UA72227C2 - Method for producing affine sorbent for extracting plasminogen from human blood - Google Patents

Abstract

The proposed method for producing affine sorbent for extracting plasminogen from human blood consists in using, for obtaining the sorbent, the synthesis of insoluble composition, which initiates photochemical reactions, by the photopolymerization of N-vinylpyrolidone with maleic anhydride.

Description

The invention relates to biochemical processes aimed at obtaining enzyme preparations, and can be used by both the chemical and pharmaceutical industries in the production of affinity sorbents intended for the isolation of enzymes of a certain group.

The closest in technical essence and practical purpose is the known method of obtaining an insoluble granular copolymer (matrix) during the copolymerization of M-vinylpyrrolidone with maleic anhydride by thermochemical initiation, subsequent granulation (crosslinking), which is carried out in two immiscible organic liquids (dimethylformamide) and organosilicon (polyethylenesiloxane liquid) of nature at 70-80" for 2-3 hours and, finally, the addition of a ligand, which means the formation of a sorbent (11.

The disadvantage of the closest of the known methods of obtaining an affinity sorbent is the low degree of reproducibility of the properties of the copolymer based on M-vinylpyrrolidone and maleic anhydride, which is a consequence of the thermochemical radical-chain method. So, for example, there is no constancy in the reproduction of such a parameter as the molecular weight from one run to another, which is characteristic of thermopolymerization due to local areas of overheating of the reaction mass in the volume (21. This drawback is typical for thermochemical ( 460-807, "-6-8 hours"), homo- and copolymerization of various unsaturated monomers (Table 1).

This goal is achieved by the fact that the process of copolymerization of M-vinylpyrrolidone and maleic anhydride in a molar ratio of 4:1, respectively, is carried out by the method of photochemical initiation at room temperature of 18-257C for (6--1) hours.

Table 1

Conditions of thermochemical synthesis of copolymers" and their physical and chemical characteristics

Conditional Reaction medium Medium and designation of reaction medium Medium (p), molecular Synthesis time (UV- Yield of copolymer, copolymer photo-copolymer g/dL mass of copolymer irradiation), hours cl» | diokkan.//// | 060 | 1832000 | sh-:Y 7 | 70 2 "Ratio of M-vinylpyrrolidone and maleic anhydride 4:1; thermal initiator - dinitrile-azo-bis-isobutyric acid; copolymerization temperature 7070.

The essence of the invention is explained by examples.

Example 1. a) Photochemical method of obtaining a linear photocopolymer based on M-vinylpyrrolidone and maleic anhydride.

10 g (0.091 mol) of M-vinylpyrrolidone, 2.23 g (0.02 3 mol) of maleic anhydride, 0.45 g (0.0027 mol or 2.95 mol) of photoinitiator (Darokuri 173 ) and 10 ml of solvent (dioxane).

The contents of the flask are thoroughly mixed until complete dissolution and homogenization of all components of the reaction medium. Then the resulting reaction mass is subjected to UV irradiation at Xmax.-300-400nm in the mode of periodic mixing for two hours. at a temperature of 18-2570.

As a result of photocopolymerization, a linear thermoplastic photocopolymer (FKP-1) is obtained based on M-vinylpyrrolidone and maleic anhydride in dioxane. The photocopolymer from the dioxane solution is planted with a solvent mixture of acetone:hexane-2:4. Solvents (acetone, hexane) are removed from FKP-1 by decantation, additionally washed with diethyl ether and dried in air. After these operations, FKP-1 is obtained in the form of a flake-like powder of white color. The yield of FKP-1 based on the initial raw material was 11.62 g (95 wt.95).

The molecular weight of FKP-1, determined by the value of the characteristic viscosity (n) is 20.69 g/dL. (Table 2) and is 233900. b) Granulation (cross-linking) FKP-1.

In a three-necked reactor with a capacity of 0.5 l, equipped with a mechanical stirrer, a reflux condenser and a thermometer, 0.3 liters of polyethylene siloxane (PES-5) are loaded and, with intensive stirring, solution-1 (10 g of FKP-1 in bOml of dimethylformamide) and solution-2 are added almost simultaneously (0.52 g or 5.2 wt.95 from the weight of FKP-1 4,4-diaminodiphenylmethane in 3 ml of dimethylformamide). During 1 hour, the temperature is raised from 18-257C to 70-757"C0, and at this temperature the reaction mass is maintained for another 1 hour, after which the temperature in the reactor is raised to 80-857C for 2 hours. Then the heating is stopped and the reaction mass is slowly cooled for 2.5 hours with constant stirring.

The resulting cross-linked granular photocopolymer FKP-1 (GFKP-1) is separated by filtration from polyethylenesiloxane and dimethylformamide and washed first with several portions of hexane, and then, to completely remove the remains of polyethylenesiloxane oil (PES-5), with sulfuric ether. GFKP-1 is dried at a temperature of 25-30 and stored in an airtight container. Yield of granulated photocopolymer GFKP-1 - 9Zmas.Oo (9.8g) (Table 3).

Table 2

Conditions of photochemical synthesis of photocopolymers and their physicochemical characteristics designation (solvent) of synthesis pi, g/dL |molecular weight| irradiation), |photocopolymer, photocopolymer photocopolymer photocopolymer h. o, o 1 FKPYA | 7 dioxgan// 069 | 2339900. | sh- 6 shshsh | 95 same Z"("

FC? 0 17111ldioxane// | 0.74 | 251200 | (b g shDsh MuYAZHV 97 2 and FKPZ | dioxgan/ | 068 | 232200 | (6 | 9

Table C

Some physicochemical parameters of photocopolymers and affinity sorbents (SFKP) 1 1 g)

Conditional | mobnnonnnnn | The relative degree of designation : : swelling by : granular | affine about sFkpa | 77778 177771717171717707777171717171717197. 17... .90 2 | 6 and sFkp3 | 77/80 17771717171750. | ...yu.93 5 Yushshch | 9 | is c) Immobilization of I-lysine on granular photocopolymer-1.

Preparation of sorbent SFKP-1.

250 ml of distilled water, 10 g of HFKP-1 and 1.0 g of I-lysine chlorhydrate (1Omas.bo from the weight of the photocopolymer) are added to a round-bottomed flask with a capacity of 0.5 l, while maintaining the pH at -7.0-7.2. Inoculation (Immobilization) of lysine hydrochloride to GFKP-1 is carried out at a temperature of 25"7C and constant stirring, maintaining a pH of 7.0 for 4 hours. After 14-16 hours, the obtained affinity sorbent is separated by filtration and washed with a 0.2M solution of Manso"z , then drain the 0.2 M Manso solution and finally wash the sorbent with distilled water to pH 7.6-8.0.

As a result of the mobilization of I -lysine, a sorbent SFKP-1 based on a photocopolymer (FKP-1) was obtained.

Data from the analysis of the first washing sample of the solution after grafting I-lysine by the method of the reaction of the c-amino group with ninhydrin show that the concentration of grafted I-lysine on the granulated photocopolymer is 8.95% w/w. The output of the SFKP-1 sorbent after thorough washing is 6b2ws.Oo (6.82g) (Table 3).

Example 2. a) Photochemical method of obtaining a linear photocopolymer based on M-vinylpyrrolidone and maleic anhydride.

25 g (0.228 mol) of M-vinylpyrrolidone, 5.6 g (0.057 mol) of maleic anhydride, 1.15 g (0.007 mol or 2.Omas.95) of photoinitiator (Darokuri 173) and 25 ml of solvent (dioxane).

Further, as in Example and.

The yield of FKP-2 based on the initial raw material is 30.8 g (97 wt.9 o). The molecular weight of FKP-2, determined by the value of the characteristic viscosity |p)50.74 g/dL, is 251900 (Table d). b) Granulation (cross-linking) FKP-2.

Everything is as in Example 16. The yield of granulated photocopolymer-2 (GFKP-2) is 9.Zg (9Zmas.9o) (Table 3). c) Immobilization of I-lysine on granular photocopolymer-2.

Preparation of sorbent SFKP-2.

Everything, as in Example 1c. Data from the analysis of the first washing sample of the solution after grafting I-lysine by the method of reaction of the x-amino group with ninhydrin show that the concentration of grafted I-lysine on the granulated photocopolymer is equal to 7.9wt.9o. The yield of SFKP-2 sorbent after thorough washing is b5ws.9o (7.15g) (Table 3).

Example 3. a) Photochemical method of obtaining a linear photocopolymer based on M-vinylpyrrolidone and maleic anhydride.

50 g (0.45 mol) of M-vinylpyrrolidone, 11.18 g (0.114 mol) of maleic anhydride, 3.22 g (0.02 mol or 2.0 wt.9 o) of photoinitiator (Darokur 1173 ) and 100 ml of solvent (dioxane).

Then everything is as in Example and.

The yield of FKP-3 based on the raw material is 59.9 g (9Zmas.95). The molecular weight of FKP-3, determined by the value of the characteristic viscosity (1)-0.68 g/dL, is equal to 232200 (table d). b) Granulation (cross-linking) FKP-3.

Everything is as in Example 16. The yield of granulated photocopolymer-3 (GFKP-3) is 9.8g (95wt.9o) (Table 3). c) Immobilization of I-lysine on granular photocopolymer-3.

Obtaining the SFKP-3 sorbent.

Everything, as in Example 1c. Data from the analysis of the first washing sample of the solution after grafting I-lysine using the method of the reaction of the x-amino group with ninhydrin show that the concentration of grafted I-lysine on the granulated photocopolymer is 8.00wt.9o. The yield of SFKP-3 sorbent after thorough washing is bimass.9o (6.71g) (Table 3).

The experimental results given in Tables 2 and 3 indicate that in the process of photochemical initiation of comonomers with different concentrations in the reaction mass, photocopolymers with good reproducibility of molecular mass values and a fairly high yield of the final photocopolymer with a narrow molecular mass distribution were obtained, as well as affinity sorbents based on them.

The obtained affinity sorbents were tested for their ability to release an enzyme such as plasminogen from human blood plasma. SFKP-2 sorbent is taken as an example for isolating plasminogen from plasma.

Isolation of plasminogen from human blood paste fraction I! according to Kon with SFKP-2 sorbent.

40g of fraction II paste is suspended in 200ml of 0.14M mass solution in 0.05M phosphate buffer pH-7.4! along Kohn and then centrifuged for 30 minutes. at 80009. The supernatant liquid is collected and suspended in it 4 g (200 ml) of the prepared sorbent SFKP-2 at a ratio of raw material: sorbent -10:1. After stirring for 7 hours at 4"С, the sorbent is separated by decantation and transferred to a column. Non-specifically adsorbed proteins are isolated with a 0.5M solution of Mass in 0.05M phosphate buffer pH-7.4 until the absorbance (optical density) at

Chl-280nm of the eluate will not be less than 0.05. Plasminogen is eluted on the column with a 0.2M solution of ε-aminocaproic acid in a 0.05M phosphate buffer, pH 7.4. Removal of ε-aminocaproic acid is carried out in the cold (47C) by precipitation of protein at the rate of 0.41 g of ammonium sulfate per 1 ml of protein solution. After centrifugation for 15 min. at 80009, the target product (plasminogen) is obtained by dissolving the precipitate in 0.1M MNaNcSO". The solution is dialyzed at 4"C against 0.0114M MNANSO"z at pH-8.0. The resulting solution is subjected to lyophilic drying. The yield of electrophoretically homogeneous plasminogen is bZmg or 15.8mg per 1g of SFKP-2 sorbent with an activity of 17 caseinolytic units (k .o.)"" per 1 mg of plasminogen.

The results of biospecific sorption of plasminogen from human blood plasma by affinity sorbents obtained on the basis of photocopolymers are shown in Table 4. These affinity sorbents were characterized by the amount of I-lysine immobilized on them, as well as by the activity and yield of isolated plasminogen (Table 4). The isolated preparation of the proenzyme has a specific activity that is consistent with the activity of plasminogen preparations obtained on other affinity sorbents. However, a positive characteristic feature of plasminogen preparations isolated by new sorbents is the absence of spontaneous activity. In addition, sorbents based on SFKHT can be used for a long time, that is, repeated regeneration of sorbents is possible, in which their chromatographic properties are not lost.

Note: x electrophoresis of lyophilized plasminogen preparations is carried out in a polyacrylamide gel in a system of trisborate buffer pH-7 4. Solutions containing 25-30 μg of plasminogen were analyzed. yah For 1 ko. take such an amount of enzyme that releases 450 μM of tyrosine dissolved in 1595 trichloroacetic acid under the conditions of the analysis.

Table 4

Biospecific sorption properties of sorbents obtained on the basis of photocopolymers for the isolation of plasminogen from human blood plasma

Relative degree !

Conditional g. swelling Specific affine Activity

Immobilized | of the cross-linking reagent, the capacity of the sorbent, mg and designation: swarms of the sorbent for and plasminogen, and Y -lysine in (4,4-diamino-; plasminogen per 1 g of sorbents: : volume, 1xO c.o./mg protein times the sorbent and sFkp3 | 80 | shХ 525 «B | 50 2 2 Sh | 158 | 7160

Sources of information 1. Rogozhyn S.V., Davdovich Yu.A., Andreev S.M., Yurtanov A.I. / Polymeric macroreticulate M-oxysuccinimide for the synthesis of peptides // Dokl. Academy of Sciences of the USSR, 1973. T.2P.-Mob. P.1356-1358. 2. S.E. Bresler, B.L. Jerusalem Physics and chemistry of macromolecules. M.-L.: Nauka, 1965. 510.

Claims (1)

The method of obtaining an affinity sorbent for the isolation of plasminogen from human blood by the method of photoinitiated radical chain copolymerization of M-vinylpyrrolidone with maleic anhydride, followed by cross-linking - granulation and grafting of the ligand I. -lysine, which differs in that the process of copolymerization of M-vinylpyrrolidone with maleic anhydride is carried out by the method photoinitiation under the influence of UV radiation at "max. - 300-400 nm and a temperature of 18-257С followed by cross-linking-granulation and grafting of I.-lysine.