SU526294A3 - Method for preparing biologically active macromolecular compounds fixed on a carrier - Google Patents

Description

g -: -; 1 s-p is found ne neBerHyos-i .fc-: a,; 1 since it is only for y-i. Speech for substances, keobho, and:, bitch, there is a blend of interaction. g .; -.:. The active protein, confined to confJni-iMGpa, manifests itself as a .Ti.iib E of a very low degree and certain conditions. These conditions: w concatom B tom. that the necessary 1/1 reactions of the process should be able to subunit through the pores of the cross-linked c-polymer and, moreover, to achieve the active flux that was found in the copolymer of the active stream; .. It is completely obvious that the high 1st substance can not penetrate copolymer, but even hkkr. substrates enter iCTiiHe very slowly, because diffusion takes place through the czoy;) This eliminates this. It creates the possibility that the whole dispersedly active protein is located in a bound state on the surface of the formed surface. Limer and only in inexpressible amount is the Vnl, -ri of this copolymer. This is achieved by nyTov complementary use of the material, which is a molecular sieve, whose size is so small that the applied biologically active protein cannot be ground into them. IIpe, iJiaraev; biH, a method of making macromole} of chromium compounds fixed to a carrier, consists in that macromolecular sedigents A are first subjected to T. interaction with compound B, which has at least one functional group capable of connecting with macromolecules Compound A and, in addition, has at least one more cnocoSnyjo functional group to the polymerisation, then the molecular sieve is added with such a degree of crosslinking (or pore size) that it does not penetrate through; ;;; macromolecular molecule connected and A, and, if necessary, supporting CoCi :: ..:;:} - co-polymerizing the monomer molecularly, Ha6vxaeT in the reaction medium, then polymerizing the enclosed alcohol inside the molecular sieve of the compounds. Thus, liiiiif-iaioi: a macromolecule. A joint is connected, fixed on a carrier, covalently; 1 hour of ITIC polymerizate, which penetrates into particles of molecular feed. The macromolecular compounds A according to the invention are protein, ophiOHHO biologically active protein, foryents, hormones, and antibodies, nucleic acids, peptides, porphyrins, hemines, phosphatides, cerebrosides, gangluosides, glucosides, etc. It is significant that the macromolecule is so large that it can hardly penetrate into the pores of the applied molecular sieves. The minimum value of the corresponding macromolecules used according to the invention therefore depends on the pore size of the molecular sieve used. Especially preferred macromolecules are biologically active proteining, in particular enzyme active proteins. A large number of compounds can be used as compound B, depending on the ability to interact with macromolecular compound A. as well as on the type of copolymers obtained. If macromolecule A contains, for example, amino acids, then compounds that have interoperable groups, such as oxirane groups, ethyleneiminges, are used. halide groups, acid halide, azido acid anhydride acid groups. Further examples 6 show the use of succinic or alkyping of amino groups into peptide and protein groups, such as cate aldehydes, hydrazides, oxazalones, dislocated chloroformate ester, carbonic ester with phosphoric ester, secondary mouse ether, hydroxynitrile ester, acetic acid, acetic acid ester, secondary phosphate ester, hydroxynitrile ester acetic acid, citrate ester, acetic acid ester, secondary mouse ether, hydroxynitrile ester acetic acid, citrate ester, acetic acid ester, secondary mouse ether, hydroxynitrile ester acetic acid, citrate ester, acetic acid ester, secondary mouse ether, hydroxynitrile ester acetic acid, citrate ester, acetic acid ester, secondary mouse ether, hydroxynitrile ester acetic acid, citrate ester, acetic acid ester, secondary cyanine ester, hydroxynitrile ester acetic acid, citrate ester, acetic acid ester, secondary mouse ether, hydroxynitrile ester acetic acid, acetic acid ester; p-nitrophenol, polyhalophenols, dicyclohexylcarbodiimide and phenyltriazine. In addition, mixed anhydrides of carbonic acid with sulfuric acid, imidazolide carbonic acid. When using other macromolecular compounds in accordance with available functional compounds, other suitable alkylating and acylating agents are used. In the case of nucleic acids, substantially the same active groups as those used with proteins and peptides are allowed. Amides of acids, such as normal carbonic acid amide, alkylamide, morofolid, etc., are particularly suitable here. Multifunctional compound B can have one or more groups, method i i-x. to compound with macromolecular compound A. It preferably has a group of one type. In addition, this compound should contain a group capable of polymerization or polycondensation. Uto, first of all. carbon-carbon double bonds, mainly, which can enter into homopolymerization and copolymer reaction. As well as carbon-oxygen double bonds, as in the carbonyl group and others; OH - groups, isocyanate groups and caprolactamuses, etc. Examples of compounds B are: 2,3-epoxypropyl ester of acrylic acid, 2,3 butene oxide, 1-acyloxy-3- (N-ethyleneimine propanol-2), maleic anhydride, amyl bromide, acryloyl chloride, 2,3-epoxy 3-methacrylic acid you, 2,3-epoxypropyl monoester of maleic acid, 1- (allyloxy) -2,3-eguxybutan, etc. The molecular sieve used should have a pore size that does not allow macromolecular compound A to penetrate and allow the polyfunctional accessory compound B. to penetrate inside. Preferably these are gel-like molecular sieves based on shields. Volimers, such as polyacrylamide gel, cross-linked carbohydrate, for example, cross-linked dextran, agar, etc. Polymerizable monomers can be acrylamide, methacrylamide, acrylic acid ester and acrylic acid methyl ester, styrene, etc. with an appropriate amount of a crosslinking agent, such as N, M-methylene bis-acrylamide, tetraethylene glycol dimethylcrylate or ethylene diacrylate, divinylbenzene, and so on. The pore size is controlled by the amount of the crosslinking agent, and with this amount increasing, the pore radius of the sieve is reduced. Molecular sieves based on crosslinked dextran and derivatives of acrylic acid and methacrylic acid, especially amide and ether, are preferred. Molecular sieves are fully or partially subjected to swelling before polymerization, since polymerization or copolymerization of compound B must take place inside the molecular sieve and therefore molecular sieve swells so that the solution is absorbed, in which the macromolecular compound A is combined with the compound B, so that only the macromolecular part of the product A remains on the surface of the molecular sieves. B. The volume of the reaction solution is chosen such that the full swelling of the molecular sieves does not roiskhodit, so that this passage is provided only inside the final polymerization molecular sieves. By appropriate combination of a molecular sieve with a compound B and a system capable of polymerization, as well as, if necessary, of a solvent, each time, if desired, complete filling and penetration of the polymerizate into the molecular sieve or only surface penetration can be carried out with the remaining unfilled hollow space. inside molecular sieves. The polymerizable system is selected each time to maintain the desired properties of macromolecular compound A, for example enzyme activity, acceptable polymerization conditions, especially polymerization temperature and polymerization properties of polymerizable compound B. If used as a macromolecular compound A thermostable nucleic acid, then it is suitable for complete condensation when heated compound Bplm nt as the only element; 1 system by limerization. If macromolecular compound A is a thermo-sensitive enzyme, then it is appropriate to work under copolymerization conditions using suitable comonomers, for example, acrylamide in the presence of low-temperature polymerization catalysts, for example, peroxides and, if necessary, accelerators and drying agents. Pulp initiators and catalysts used in polymer chemistry, which do not adversely affect the activity of macromolecular compound A, are used as initiators. Inorganic or organic peroxides, azo compounds, etc. are used as initiators or catalysts. Additionally, accelerators of the reaction, such as amines, etc., can be used. When using derivatives of acrylic or methacrylic acid as compound B, which has the ability to polymerize, it turned out to be most convenient to use combinations of peroxydisulfate and amine initiators. The use of crosslinking agents in the polymerization system is not necessary, since the polymerization inside the molecular sieve wells imparts the properties of the reticulated polymer to the resulting polymer. However, it is still advisable to use a crosslinking agent to modify the physical properties. The resulting macromolecularly fixed on the carrier - - :: :: - mie has an excellent rotation .1:: tivnost. Exit.: I ..: Rolot spts molecules. iiO on the basis of the yuleku net-forming "; s dextran. Gluxel-olottdaz (22O U / c-fr) N, N-methylene bis-acrylamide Acrylic acid chloride Methylene chloride Ammonium peroxide disulfate. M-Dimethylaminopropionitrile. 1 mg of glucose oxidase is dissolved in 5 ml of 0.5 M triethanolamine buffer, pH 8.0 is cooled to 10 ° C under nitrogen. OAOm of acrylic acid chloride in 2 ml of methylene chloride is added to this reaction mixture and stirred for 30 minutes. In this protein solution treated with acrylic acid chloride, 0.8 g of acrylamide and 0.05 g of N, N-methylene bis-acrylamide are dissolved. Polymerization is carried out with 0.05 ml of a 5% N-dimethylaminoproshgonitrile solution, as well as with 0.05 ml of a 5% solution of ammonium disulfate peroxide. Immediately after this, 5 g of molecular sieves (sephadex Q -2.5., Medium) are added to the reactive mixture and stirred under nitrogen. The volume of the reaction solution is chosen in such a way that it is not enough to completely swell the molecular sieves and a single polymerization unit does not form, but the polymerization has taken place exclusively in the molecular sieve granules. The obtained particles are suspended in 5O ml of distilled water, stirred, filtered, and aiofiliated. Ca. 5 g (per Exit: Molecular sieve recalculated and lyophilized). The specific activity is 160 and / g of lyophilicate. Example 2 (for comparison). Starting materials: Sephadex Q- -25, medium Glucose - oxidase (220 U / mg) Bromtnan, 75 ml of water are added to 3 tons of Sephadex Q -25 medium; 1OO ml 2 5% aqueous solution of bromozzana. The pH value becomes ra 1P-1 M 11 and by dosing 2 n. natro |: si sh.To-chi, 1 keep pH for 6; constant. Washed OD M rast1. sodium hydrocarbocate. Sephadex, aktiBiroi.-Other bromine cyan, is mixed with 200 mg of chykoza - ase oxide, dissolved in 10 V .--; 0.3 M griethanolamine buffer, pH 8.0. The HL-acaioin mixture was left for 18 h at 4 ° C. Washed with 0.2 M dnophosphate buffer, pM 7.5. The specific activity obtained; 1) In an extremely simple manner, the enzyme coupled to the nose; IC: LEM is 38 O / g. Generally, fixing proteins by cross-linking the act ;; th; 5 by policon.hari cyan bromine,:. (- jiaccjMaTpsibaeTCH as a method with maintaining nanoparticle activity. Comparison of examples 1 and 2 shows that despite the amount of enzyme contained in according to a known method, a product was obtained with an activity 25% lower than the activity obtained by the proposed method. The output of the enzyme obtained was 8 times higher. Example 3. Starting materials: Polyacryl glucose oxidase-based molecular sieves (220 U / mg ) Acryl amide N, N -methyl e -bis-acrylamide Acrylic acid chloride Methylene chloride Ammonium peroxide N-dimethylaminopropienitrile disulfate. The enzyme solution was incubated as indicated in Example 1. Immediately after adding the initiator solution, 5 g of molecular sieves (biogel P6 5010O mesh) were added and mixed under nitrogen atmosphere Molecular sieve swelling occurs within a few seconds. The volume of the reaction solution is chosen so that the molecular sieves are not completely swollen, a single polymerization block is not formed. And polymerization proceeded exclusively in the granules of molecular sieves. The particles obtained are suspended in 5OO ml of distilled water, stirred vigorously and fractionated into two types of screens with a cell width of 0.4 and 0.2 mm. Particles of 0.2-0.4 mm fill the column with a diameter of 20 mm and elute with 4 liters of 0.2 M phosphate buffer; pH 7.5. Output. Ca molecular sieves. 5 g (in terms of dry weight, specific activity of 16 O and / g (in terms of dry weight). Example 4. Fixing of polyadenylic acid (poly A) Initial products: Poly L Triethanolamine buffer (0.5 M, pH 8.0 ) Acryloyl acrylamide Ammonium peroxide disulfate (5%) N, m-bis-acrylamyl Dimethylaminopropionitrile (5%) Sephadex G-25, Wednesday 27.4 mg of Poly A was dissolved in 2 ml of triethanolamine buffer and cooled to 10 C. This the reaction solution is added under a nitrogen atmosphere in 0.01 ml of akoiloyl chloride dissolved in 1 ml of ether and stirred for 30 minutes, finally mixed with 0.3 g of 910

acrylamide and 0.2 g of N, N-methylene - bis-cular compounds by reacting

-acrylamide and 2 g Sephadex -25 and on-biologically active macromolecular

start polymerization with 0.02 ml of dimethyl compound A with a low molecular weight compound aminopropionitrile and 0.02 ml disulfation B, but containing at least one

non ammonium ammonium. The resulting product in gra-5group, capable of polymerization, and

pools are introduced for about 8 hours into the column and washed-at least one group capable of taking 2, 5 liters of a 0.5 M solution of common salt with a macromolecular compound to prove fixation with suspension A, followed by polymerization, prolonged Sephadex combined Poly A with AB in solution, about t 0, 3 n. potassium hydroxide solution and hydrolyzed 10 persons in that, in order to reduce darkness at 37 ° C., biological activity is fixed. After hydrolysis, nucleic compounds are obtained on the carrier, the proacid is added to the solution, the concentration of which (10%) is calculated by dB before polymerization.

weight gain by weight Poly A. Molecular sieve with pore size in the sweep. The proposed method allows 15 states smaller than the size of the molecule

saving expensive and, in many cases, a hard-macromolecular compound A, into a bioavailable protein that is sufficient for incomplete swelling, since it is all bound on the surface of the product A. B. The method according to claim 1, Claim 1. The formula of the invention is that the polymerization is product. The method of obtaining fixatives: g -; u) and AB is carried out in the presence of a biologically active poppy in the presence of a copolymer carrier; . -... connection with him.

526294