SU1634672A1 - Method of preparing polymeric hydrogel - Google Patents

Abstract

The invention relates to -iCH2-CH) r CH2-CHV CH2-CH2 polymer chemistry and chemical enzyme 1 and can be used in chemical analysis and medicine to determine cholinesterase substrates, biological fluids detoxification and medical diagnostics. The invention makes it possible to create a polymer hydrogel with a covalently immobilized cholinesteraea, which has an increased (by a factor of 2.0-3.3 times) resistant to the action of organophosphorus compounds while maintaining the total enzymatic activity. This is achieved by an acylated aqueous solution of cholinesterase with an unsaturated acid chloride at pH 7.5, 9.0 and a molar ratio of enzyme and acid chloride 1: (10- and subsequent radical copolymerization of the acylated enzyme with a hydrophilic monomer and a bifunctional comonomer — binder in the presence 1-4% by weight of the reaction mixture of a linear water-soluble copolymer of the general formula V CH2-CH NHo Ј (L o CO - and 0 J o

Description

where U. 50-55 mol. 7, at 10-15 mol.%, Z 40-40 MO-T L, mol. m. 50000-100000.

2 tab.

This invention relates to polymer chemistry and chemical enemology, in particular, to a process for the preparation of cholinesterase immobilized on synthetic polymers.

The purpose of the invention is to increase the sustainability.

immobilized enzyme to the action of organophosphorus compounds while maintaining the overall enzymatic activity.

Example 1. A mixture of monomers consisting of 63.1 g (0.6 M) 4-vinylpyridine (4-VP) and 7.1 g (0.1 M) of acryl-amide (AA) is dissolved in 120 ml of dimethylformamide, add 0.12 g of azobisisobutyric acid dinitrile, degassing and polymerise at 60 ° C for 24 hours. The synthesized copolymer is precipitated with an excess of diethyl ether, dried, dissolved in 100 ml of methanol and reacted with 2 , 8 g (0.014 M) of phenacyl bromide at 60 ° C for 6 hours. The resulting product is isolated by precipitation in diethyl ether and, after drying, dissolved in 40 ml of absolute ethanol, 1.1 g of hydrochloric acid is added. to (0.016 mol), 40 ml of pyridine and the mixture is kept at 80 ° C for 5 hours. The copolymer was precipitated vym diethyl ether and dried. The content of AA and 4-VP units in the copolymer was determined according to the elemental analysis of the unquaternized sample, the number of 4-vinyl-L-phenacyl-sympyridinium bromide units (4-VFP) was calculated according to IR-spectroscopy and potentiometric titration of the oximated sample and model systems . For this copolymer, the parameters x, y and z are 52.13 and 35 mol%, the molecular weight determined by the light scattering method is 85,000.

Table 1 presents the data of the synthesis of copolymers of 4-vinyl-phenacyloxympyridinium bromide, 4-vinylpyridine and acryl amide.

Example 2 g acetylcholine

terases are dissolved in 100 ml of 0.1 M

phosphate buffer (pH 8.5), cooled before and 21 mg of methacrylic acid chloride was added dropwise, after which the solution was stirred for 15-20 minutes. To the same solution, 29.4 g AA are added with stirring; 13.2 g of N N-methylene bisacrylamide; 2.2 g of the copolymer obtained in the example; 0.008 g ammonium persulfate, blowing

about Q

five

0

with argon for 20-30 minutes, then 0.008 g of H, S, AND-tetramethylethylenediamine is added and left for 1 hour. The resulting gel is crushed on nylon screens, washed with deionized water and water-alcohol mixture and left to swell in water. The activity of immobilized cholinesterase is determined by spectro & nood-on methyl acetate. DDVF is used as a cholinesterase inhibitor. The time of half-inactivation of the starting acetylcholinesterase is 19 minutes, immobilized - 39 minutes.

Table 2 shows the properties of a polymeric hydrogel with a covalently immobilized cholinesterase.

Example 13 (control). All operations were carried out analogously to example 6, using the same ratios of the reagents, but instead of the copolymer obtained in example 1, the copolymer obtained in example 4 was used. The half-inactivation time of immobilized cholinesterase DDVF was 19.5 minutes, and the original 19 min, residual activity - 97% of the original.

Example 14 (control). All operations are carried out analogously to example 7, the same amounts of starting materials are taken, but the copolymer obtained according to example 5 is used. The half-inactivation time of immobilized cholinesterase of phosphacol is 16 minutes, the starting time is 16 minutes, the residual activity is 98% of the original.

Example 15 (control). All operations are carried out analogously to example 6, using the same ratios of reagents, but taking 0.7 g (0.5 wt.%) Of the copolymer obtained in example 1. The half-inactivation period of immobilized cholinesterase DDVF is 19.3 minutes, the original 19 min, residual activity - 98% of the original.

Example 16 (control). All operations are carried out analogously to example 7, using the same ratios of reagents, but taking 5.9 g (5 wt.%) Of the copolymer prepared according to example 2. The semi-inactivation time of the immobilized cholinesterase with phosphacol is 16.1 min, the original - 16 min. Residual activity - 67% of the original.

Example 17 (comparative). All operations are carried out analogously to example 6, using the same ratios of the reactants, but no copolymer of the specified structure is added to the reaction mixture. The time of semi-inactivation of immobilized cholinesterase - 19.2 minutes, the original - 19 minutes

Thus, the proposed method allows to obtain preparations of immobilized cholinesterase, which have an increased resistance of the immobilized enzyme to the action of phosphorus-organic compounds (2.0-3.3 times), while maintaining the enzymatic activity.

Claims (1)

The invention The method of obtaining a polymer hydrogel with a covalently immobilized enzyme - cholinesterae by acylation of an aqueous solution of the enzyme an unsaturated acid chloride at pH 7.5-9.00 and a molar ratio of enzyme: acid chloride 1: 10-200, and the subsequent radical copolymerization of the acylated enzyme with a hydrophilic monomer and a bifunctional comonomer-binder, characterized in In order to increase the stability of the immobilized enzyme to the action of organophosphorus compounds while maintaining the overall enzymatic activity, the acylated enzyme is copolymerized in the presence of 1-4% by weight of the reaction mixture of a linear water-soluble copolymer of the general formula - sngsn) x- (sn2-sn) sn-sn2-sn) g where x y 50-55 mol%; 10-15 mol%; 4 (control) 5 (control)  Copolymer of 4-vinylpyridine and acrylamide prior to FAB treatment. Note. Dinitrile azobisomethyl acid is used in the following quantities: example 2 - 0.2 g; 3 - 0.06 g; 4 - 0.04 g; 5-0.25 g Abbreviations: 4-VP - 4-cnnlinridin; AA - acrylamide; FAB - Phenacilum; ; GAM - SULPHATE HYDROXYLVMY. : NFU СН9Вг I L ) H z 30-40 mol.%, mol. m. 50,000-100,000. Table Note. C / A - half-inactivation period; T is the temperature; HE-holinasteraa; AChE-acetnlcholinesterase; BUHE - butylrhlnnesteraa; DDVF - 0,0-dimethyl-0-dichloro8 vinyl phosphate; FC - phosphacol; AH - acrylic acid chloride; THEM - methacrylic; AA - acrylamide; IDPs - H-vnnylpyrolidone; MA, methacrylamide; BIS - NY-Hvti - bisacrylamide; TGM-3 is a diester of methacrylic acid and ethylene with a degree of polymerization; tgm-iso degree of polymerization 13.