SU730690A1 - Method of preparing immobilized nucleases - Google Patents

Description

one

The invention relates to a method for producing immobilized enzymes by binding them to a soluble matrix, which can be used in medicine for the production of enzyme-based drugs (enzyme therapy), as well as in industry. Enzymes immobilized on dextrans can be used in industrial-type reactors when squatting cyclic and continuous technological processes using. semipermeable membranes to remove the reaction products from the mixture of the enzyme with the substrate.

The prospect of using enzymes as drugs is very high, but their use is currently quite limited and does not correspond to the potentialities of such effective catalysts. One of the disadvantages of enzymes that limit their use as drug Heparas is their low stability, as a result of which the enzymes introduced into

the body, as a biologically active substance or drug, is very rapidly destroyed.

There are several ways to increase the stability of enzymes; physico-chemical methods based on selection of conditions optimal for storage of enzymes (ju, pH, t ° activators, etc.) C, immobilization of enzymes on insoluble matrices 2 and microencapsulation, i.e. their inclusion inside the smallest capsules, impermeable to the enzyme Z.

To enhance the biological activity of enzymes, methods for selecting optimal conditions are unsuitable, since these conditions

15 can only be deprived of in vitro, but not in the body, the homeostatic mechanisms of which change these conditions in accordance with their internal parameters.

20

Immobilization of enzymes on insoluble matrices may be suitable for enhancing the biological activity of enzymes in those rare cases when they perform their functions in the body in an insoluble form (for example, enhancing the digestive function of the gastrointestinal tract).

Microencapsulation makes it possible to obtain stable enzymes in an injection form, and, therefore, can be regarded as a way to enhance the biological activity of enzymes. However, the walls, microcapsules are impermeable to the macromolecules of the enzyme. Equally, substrates with molecules of p - weight, commensurate with the molecular weight of the enzyme, and even more superior to it (proteins, nucleic acids, poly: Saccharides), cannot penetrate into the capsule. Therefore, microencapsulated enzymes do not catalyze the conversion of such substrates, and this method of enhancing biological activity is limited to enzymes that convert low-molecular-weight substrates that penetrate the microcapsule membrane.

The closest to the present invention is a method of immobilizing enzymes by binding them to water soluble matrices. According to this method, the immobilization of enzymes is carried out as follows. Dextran, DEAE dextran or soluble KM-cellulose is dissolved in water, pH 11.0. With stirring, a water-acetone solution of 2-amph-4,6 dylloro-S-triazine is added. Reactor scrubber is acidified by acidifying the reaction mixture to pH 3, O-4.0. The activated polymers are precipitated with acetone, reprecipitated, and used for binding to enzymes. Binding is carried out by adding an enzyme solution to the solution of activated polymer in borate buffer (pH 8.8) and keeping the reaction mixture at room temperature for 16 hours. The reaction is stopped by adjusting the pH of the mixture to 6.0 and removed unbound by means of an ultrafiltration enzyme. Dextran and DEAE-dextran preparations with molecular weight of about 20 Ooooo are used to immobilize the enzyme.

The purpose of the invention is to enhance the biological activity of nucleases while preserving their injection form, i.e. ensuring their permeability and creating effective / concentration in the internal environment of the body for a long time and increasing the antiviral and antitumor effects of drugs.

This goal is achieved by the fact that nucleases isolated from organisms phylogenetically distant from the target organism are covalently linked by a diazo combination with a soluble matrix, m-aminobenzyloxymethyldextran, and m-aminobenzylloxymethyldextran mol% is used. 400OO-60000.

In order to find the optimal parameters for preparations of nucleases possessing antiviral and anti-salt action.

We study the relationship between the masses. immobilizing dextran and the biological activity of modified nucleases. Established that a pronounced biological activity, within the limits of the

lichin, are given in table. 1, possesses only a nuclease associated with dextran mol. Weight. 40000. Enzyme associated with dextran mol. Weight. 20,000 and 60 ooo, has stability, almost oshshako-

weight of the enzyme bound to the dextran mol. weight. 4OOOO. OmiaKO has an antitumor effect only when the contact (direct) method of influence on the tumor (the tumor develops intraperitoneally - the enzyme is injected intraperitoneally). With an indirect method of exposure (the tumor develops intraperitoneally - the enzyme is injected intramuscularly; the tumor develops subcutaneously - the enzyme is injected intramuscularly)

the biological activity of the modified enzymes does not differ from that of the thioid.

This is due to the fact that the enzyme

associated with dextran mol.ves. 60,000, does not spread in the body, remaining localized at the site of administration to, therefore, does not reach the tumor when attempting to mediate

on her. The rate of propagation in the body of an enzyme associated with the mol. ALLOCORA, does not differ from the rate of propagation of the native enzyme. Thus, the modified enzyme

is rapidly excreted from the body, its efficacy 1a concentration is created only at the site of administration and rapidly decreases due to the removal of the enzyme from the body.

Thus, along with the stability of the modified enzyme, the molecular weight of the modifying matrix is of great importance for increasing the biological activity of the enzyme. The molecular weight of the matrix must be such as to ensure the permeability of the enzyme and the longest time to maintain its effective concentration in the body. For nuclease Ser. gpc sease,;) modifiable dextran, the most optimal is its mol, wt., equal to 40OO. For ribonuclease somewhat wider than 40,000-60000. Enhancing the biological activity of enzymes is illustrated by examples of Sep.mohcescena nuclease, and Act pH. Since they are isolated from organisms that are far phylogenetically distant from the target organism (mammals), Covalent binding to dextrans of nucleolytic enzymes isolated from the organism of mammals, for example, pancreatic DNA and RNA, is accompanied by their stability. - but does not lead to a noticeable increase in the biological activity of these enzymes, which remains at the level of the biological activity of the active enzymes. The mechanism of this phenomenon is directly related to the issue of enhancing the biological activity of enzymes. Pancreatic nucleases isolated from the pancreas of bovine cattle are effectively inhibited by the target inhibitors when introduced into the target mammal organism. Due to their toxicity to nucleases isolated from phylogenetically distant organisms, the inhibitory mechanisms of the target have little effect because of their phylogenetic distance from the enzymes. Therefore, an introduction to

The mammalism of the same dosage of equally stabilized enzymes, but isolated from different sources, is accompanied by a different level of increase in the catalytic activity of this type in the body. The highest level of elevation of activity is observed when a stabilized enzyme is introduced into the body, phylogenetically distant from the target organism. As a result, the biological effect of the unit of catalytic activity introduced into the body (antitumor and antiviral effect per unit of activity) for this enzyme has a higher value.

The observed phenomenon is of a general nature, t, e, in any case, an increase in the biological activity of enzymes is maximal with the stabilization of enzymes phylogenetically distant from the organism of the target. There may be some cases of an increase in biological activity when phylogenetically related enzymes stabilize, if this violates their relationship with the inhibitor.

using gelfilter on Sephadex Q -75 or Q-1OO. As a result, yBeniraH-

3-5 times the stability of the enzymes, characterized by thermal stability, storage stability, stability in urea solutions and in the internal environment of the body. Peeled covalent

but enzymes associated with dextran are further used as drugs.

Example 1, Preparation and study of tumor-related action covalently

in a nagash with dextran nuclease Ser, marcescens.

but). Preparation of covalently bound dextran nuclease S, er. marceGct.ns and study its stability.

12O mgm-aminobenzyloxymethyldextran, nitrogen content 0.7%, dissolved in cold in 1 ml O, 5 n. of HCl, To the resulting solution is added DL mg laMOg, the diazototal is carried out for

Claims (3)

5 MP About, 06 ml of NaOH (3.5 N,) was added, adjusting the pH of the reaction mixture to 3-4. To the solution of the formed shlodextran with constant stirring of the target organism systems, however, at this stage of development they are not regular. Enzymes are immobilized by their binding to a soluble matrix by the diazo-combination method. By means of the diazo-co-chaining method, it is possible to bond through amino, sulfhydryl, cyclic and heterocyclic groups of amino acids, which allows for binding through the maximum possible number of points and , to obtain the most stable preparations of enzymes. The multipotency of the diazo coupling reaction also makes it possible to directionally change the assortment of immobilized enzymes bound to dextran through qualitatively different acid residues. The process is carried out as follows. Activated by introducing the m-amylobpsyloxy radical of the radical in them, the dextrans are treated with a 0.5 K solution of N ANO. HC1 at a temperature from 0 to the pH of the resulting solution of diazodextran is adjusted to 3-4, the enzymes are dissolved in 0.2 M Tris-HC1 buffer (pH 8.5) and added, with stirring, to diazodextrai. The mixture is not left for 10-14 hours at. Enzymes covalently bonded to dextral enzymes are separated from the reaction products 773. 25 ml of the enzyme solution in 0.2 M Tris-HC1 buffer, pH 8.5, are added. The total amount of added protein is 97.5 mg. The reaction mixture is stopped at 1O-14h at and after the indicated time is separated by covalently bound dextran nuclease from unreacted dextran and the enzyme by gel filtration on Sephadexag Q-75 or C-100 balanced with phosphate buffer pH 7.2 O, O. M. Stability The dextran-linked nuclease is checked by heating for 30 minutes and treated with urea at a final concentration of 2 M and 4 M (contact with urea for 30 minutes and then removing it with dialysis against phosphate buffer for 1.5 days). The thermostability of the bound enzyme, which is scored by the percentage of preservation of activity after 30 min of warming up, is 66% (for a native 10%). After treatment with 2 M urinary, 94% of the activity of the bound enzyme is retained (for the native 45%), after treatment with 4 M urinary, 42% is maintained (for the native 10%). Nuclease associated with dextran is stored in solution for 1.5 months at 10-15 ° C without loss of activity, while the native enzyme loses 5O% of activity under these conditions. Nuclease associated with dextran is preserved in the culture medium of a tissue culture for about a week without loss of activity, whereas the native enzyme loses its activity with an average of 24 hours. (depending on the initial concentration) Saturated enzyme injected into the abdominal cavity of mice infected with ascitic Ehrlich carcinoma disappears completely within 3-4 hours, while the associated enzyme - 12-24 hours. b) The study of the antitumor activity associated with dextran nuclease. The study of the antitumor effect is carried out on two mouse transplantable tumors: ascitic form of Ehrlich carcinoma and solid form of sarcoma 37. The protin activity of drugs is taken into account after the end of the course of treatment, which is identical in dose and method of administration for natiyugo and correspondingly immobilized enzymes, and is evaluated by the dose of the method of administration for natyyugo and respectively immobilized enzymes and is evaluated by the dose of the method of administration for natyyugo and respectively immobilized enzymes and is evaluated by treatment These antitumor activity of the native and the modified nuclease preparation are given in table. 1, 0 Table. The enzyme Ascites with in dex-pm-Aminobenzyloxymethyldextran in amounts contained in the preparation covalently associated with dextran does not have an antitumor effect. Studies show that the biological activity of the nuclease, which exhibits an antitumor effect, increases significantly after the enzyme is bound to soluble dextran. Example 2. Preparation of covalently bonded dextran ribonuclease Aci. PimoSus and study of its antiviral effect. a) A method for making ribonuclease covalently bound to dextran and studying its stability. 11.0 gm-hmsh-jenzyloxymethyldextran is dissolved in cold in 100 ml 0.5 n. HC1. To the resulting solution was added 400 mg of NaNOn. Diazotipia carried out for 5 minutes. 3 ml of NaOH (3.5 N) are added and the pH of the reaction mixture is adjusted to 3.0. With constant stirring, 7.5 ml of a solution of ribonuclease in 0.4 M tris-pH of 8.5 (the total amount of the added enzyme is 1.0 g) is poured into the solution of the formed diazodextran. The reaction mixture is left for 10-14 hours at and, after a specified time, is separated covalently bound to the dextran of ribonucleases from the unreacted plant and the enzyme by gel filtration in Sephadex Q-75 or G-100, phosphate-boosted with pH 7, 2 0.05 M. Stabilized ribonuclease bound to dextran is checked by heating at 60 ° C for 30 minutes and also with urea at a final concentration of 6. M (contact with urea for 30 minutes with its subsequent removal against phosphorus buffer for 1.5 sou t.). The thermostability of the bound enzyme, expressed as the percent retention of activity after 30 min of warming up, is 54% (for a native 6–2O%), depending on the time of the study of the enzyme after dissolution. In the freshly dissolved enzyme, the thermostability is about 20%, when stored after dissolving in a frozen form or for a period of 1.5-2.0 days. thermal stability is reduced to 3-6%. After treatment with 6 M urea, 56–6% of the activity of the bound enzyme is protected (for a native 22%). b) Investigation of the antiviral effect of dextran-linked ribonuclease. Antiviral activity is studied on a schur virus of strain AG-22 adapted to white, outbred mice. Animals are infected by intravital injection of a dose of lOOLDgj, after which they immediately begin treatment with native and parallel immobilized farms. Enzymes are administered twice a day .; The antiviral effect is assessed on the percentage of surviving animals. The results of the research are given in table. 2. Table2 7 O From the data presented, it follows that the biological activity of Aci ribscnuclease. manifesting in its antiviral effect, immobilization with dextran increases substantially. m-Aminobenzyloxymethyl dextran: in an amount corresponding to its content in the preparation of ribonuclease covalently bound to dextran, has no antiviral effect. Claim 1. Method for obtaining immobilized nucleases by binding them to an extensible matrix, characterized in that, in order to enhance the biological activity of the nucleus while preserving their injection form, use of nucleases isolated from organisms phylogenetically distant from the target organism that are covalento bind by diazoconnection with m-aminobyenz p. hydroxymethyldextran. 2. The method according to claim 1, characterized in that m-aminobenzyloxy-dextran with mol. Weight is used to immobilize nucleases. 400OO-600OO. Sources of information taken into account in the examination 1. Dixon N1, Webb E., Enzymes, Mir, 1966, p. nineteen. 2.Kestner A.I. Chemistry Advances. 1974, t. X, Vyp.8. 3.T. M. S. C1iein, Nc -tuv-e, 1971, 229, No. 5280, p. 117. 4 / Wypes J.R. , Donu (i P., (lv M-D. YusK.Wyuruv. ActQ, / 1971, 25O; p. 522 (prototype).