SU1419717A1 - Method of producing biospecific adsorbents for extracting fibronectin and colagenoses - Google Patents

Abstract

The invention relates to the chemistry of polymers and allows the preparation of biospecific adsorbents for the release of fibronectin and collagenases, having a capacity of 2.0-2.6 mg / mg of ligand for the substances to be emitted. This is achieved by using a method for producing biospecific adsorbents to isolate fibronectin and collagenases by activating an insoluble matrix and then covalently linking it with a specific ligand of fragments of the oL-chain of the collagen molecule or about collagen 1CB7-bromo cyanogen peptide as ligands. 1 tab. and “e (L

Description

with

Immersion relates to the chemistry of polymers, namely, the method of obtaining biospecific adsorbents for the isolation of fibronectin and collagenases, and can be used in biochemistry and medicine.

The aim of the invention is to increase the capacity of adsorbents for fibronectin and collagenases.

The specific ligands used in the method are of. -Collagen chains or oL 1CB7 peptide obtained by cyanide splitting oi. J-chains of collagen.

Example 1. 140 mg of lyophilized collagen from rats' skin is dissolved in 70 ml of 0.5 M acetic acid for 16 hours at 4 ° C. The solution is dialyzed against 8 liters of 0.06 M sodium acetate buffer, pH 4.8, for 16 hours at and then heated at 45 ° C for 30 minutes. The resulting solution is filtered, injected on a KM-52 cellulose column (2.5x23 cm), equilibrated with 0.06 M sodium acetate buffer, pH 4.8, and eluted with a linear gradient of NaCl (1 M) in the same buffer . A two-chamber gradient mixer, 400 ml in each chamber, total volume 800 ml. Chromatography

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conducted at 42 C. The flow rate is 200 ml / h. Fractions analyzed by absorption at 230 nm. The corresponding fractions containing the oi,, oi, + ft 2, and o, -components are dialyzed against 0.1 M acetic acid and lyophilized. The total protein yield of 50.2% (od | chain - 9.52 mg, - 18.4 mg, / J „- 7.76 mg, - 22.5 mg oi., + / B„ - 12, 09 mg).

Lyophilized, - or / 3, - fragments (180 mg each) are dissolved in 40 ml of 70% merabic acid and incubated for 4 hours at 37 ° C under nitrogen with 0.5 g of BrCN. The mixture is then introduced onto a Sephadex C-25 column (1.8 x X 60 cm), equilibrated with 0.1 M acetic acid. The flow rate is 45 ml / h. The fraction exiting the column exclusion volume is lyophilized and 165 mg of a mixture of bromine cyanogen odi-collagen peptides are obtained. Obtained 7.5 mg oi 1 CB7 peptide collagen

Lyophilized collagen oL 1CB7 peptide (20 mg) was dissolved in 10 ml of 0.1 M NaHCOj, pH 8.4, containing 0.15 M NaCl, 10 ml of bromine cyan-activated Sepharose 4B and

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the suspension is stirred for 16 hours at 4 ° C. Then the sorbent is washed with 50 ml of the same buffer, 50 ml of water and stirred for 2 hours at 20 ° C with 10 ml of 1 M solution of ethanolamine, titrated with 6N. HC1 to pH 9. The sorbent is separated and washed with 50 ml of 0.1 M sodium acetate buffer with 1 M NaCl, pH 4.5 and 0.1 M sodium borate buffer with 1 M NaCl, pH 9.0 (operation is repeated three times). The sorbent is then washed with water and stored as a suspension in water at 4 s in the presence of 0.02% sodium azide.

The amount of added od, 1CB7 peptide is determined by the content of hydroxypropin in the hydrolyzate of an aliquot of the adsorbent.

Examples 2-9. Similarly, adsorbents based on sephrosis 4B or CL-4B, spheron 1000, and toyopal HW-65 with oi, and on chains, and / 3,2 components, oi ICC8-bromine cyan peptide and gelatin, are obtained.

The properties of the resulting sorbents are presented in the table.

Example 10. A cluster of silica gel ACA-750 is added with 35 ml of toluene and 2.5 ml of 3- (2,3-epoxypropoxy) propyltrimethoxysilane, evacuated with a rotary evaporator for 5 minutes and boiled under stirring for 10 hours at 110 C. Sorbent washed on the filter with 70 ml of toluene, 140 ml of acetone and dried. To the resulting product were added 167 ml of 0.1N.HCl in acetone and stirred for 2 hours at 20 seconds. The sorbent is washed with 100 ml of water to pH 7 of the wash water and dried in vacuum at 80 ° C.

The resulting sorbent is suspended in a solution of 0.256 g of periodate sodium in 200 ml of water and stirred for 1 hour at 20 ° C. Then the sorbent is washed with 300 ml of water and stirred for 16 hours at 20 ° C with a solution of 100 mg of C, -chain in 50 ml of water, after which the product is washed with 100 ml of water, suspended in 50 ml of 0.1 M bicarbonate buffer, pH 8.5, add 0.05 g sodium bromide and stir for 1 h at 20 ° C. The resulting adsorbent is washed with 50 ml of 0.1 M sodium acetate buffer, pH 4.5, containing 1 M NaCl, and 50 ml of 0.1 M sodium borate buffer, pH 9.0, containing 1 M NaCl (the operation is repeated three times ). Then the sorbent is washed with water and stored at 4 ° C.

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Adsorbent properties are given in

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the table.

Example 11. (Human plasma plasma fibronectin chromatography on adsorbents containing collagen molecule fragments).

A solution of human blood plasma is incubated for 1 h at 20 ° C (lyophilized plasma from 60 ml of blood is dissolved in 30 ml of 0.05 M Tris buffer, pH 7.4, with 0.15 M NaCl, 0.5 mM PMSF and 0 , 02% sodium azide) with 10 ml oi, -sepharose 4B. The adsorbent is then packaged in a siliconized glass column, which is washed with the same buffer until absorbed at 280 nm in the eluate. After this, the column is washed with a equilibrating buffer solution containing 1 M urea (50 ml) and the fibrinectin is washed out with the same buffer with 4 M urea (14 ml). In conclusion, the column is washed with the same buffer with 8 M urea. Functions analyzed by absorption at 280 nm. The fractions containing fibronectin are dialyzed against 0.1 M acetic acid and lyophilized. Exit fibronectin 17 mg (94.4%). The purity of the resulting product is confirmed. polyacrylamide gel electrophoresis data in the presence of sodium dodecyl sulfate.

Example 12. (Chromatography of collagenase from the culture medium of human skin fibroblasts on o (1CB7-sepharose).

A. Collagenase is isolated from 5 L of medium by precipitation with ammonium sulfate (60% of our stage). In the case of a fermentation free medium, precipitation is carried out in the presence of serum albumin at a concentration of 0.5%. The precipitate was separated by centrifugation, dissolved in 0.01 M Tris-HCl buffer, pH 7.5, content of 0.001 M CaCl2, and after dialysis against the same buffer, a solution containing 300 mg of protein was injected onto a KM-cellulose column, equilibrated with the same buffer, the elution of collagenase was carried out with a NaCl gradient (3 M) in 0.01 M tris-HC buffer, pH 7.5, with 0.001 M CaCl2Co current rate 0.1 ml / min. The degree of purification at the stage of ion-exchange chromatography is 30 times. The resulting collagenase preparation is further used for affinity chromatography. Collagenase activity is determined by hydrolysis of c-collagen.

B. Sorbent ob1CB7-sepharose (5 ml of packaged volume) is incubated for 1 hour at 4 ° C with a solution of 10 mg of the collagenase preparation obtained in item A in

10 ml of 0.05 M Tris-HC buffer, pH 7.4, containing 0.1 M NaCl, 0.001 M CaSC, 0.005 M PMSF and 0.05% Brij 35.

Then the sorbent is packaged in a column (1.2 X 4 cm), which is washed at 4 C with 25 np of buffer A, buffer A with 1 M NaCl, 0.05 M sodium acetate buffer, pH 5.2, containing 0.2 M NaCl,

0.001 CaClj, 0.005 M PMSF and 0.05% Brij 3. Chromatography was carried out at a flow rate of 20 ml / h, the volume of the fractions was 1 ml. Fractions analyzed by absorption at 280 nm and collagenase activity. Collagenolytic activity was distributed in two fractions eluted with buffer A containing 1 M NaCl and sodium acetate buffer. The output activity 40%,

cleaning degree 10 times.

Claims (1)

Invention Formula A method of producing biospecific adsorbents for isolating fibronectin and collagenase by activating the insoluble carrier and subsequently covalently coupling it to a specific ligand, otlichayuschiy- the fact that, in order povppeni capacity adsorbents fibronectin and kolla- dehydrogenase, as a specific ligand used oi -chain Collagen molecules ICO ot 1CB7-cyan bromide peptide. 514197176 Properties of adsorbents with immobilized fragments of the molecule collagen t, / in mg / g dry weight of the matrix