The method of obtaining the primary inhibitor of the protease of the lung and pancreas of cattle

 

(57) Abstract:

The invention can be used in medicine, Enzymology, cell and molecular biology. Technical problem: simplify technologies, reduction stages, the reduction of the target product, increasing its yield and specific activity. The inventive frozen bodies of cattle homogenized, extracted from them the main inhibitor of the prosthesis aqueous acid solution at pH 1-3, the extract obtained fractionary and concentrate ultrafibre in a tangential flow of the first hollow fibers with a pore size of 15-50 KDa, 5 KDa, the final cleaning is performed by cation exchange chromatography. Effect: simplified technology, cheaper product, increase the yield of 8 times, the specific activity of 10.8 times. 1 C.p. f-crystals.

The invention relates to medicine and Enzymology, in particular to the technology of production of the main inhibitor of the prosthesis (IPRs) trypsin, kallikrein, plasmin. IUP is the current top drugs: kontrikala, Gordons, and other drug, is used in biochemistry, cell and molecular biology [1, 2]

A method of obtaining the IPRs of the lungs, pagelogo, fractionation with ammonium sulfate, gel filtration and ion-exchange chromatography [3] However, this method requires a significant consumption of ammonium sulfate and environmentally harmful.

A method of obtaining the IPRs of organ tissues of cattle by grinding, acid extraction and subsequent affinity chromatography on trypsin-sepharose [4] This method gives the desired product with a specific activity of 7,000 Kiu/mg, but used resin is too expensive for industrial applications.

Closest to the claimed is a method of producing an inhibitor of the prosthesis and the pancreas of cattle [5] the Method of obtaining the primary inhibitor of the prosthesis, is as follows: lung, or pancreas of cattle crushed, extracted with 1% solution of oxalic acid, removing the precipitate by filtration, perform chromatography on silicagel, target product elute with acetone followed by alkalization, IPRs precipitated 4-6 volumes of acetone, absoluut and dried. The output of the IPR is 40000-57000 E/kg specific activity 500-600 Kiu/mg

Comparative analysis of the proposed method and the prototype showed that the defining significant ex is corruption in tangential flow, instead chromatography and precipitation of the product with an organic solvent (prototype).

An object of the invention is the simplification of the method of obtaining the IPRs reduction process, increase the yield and specific activity of the target product.

The goal of the project is achieved by the proposed method lies in the following: frozen lungs, pancreas, submandibular or parotid gland of cattle crush grinder, homogenize, add or trichloroacetic acetic acid to pH 1-3 and cake separated by centrifugation. Bring the pH of the supernatant to 6.2 to 7.2 using NaOH. The extract is subjected to ultrafiltration hollow fibers with a pore size 15-50 KDa and concentrate on hollow fibers with a pore size of 5 KDa. The concentrate is applied on a column with carboxymethylcellulose conducting sorption of the target product at a pH of 6.2 to 7.2, and desorption of 0.45 M solution of sodium chloride. Output accounted for pancreatic 104,500 E/kg specific activity 5,500 Kiu/mg, which is higher than the method prototype 1.8 and 9 times respectively. When the allocation of IPRs from the light output was $ 455,000 E/kg specific activity 6500 Kiu/mg, which is higher than the method prototype in 8 and 10.8, respectively.

Ulltravioleta in a tangential flow of the first hollow fibers with a pore size 15-50 kDa, and then with a pore size of 5 kDa, and then with a pore size of 5 kDa, which allows sequentially to first carry out the fractionation of proteins after acid extraction, and then to carry out the concentration of the extract, allowing for an easier way. The study of the protein composition of the extract shows that the main number of synthesized protein has a molecular mass of 15 kDa, so the optimal mode of fractionation (separation IPRs) is ultrafiltration in hollow fibers with a pore size 15-50 kDa. The concentration of the obtained filtrate is carried out by ultrafiltration in hollow fibers with a pore size of 5 kDa, which allows to get rid of low molecular weight impurities, 20-30 times to reduce the volume of the working fluid before chromatographic purification of IPRs, and, thereby, to further simplify procedures and improve the yield of the target product.

Purification of the target product are carried out by sorption on the carboxymethyl cellulose at a pH of 6.2 to 7.2 with subsequent desorption IPRs 0.45 M solution of sodium chloride, which allows to further purify the desired product 30-50 times.

The above mentioned distinguishing features allow you to get a new positive effect on sravnenie activity 2.2 times, on the specific activity of 10 times, and from the lungs of 9.5 times and 11.8, respectively. In addition, the proposed method allows to reduce the cost of the target product by reducing the consumption of chemical reagents, to improve fire safety and environmental cleanliness of the process by eliminating the use of large quantities of acetone.

The invention is illustrated by the following specific examples:

Example 1. One kg of frozen lungs of cattle grind in a meat grinder, then homogenizer, add water to a volume of 3 l and THU-150 ml of 50% solution, stir and leave for 1 h at room temperature. Further, all operations can be performed at room temperature. The extract is separated from the precipitate by centrifugation at 5,000 g for 10 minutes and the pH adjusted to 6.2 to 7.2 using NaOH. The extract is filtered at the facility UPL-0.6 hollow fibers AR-0.2 to 50 PA. The obtained filtrate is concentrated by hollow fiber AR-0,2-5 PA to 100 ml. of the Concentrate is applied on a column with carboxymethyl cellulose CM-52 (3x7) firm "Whatman", balanced water. The column is washed with water and inhibitor elute with 100 ml of a 0.45 M solution of sodium chloride.

The output was 455,000 E/kg activity 6,500 Kiu/mg

Example 2. All operations proteostasis 104,500 E/kg activity 56500 Kiu/mg

Example 3. All operations were performed as described in example 1, using instead of THU glacial acetic acid, bringing the pH to 1-3.

The output amounted to 235,000 E/kg activity 5,000 Kiu/mg

Thus, the proposed method of obtaining the IPRs in comparison with the method of the prototype allows you to:

1. To reduce the number of process steps, its duration.

2. To reduce the cost of the target product by reducing the consumption of chemicals.

3. To increase the output of the IPR from the pancreas activity 1.8 times, on the specific activity of 9 times, and from the lungs 8 times and 10.8, respectively.

Sources of information:

1. Frifz H, G. Wunderer Drug Res. 1983, 33 (1), N 4, pp. 479-494.

2. The state of drug development IUP, pharmacy, 1992, No. 2, pp. 66-69.

3. Vogel R. Trantschold I. Worle E. ads. Natural Rcoteinase Inhibitors, pp. 76-95, Academie Press, New York, 1968.

4. Chauvet J. Acher R. FEBS Lett, 23, 317, 1972.

5. DE, patent N 1492114, CL ANC 17/00, 1972

1. The method of obtaining the primary inhibitor of proteases from the lungs and pancreas of cattle, including grinding of raw materials, extraction of the aqueous acid solution, separation and purification of the target product, characterized in that the separation of the target product ossei mol. m 15 50 KDa, then mol. m 5 KDa, and purification of the target product are carried out by chromatography on carboxymethyl cellulose by sorption at pH of 6.2 to 7.2, and desorption of 0.45 M sodium chloride solution at the same pH.

2. The method according to p. 1, characterized in that the extraction is carried out in an aqueous solution of acetic acid or trichloroacetic acid at pH 1 to 3.