Elution is carried out with TRIS-HCl buffer containing a linear gradient of NaCl concentration from 0.0 to 0.4 M, at a rate of 450 ml / h. The volume of collected fractions is 10 ml.

Step 7. The TF-containing eluate fractions are identified (Step 2), mixed and diluted with distilled water to an activity of 12-15 seconds in a thrombin time test.

Step 8. Sucrose up to 2%, sodium azide up to 0.01% are added to the pool of active fractions, 1.0 ml are poured into penicillin vials and freeze-dried.

The result is 10-20 ml (depending on the activity of the initial batch of Agkistrodon halys poison) of the purified thrombin-like enzyme.

Thus, from 100 mg of the crystalline venom of the Agkistrodon halys snake, 0.3-0.5 mg of TF is obtained.

Verification of the coagulating properties of TF

To test the ability of the obtained TF to induce coagulation, the coagulation time of RNP, platelet-poor plasma of patients with hypofibrinogenemia (n = 12) and 0.2% fibrinogen solution was studied. For this purpose, 0.1 ml of the studied plasma was added to the recording cell of the coagulometer, the plasma was incubated for 30 seconds at + 37 ° C and 0.1 ml of TF solution (or 0.1 ml of Reptilase®), and the time of clot formation was recorded. In the control, the same volume of distilled water was added instead of TF (or 0.1 ml of Reptilase®).

As can be seen from the table, the clotting time of RNP and fibrinogen solution after adding TF does not differ from the clotting time with Reptilase® (prototype). At the same time, the addition of distilled water did not cause a coagulating effect. Moreover, in the plasma of patients with hypofibrinogenemia, the coagulation time with TF was lengthened and did not differ from the coagulation time with Reptilase® reagent.

Thus, the obtained TF from Agkistrodon halys snake venom has coagulating properties similar to the properties of the known thrombin-like fraction from Bothrops atrox snake venom (Reptilase®).

Claims (1)

A method of obtaining a thrombin-like coagulating enzyme, which consists in chromatographic purification of snake venom, identification and mixing of fractions containing a thrombin-like enzyme, followed by isolation of the target product by chromatography on Sepharose, balanced Tris-HCL buffer solution, characterized in that Agkistrodon halys snake venom is 0.05 M solution of sodium chloride is chromatographed on a column filled with Sephacryl S-300 HR, after identification and mixing of the fractions with a thrombin-like enzyme, they are concentrated by ultrafiltration to 1/10 of the original one volume on cellulose acetate membranes with a molecular weight selection of 10 Kd, the resulting solution is chromatographed on a DEAE Sepharose FF column equilibrated with acetate buffer solution, the fractions are taken and thrombin-like enzyme, then concentrated by ultrafiltration to 1/20 of the original volume, the concentrate after dilution in Tris-HCL buffer solution chromatographic on a column with SP Sepharose FF gel, the fractions containing the target product are combined, diluted with distilled water to an activity of 12-15 s in the thrombin time test nor contribute sucrose, sodium azide to 0.01%, poured into vials and freeze-dried.